Academic literature on the topic 'Non-OR copper binding site'
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Journal articles on the topic "Non-OR copper binding site"
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Erales, Jenny, Brigitte Gontero, Julian Whitelegge, and Frédéric Halgand. "Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis." Biochemical Journal 419, no. 1 (March 13, 2009): 75–86. http://dx.doi.org/10.1042/bj20082004.
Full textAbstract:
CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12–Cu2+ complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp16–Asp23, Asp38–Lys50 and Asp70–Glu76, with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.
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McArdle, H. J., S. M. Gross, D. M. Danks, and A. G. Wedd. "Role of albumin's copper binding site in copper uptake by mouse hepatocytes." American Journal of Physiology-Gastrointestinal and Liver Physiology 258, no. 6 (June 1, 1990): G988—G991. http://dx.doi.org/10.1152/ajpgi.1990.258.6.g988.
Full textAbstract:
It is possible, in vitro, to label albumin with copper either exclusively on the specific binding site or partly on the specific site and also on other sites by altering the pH at which the two ligands are mixed. Copper attached exclusively to the specific site is taken up more rapidly than copper attached to that site and others on albumin. The effect is proportional to the amount of copper on the specific site. Additional histidine stimulates uptake irrespective of the copper binding site on albumin. The effect is related to the histidine on position 3 of the albumin, since it is not seen when dog albumin is labeled under the same conditions. The data suggest that the cell recognizes and presumably binds the copper-albumin (CuAlb) complex but may preferentially recognize the ternary complex formed by CuAlb and histidine. We suggest that, in vivo, copper is bound mainly as the ternary complex and that the structure formed, presumably similar to that formed by a copper-histidine complex, is what is actually recognized by the cell. After binding, the albumin and histidine are released, possibly by a reduction step, and the copper is transported across the membrane. If the copper cannot be transported (as occurs when the cells are incubated at 4 degrees C), it blocks further binding of the ternary complex.
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Cater, Michael A., Sharon La fontaine, and Julian F. B. Mercer. "Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)." Biochemical Journal 401, no. 1 (December 11, 2006): 143–53. http://dx.doi.org/10.1042/bj20061055.
Full textAbstract:
The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hep-atocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular traf-ficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mut-ation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the consti-tutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.
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Calabrese, L., and M. Carbonaro. "An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin." Biochemical Journal 238, no. 1 (August 15, 1986): 291–95. http://dx.doi.org/10.1042/bj2380291.
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The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent ‘oxidative’ attack of proteins and lipids.
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Sinopoli, Alessandro, Antonio Magrì, Danilo Milardi, Matteo Pappalardo, Pietro Pucci, Angela Flagiello, Jeremy J. Titman, et al. "The role of copper(ii) in the aggregation of human amylin." Metallomics 6, no. 10 (2014): 1841–52. http://dx.doi.org/10.1039/c4mt00130c.
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Copper(ii) coordination to human amylin has an influence on the aggregation and cytotoxic features of the polypeptide. Comparative investigations, carried out on a model peptide encompassing the 17–29 aminoacid region of amylin containing the putative metal binding site, support the non-fibrillar nature of the copper(ii) complexes.
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Kekez, Ivana, Mihovil Faletar, Mario Kekez, Laura Cendron, Maya Wright, Giuseppe Zanotti, and Dubravka Matković-Čalogović. "Copper Binding and Oligomerization Studies of the Metal Resistance Determinant CrdA from Helicobacter pylori." Molecules 27, no. 11 (May 24, 2022): 3387. http://dx.doi.org/10.3390/molecules27113387.
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Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoCbut lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal–protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.
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NAKAMURA, Motoyoshi, Tasuku NAKAJIMA, Yasunori OHBA, Seigo YAMAUCHI, Byung Rho LEE, and Eiji ICHISHIMA. "Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis." Biochemical Journal 350, no. 2 (August 23, 2000): 537–45. http://dx.doi.org/10.1042/bj3500537.
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Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g‖ and CuA‖, we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).
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Maghool, Shadi, Michael T. Ryan, and Megan J. Maher. "What Role Does COA6 Play in Cytochrome C Oxidase Biogenesis: A Metallochaperone or Thiol Oxidoreductase, or Both?" International Journal of Molecular Sciences 21, no. 19 (September 23, 2020): 6983. http://dx.doi.org/10.3390/ijms21196983.
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Complex IV (cytochrome c oxidase; COX) is the terminal complex of the mitochondrial electron transport chain. Copper is essential for COX assembly, activity, and stability, and is incorporated into the dinuclear CuA and mononuclear CuB sites. Multiple assembly factors play roles in the biogenesis of these sites within COX and the failure of this intricate process, such as through mutations to these factors, disrupts COX assembly and activity. Various studies over the last ten years have revealed that the assembly factor COA6, a small intermembrane space-located protein with a twin CX9C motif, plays a role in the biogenesis of the CuA site. However, how COA6 and its copper binding properties contribute to the assembly of this site has been a controversial area of research. In this review, we summarize our current understanding of the molecular mechanisms by which COA6 participates in COX biogenesis.
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D’Angelo, Paola, Stefano Della Longa, Alessandro Arcovito, Giordano Mancini, Andrea Zitolo, Giovanni Chillemi, Gabriele Giachin, Giuseppe Legname, and Federico Benetti. "Effects of the Pathological Q212P Mutation on Human Prion Protein Non-Octarepeat Copper-Binding Site." Biochemistry 51, no. 31 (July 27, 2012): 6068–79. http://dx.doi.org/10.1021/bi300233n.
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Eakin, Catherine M., Jefferson D. Knight, Charles J. Morgan, Michael A. Gelfand, and Andrew D. Miranker. "Formation of a Copper Specific Binding Site in Non-Native States of β-2-Microglobulin†." Biochemistry 41, no. 34 (August 2002): 10646–56. http://dx.doi.org/10.1021/bi025944a.
Full textDissertations / Theses on the topic "Non-OR copper binding site"
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Mai, Phuong Thao. "The potential role of copper binding sites in prion propagation." Doctoral thesis, SISSA, 2014. http://hdl.handle.net/20.500.11767/3905.
Full textAbstract:
Transmissible spongiform encephalopathies (TSEs) or prion diseases are caused by a post-translational conversion of the normal cellular form of the prion protein (PrPC) into the pathological and infectious isoform denoted as prion or PrPSc. PrPC has been shown as a high-affinity copper-binding protein, and to a lesser extent binding to other divalent cations through the octarepeat region (OR) and the non-OR copper binding sites located in the disordered N-terminal domain. Studies on the role of copper in promoting prion conversion and infectivity yielded controversial results. In this work, we explored the role of histidine residues which are crucial for copper coordination in prion conversion using a combination of cell culture and cell-free approaches. The first evidence was derived from chronically prion-infected neuronal murine cells (ScN2a) transiently expressed in murine PrPC carrying artificial mutations at histidines located both at the OR and non-OR regions. We found that the lack of each histidine in the OR has neither effect on prion replication nor protein maturation and trafficking. Intriguingly, mutagenesis of histidine 95 (H95Y) does enhance prion conversion leading to de novo infectious material formation and cause aberrant accumulation during protein trafficking. Thus, we hypothesize that H95 could function as molecular switch for prion conversion, and copper bound to this residue may function in protein conformation stabilization. We also propose a cellular model for prion formation in cells expressing the H95Y mutant. Interestingly, our data may establish a platform for rationally designed experiments aimed at elucidating whether the H95Y mutation may cause de novo prion diseases in transgenic mice.
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Tran, Thanh Hoa. "The non-octarepeat copper-binding site of the prion protein and its potential role in prion conversion." Doctoral thesis, SISSA, 2016. http://hdl.handle.net/20.500.11767/4870.
Full textAbstract:
Transmissible spongiform encephalopathy (TSE) or prion diseases are fatal neurodegenerative disorders caused by a change in conformation of the prion protein from the normal cellular form (PrPC) to a misfolded form (PrPSc). Prion protein has long been known as a copper binding protein. Although the functional implication of copper binding to PrP is not yet clear, it is believed that copper is an important cofactor in prion disease. Therefore, the aim of this work is to determine the potential role of copper in prion conversion. Copper can effectively bind to PrPC via histidine residues in the octapeptide repeats (OR) and the non-OR region located in the disordered N-terminal of the protein. Our hypothesis is that if copper binding plays a role in prion disease, removal of histidine residues in N-terminal domain may affect the prion conversion process. To examine this, we created a series of mutant murine PrP (MoPrP) molecules by replacing histidine residue at OR and non-OR region by tyrosine. These constructs were transfected into ScN2a cells and the efficiencies of prion conversion were evaluated. The results showed that replacing histidine by tyrosine at non-OR region led to an increasing of PrPSc conversion. When copper was removed by cuprizone, the construct with tyrosine at non-OR site (MoPrP H95Y) did not alter the level of PrPSc which meanwhile increased in case of the MoPrP wild-type (WT). To test these mutants in vitro, we produced recombinant protein, did the fibrilization assay and compared the lag phase. The result clearly showed that these constructs with tyrosine (H95Y, H110Y, H95Y/H110Y) need a shorter time to aggregate making fibrils faster than WT MoPrP. In particular, N2aPrP-/- cells expressing non-OR mutations PrP spontaneously cause prion formation that can be detected by ASA or PMCA. Moreover, transgenic mice overexpressing MoPrP H95Y showed clinical signs and died at 100 days with PK-resistant PrP in their brain. Based on these data, we can concluded that the substitution of histidine by tyrosine at non-OR region can enhance PrPC-PrPSc conversion process, and the non-OR copper binding site may have a critical role in this process.
Books on the topic "Non-OR copper binding site"
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van Dorp, Eveline L. A., Douglas Eleveld, Erik Olofsen, and Jaap Vuyk. Drug distribution and elimination in anaesthetic practice. Edited by Michel M. R. F. Struys. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780199642045.003.0012.
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An understanding of pharmacokinetics is vital for the practice of anaesthesia. Drugs are, after administration, distributed throughout the body to the effect site (mostly the brain) to exert their effects. This can be influenced by differences in protein binding, systemic blood flow, and concomitant medication. Elimination of drugs from the body is through two main routes: either unchanged through the kidneys or through metabolism by the liver (and consecutive excretion through the kidneys). This process depends on the amount of hepatic blood flow and the amount of hepatic extraction. This in turn depends on the amount of protein binding and the intrinsic hepatic clearance. The cytochrome P450 enzyme family also plays an important role in drug elimination. Individual differences in enzyme activity can lead to differences in drug effect and clearances. Changes in enzyme activity by enzyme induction and inhibition can also be of influence on drug clearance. Compartmental, non-compartmental, and physiologically based models, and various statistical approaches to estimate these models, may be used to analyze the distribution and elimination of anaesthetic agents.
Book chapters on the topic "Non-OR copper binding site"
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Nauen, R., and P. Jeschke. "Basic and Applied Aspects of Neonicotinoid Insecticides." In Green Trends in Insect Control, 132–62. The Royal Society of Chemistry, 2011. http://dx.doi.org/10.1039/bk9781849731492-00132.
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Neonicotinoid insecticides are considered the the fastest-growing class of insecticides in modern crop protection since the introduction of pyrethroids, with widespread use against a broad spectrum of sucking and several chewing pests. Seven structurally different neonicotinoid insecticides are commercially availabale. They act selectively as agonists on insect nicotinic acetylcholine receptors (nAChRs), their molecular target site, with little or no binding to vertebrate receptors. Because of the relatively low risk for non-target organisms and environment, the high target-specificity of neonicotinoid insecticides and their versatility in application methods, this important class has to be maintained globally for sustainable integrated pest management strategies and insect resistance management programmes. Combined with innovative concepts for life-cycle management such as optimized formulations, neonicotinoids will be the most important chemical class within the next few years for the control of some of the globally most destructive pest insects in many agronomic cropping systems.
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Angal, S., and Simon D. Roe. "Purification by exploitation of activity." In Protein Purification Techniques. Oxford University Press, 2001. http://dx.doi.org/10.1093/oso/9780199636747.003.0013.
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Proteins carry out their biological functions through one or more binding activities and, consequently, contain binding sites for interaction with other biomolecules, called ligands. Ligands may be small molecules such as substrates for enzymes or larger molecules such as peptide hormones. The interaction of a binding site with a ligand is determined by the overall size and shape of the ligand as well as the number and distribution of complementary surfaces. These complementary surfaces may involve a combination of charged and hydrophobic moieties and exhibit other short-range molecular interactions such as hydrogen bonds. This binding activity of a protein, which is stereoselective and often of a high affinity, can be exploited for the purification of the protein in a technique commonly known as affinity chromatography. The operation of affinity chromatography involves the following steps: (a) Choice of an appropriate ligand. (b) Immobilization of the ligand onto a support matrix. (c) Contacting the protein mixture of interest with the matrix. (d) Removal of non-specifically bound proteins. (e) Elution of the protein of interest in a purified form. At best, affinity chromatography is the most powerful technique for protein purification since its high selectivity can, in principle, allow purification of a single protein of low abundance from a crude mixture of proteins at higher concentrations. Secondly, if the affinity of the ligand for the protein is sufficiently high, the technique offers simultaneous concentration from a large volume. In practice, such single-step purifications are not common and successful affinity chromatography requires careful consideration of a number of parameters involved. The remainder of this chapter attempts to guide the experimenter in the selection and use of affinity adsorbents for protein purification. For more extensive information on this technique the reader is advised to consult the many excellent texts on this subject as well as proceedings of symposia. The construction of an affinity adsorbent for the purification of a particular protein involves three major factors: (a) Choice of a suitable ligand. (b) Selection of a support matrix and spacer. (c) Attachment of the ligand to a support matrix. The criteria for making these decisions are discussed in the following sections.
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Abulafia, David. "Towards the Garden of the Hesperides, 1000 BC–400 BC." In The Great Sea. Oxford University Press, 2011. http://dx.doi.org/10.1093/oso/9780195323344.003.0016.
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The impact of contact with the eastern Mediterranean was felt in very different ways within what we now call Italy. Greek culture seeped more slowly into the everyday life of the native peoples of Sicily – Sikans, Sikels and Elymians – than into the life of the peoples of Tuscany and Latium. In Sicily, both the Greeks and the Carthaginians kept themselves largely apart from the native population. Sardinia, rich in minerals, had for centuries been the seat of a lively civilization characterized by the stone towers known as nuraghi, of which many thousands still dot the island; they were surrounded by what seem to have been prosperous villages, firmly rooted in the rich agricultural resources of the island. They began to be built around 1400 BC, but new nuraghi were still being constructed well into the Iron Age. In the Mycenaean era, there had been some contact with the outside world, as eastern Mediterranean traders arrived in search of copper. The wealth of the native elite as far back as the second millennium BC can be measured from the tombs of Anghelu Ruju, near Alghero in north-western Sardinia; these are among the richest to have been unearthed in late Neolithic and early Bronze Age western Europe, and they indicate contact with Spain, southern France and the eastern Mediterranean. The Spanish influence can be traced in the bell beaker jars found at this site. Another Spanish connection was linguistic. The Sardinians left no written records, whether because they did not use writing or because they used friable materials that have failed to survive. But place-names, many in current use, provide suggestive evidence, as does the Sard language, a distinctive form of late vulgar Latin that incorporates a number of pre-Latin words within its many dialects. It appears that the nuraghic peoples spoke a language or languages related to the non-Indo-European language Basque. Thus a Sard word for a young lamb, bitti, is very similar to a Basque term for a young goat, bitin.
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Giacovazzo, Carmelo. "Isomorphous replacement techniques." In Phasing in Crystallography. Oxford University Press, 2013. http://dx.doi.org/10.1093/oso/9780199686995.003.0019.
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The isomorphous replacement method is a very old technique, used incidentally by Bragg to solve NaCl and KCl structures: it was later formulated in a more general way by Robertson (1935, 1936) and by Robertson and Woodward (1937). Its modern formulation is essentially due to Green et al. (1954) and to Bragg and Perutz (1954), who applied the method to haemoglobin. The technique has made possible the determination of the first three macromolecular structures, myoglobin, haemoglobin, and lysozyme. The approach may be summarized as follows. Suppose that the target structure is difficult to solve (e.g. it is a medium-sized structure, resistant to any phasing attempt, or it is a protein with bad data resolution) and we want to adopt isomorphous replacement techniques. Then we should perform the following steps: (a) Collect the diffraction data of the target structure; in the following we will suppose that it is the native protein. (b) Crystallize a new compound in which one or more heavy atoms are incorporated into the target structure. This new compound is called derivative. (c) Check if the operations in (b) heavily disturb the target structure. If not, the derivative is called isomorphous; then, only local (in the near vicinity of the binding site) structural modifications are induced by the heavy atom addition. Non-isomorphous derivative data are useless. (d) Use the two sets of diffraction data, say set {|FP|} of the target structure and set {|Fd|} of the isomorphous derivative, to solve the target structure. The above case is referred to as SIR (single isomorphous replacement). The reader should notice that redundant experimental information is available; indeed, two experimental sets of diffraction data relative to two isomorphous structures may be simultaneously used for solving the native protein. The redundancy of the experimental information allows crystal structure solution even if data resolution is far from being atomic (e.g. also when RES is about 3 or 4 Å, and even more in lucky cases). Imperfect isomorphism may hinder crystal structure solution. Then, more derivatives may be prepared; their diffraction data may be used in a combined way and may more easily lead the phasing process to success.
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P. James, Steven, and Dena Bondugji. "Gamma-Aminobutyric Acid (GABA) and the Endocannabinoids: Understanding the Risks and Opportunities." In Gamma-Aminobutyric Acid - Neuropsychiatric and Therapeutic Implications [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.99242.
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The Gamma-aminobutyric acid (GABA) system is the main inhibitory neurotransmitter system in the central nervous system (CNS) of vertebrates and is involved in critical cellular communication and brain function. The endocannabioid system (ECS) was only recenty discovered and quickly recognized to be abundantly expressed in GABA-rich areas of the brain. The strong relationship between the GABA system and ECS is supported both by studies of the neuraoanatomy of mammalian nervous systems and the chemical messaging between neurons. The ECS is currently known to consist of two endocannabinoids, Anandamide (AEA) and 2-Arachidonyl Glycerol (2-AG), that function as chemical messengers between neurons, at least two cannabinoid receptors (CB1 and CB2), and complex synthetic and degradative metabolic systems. The ECS differs from the GABA system and other neurotransmitter systems in multiple ways including retrograde communication from the activated post-synaptic neuron to the presynaptic cell. Together, this molecular conversation between the ECS and GABA systems regulate the homeostasis and the chemical messaging essential for higher cortical functions such as learning and memory and may play a role in several human pathologies. Phytocannabinoids are synthesized in the plant Cannabis sativa (C. sativa). Within the family of phytocannabinoids at least 100 different cannabinoid molecules or derivatives have been identified and share the properties of binding to the endogenous cannabinoid receptors CB1 and CB2. The well-known psychoactive phytocannabinoid Δ9-tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) are just two of the many substances synthesized within C. sativa that act on the body. Although the phytocannabinoids THC and CBD bind to these endogenous receptors in the mammalian CNS, these plant derived molecules have little in common with the endocannabinoids in structure, distribution and metabolism. This overlap in receptor binding is likely coincidental since phytocannabinoids evolved within the plant kingdom and the ECS including the endocannabinoids developed within animals. The GABA and ECS networks communicate through carefully orchestrated activities at localized synaptic level. When phytocannabinoids become available, the receptor affinities for CB1 and CB2 may compete with the naturally occurring endocannabinoid ligands and influence the GABA-ECS communication. In some instances this addition of phytocannabinoids may provide some therapeutic benefit while in other circumstances the presence of these plant derived ligands for the CB1 and CB2 receptors binding site may lead to disruption of important functions within the CNS. The regulatory approval of several THC products for nausea and vomiting and anorexia and CBD for rare pediatric seizure disorders are examples of some of the benefits of phytocannabinoids. Concerns regarding cannabis exposure in utero and in the child and adolescence are shrill warnings of the hazards associated with disrupting the normal maturation of the developing CNS.
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"those from Rendina (no. 38) and Fonti di San Callisto (no. 36) (fig. 7, 1 and 2) to the almost abstract, as on the three figurines from Riparo Gaban (nos 8, 9 and 10) (fig. 5, 3 and fig. 7, 3 and 4). In these last cases, the depiction of the sexual organs is so stylised that they could perhaps be included in our third category, of sexual symbols. However, we have included them here because, however stylised, the sexual organs are shown on these figurines in approximately correct anatomical relation (i.e. breasts are shown below heads and vulvas below breasts), so as to suggest that whole female figures are being represented; as will be seen this is not the case with the other examples of sexual symbolism. If, for the purposes of this discussion, we ignore the great typological diversity of the figurines and consider them all together, we find an overwhelming preponderance of female figures over male ones. In fact there are only two specifically male figures, both probably from Copper Age contexts: the surface find from the Copper Age settlement site of Ortucchio in central Italy (no. 35) (fig. 8) and the large figure from a votive pit in the Sicilian Copper Age cemetery of Piano Vento (no. 58) (fig. 9). The significance of the dating of these figurines will be discussed below. In contrast, the number of female figurines is at least 30, and possibly 35, if the 'probably female' examples are included. Moreover, if we are right in attributing some of the north Italian heads (particularly nos 16, 20 and 21) to figurines of specifically female type, the number would go up still further. It is worth making the point here that among the Italian figurines we do not find a specific category of sexless figures, as occurs elsewhere, e.g. at Knossos, where in Ucko's analysis (1962: 40), there were more sexless figures than sexed ones. In our list, the figurines with 'no indication of sex' are almost all fragmentary and represent body parts, especially heads, which are not sexually specific. The only complete figurines which have no sexual features shown are the two stone figurines from Cerno and Arnesano (nos 1 and 46) (fig. 6) and these in fact represent heads on largely unworked cylindrical shafts. There are also four cases of heads which do not seem to be broken off, but complete in themselves (nos 39 (fig. 10), 40, 50 and 51); this category represents a special case and will be discussed below. It is likely that most of the figurines were originally specifically sexed and that the majority was female. Female figurines occur in both the earlier and later chronological periods, in all areas of Italy and on all the types of sites where figurines are represented. Although the female sex of the figurines is not in doubt, there seems to be little emphasis on fertility. None of the Italian figurines is shown as pregnant and, although V Tinè has claimed that the example from Favella (no. 47) might have been in the birthing position, this is far from clear. None of the figures is shown doing anything; they are mostly depicted as standing, with a few shown seated (nos 4, 25, 38 and possibly 47). In as far as there is emphasis on the sexual organs, it is possible that sexuality is being emphasised rather than fertility. In any case, while there seems to be little emphasis on the limbs and other 'non-sexual' body parts, heads and faces are given at least as much attention as bodies — in contrast to the the Upper Palaeolithic 'Venuses' — and we should be careful about placing too much emphasis on the sexual organs depicted. Cultural indicators of gender Most of the figurines appear without indications of dress or any associated artefacts. The only exceptions are the clay head from Grot ta Pacelli (no. 40), which has an apparent elaborate headdress and four, or possibly five, figurines which have V-shaped features, incised, impressed or in relief, on the neck, which are sometimes interpreted as necklaces. One example is the bone figurine from Riparo Gaban (no. 8) which has a 'necklace' and a possible 'belt', both incised, on a female figurine with both breasts and vulva marked (fig. 7, 3). The other two definite incised Vs occur on figurines from Vhò (no. 14), which is a clear female figure with breasts shown in relief (fig. 2, 2)and from Arnesano (no. 46), where it occurs on a stone figurine without indications of sex (fig. 6, 1). One of the clearly female figurines with breasts from Passo di Corvo (no. 44) has a series of impressed dots in a V-." In Gender & Italian Archaeology, 116–45. Routledge, 2016. http://dx.doi.org/10.4324/9781315428178-22.
Full textConference papers on the topic "Non-OR copper binding site"
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Kaufman, Randal J., Debra D. Pittman, Louise C. Wasley, W. Barry Foster, Godfrey W. Amphlett, and Alan R. Giles. "DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644769.
Full textAbstract:
Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.
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Monroe, D. M., D. W. Deerfield, D. L. Olson, T. N. Stewart, H. R. Roberts, R. G. Hiskey, and L. G. Pedersen. "BINDING OF CALCIUM TO HUMAN AND BOVINE FACTOR X." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643835.
Full textAbstract:
Human and bovine factor X contain 11 and 12 glutamyl residues respectively within the first forty amino terminal residues that are posttranslationally modified to y-carboxyglutamyl (Gla) residues. Calcium binding to these Gla residues and at other sites is critical for activity in factor X. We have measured calcium binding to human factor X by equilibrium dialysis for the first time. We have also re-examined calcium binding to bovine factor X in order to compare the two species. Factor X (10 μM) was incubated with 45Ca in 20 mM Tris (pH 7.5), 100 mM NaCl in a half cell separated by a 12-14000 molecular weight fast-equilib-rium disk membrane at 25°C for 24 hours. Four aliquots (100 μL each) were removed from each side of the cell and counted. Data were analyzed with a variety of models that allow for more than one class of binding site and for cooperativity among binding sites. Calcium binding to bovine factor X was best simulated by a model that assumes 1 very tight site, 3 cooperative tight sites, and 18 equivalent, non-interacting sites. Based on data from des(Gla)factor X, the first site is probably a high affinity non-Gla binding site. Our results differ from two previously published reports that indicated either 1 tight and 39 loose noncooperative sites (R.H. Yue & M.M. Gertler (1978) Thrombos. Haemostas. (Stuttg.) 40, 350) or 20 calcium binding sites with the first 4 being cooperative (M.J. Lindhout & H.C. Hemker (1978) Biochimica Biophysica Acta 533, 318). Our data on human factor X fit the same model as used for bovine factor X; however, coop-erativity is less in the 3 cooperative sites. Shown below are the first six thermodynamic equilibrium constants derived from a Scatchard analysis of binding data (values are M−1).Both proteins demonstrate the same total number of binding sites and essentially the same value for the first, tight binding site. Bovine factor X exhibits cooperativity, whereas human factor X has reduced cooperativity.
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DePoli, Patricia, Theresa Bacon-Baquley, and Daniel A. Walz. "THROMBOSPONDIN INTERACTION WITH PLASMINOGEN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643824.
Full textAbstract:
Platelet thrombospondin (TSP) interacts with plasminogen in a specific and saturable manner. TSP can form a trimolecular complex with histidine-rich glycoprotein and plasminogen and the plasminogen within such complexes can reportedly be activated by tissue plasminogen activator. We have studied the interaction of TSP with plasminogen using Western blotting of plasminogen, reduced plasmin and the elastase-generated fragments of plasminogen and their binding of iodinated TSP. TSP was found to specifically bind to plasminogen and the heavy (non-enzyme) chain of plasmin in a calcium-independent manner. Binding could be blocked by preincubation of the immobilized plasminogen or plasmin with an excess of unlabeled TSP. Plasminogen domains (kringles) were generated by limited eTastase proteolysis. TSP bound specifically to a single 51 kDa plasminogen fragment. The elastase-generated fragments were separated by lysine-Sepharose chromatography and their identities established by amino acid composition and amino-terminal sequence analysis. The 51 kDa plasminogen fragment bound to lysine-Sepharose and had an amino-terminal sequence corresponding to kringle 4 (K4) and a composition consistent with that of K4-K5-plasmin. TSP binding to this fragment was not blocked by the presence of an excess of the fragment K1-K2-K3, K4, nor miniplasminogen (K5-plasmin). Binding does not appear to be directly dependent upon the specific high-affinity lysine binding site of the 51 kDa fragment. Our data suggests that thrombospondin interacts with plasminogen at a single distinct site, and that this recognition site is at or near the K4-K5 contiguous region of plasminogen.
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Broeseker, T. A., M. D. P. Boyle, and R. Lottenberg. "PATHOGENIC BACTERIA HAVE HIGH AFFINITY RECEPTORS SPECIFIC FOR PLASMIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644391.
Full textAbstract:
Binding of the key fibrinolytic enzyme, plasmin, to certain pathogenic group A streptococci was studied. In these experiments the ability of a group A streptococcal strain, 64/14, to bind either 125I-human plasminogen or the same label following activation with urokinase was measured. It was found that this strain bound <10% of the labeled plasminogen but >70% of labeled plasmin. This property distinguishes the plasmin receptor from streptokinase. These bacteria did not express a common serine protease receptor/inhibitor since they failed to bind labeled trypsin or urokinase. Maximal binding of plasmin occurred between pH 6.0 and 8.0 and in the ionic strength range of 50-200 mM salt. The Kd of plasmin binding to bacteria was approximately 10-10 M at pH 7.4 in 150 mM salt. This was determined by a non-linear least squares analysis of equilibrium binding data. Binding was reversibly inhibited by either epsilon aminocaproic acid (I50 of 0.2 mM) or lysine (I50 of 3.0 mM) suggesting the involvement of the high affinity lysine binding site of plasmin in its binding to bacteria. Bacterial bound plasmin retains its enzymatic activity, being capable of cleaving chromogenic substrates and solubilizing a fibrin- clot. The bacterial bound enzyme activity was inhibited by the low molecular weight inhibitors aprotinin and phe-pro-arg chloromethyl ketone but not by alpha-2 plasmin inhibitor. The ability of bacteria to acquire membrane associated proteolytic activity which cannot be physiologically inhibited may contribute to their tissue invasive properties.
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Berndt, M. "STRUCTURE AND FUNCTION OF THE GLYCOPROTEIN Ib-IX COMPLEX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643729.
Full textAbstract:
At high shear flow, the adhesion of platelets to the exposed vascular subendothelium requires von Willebrand factor (vWF) and is dependent upon a specific platelet membrane adhesion receptor, the human platelet membrane glycoprotein (GP) Ib-IX complex. Recent evidence suggests that vWFbinding to the GP Ib-IX complex plays an important role in other key aspects of hemostasis and thrombosis such as shear-induced platelet aggregation and the interaction of platelets with fibrin.Studies in our laboratory with a seriesof murine monoclonal antibodies directed against epitopes on GP lb, GP IX, or against complex-specificepitopes indicate that GP lb and GP IX exist in the intact platelet membrane as a native heterodimer complex(-25,000 copies/platelet). By analysis onSDS-polyacrylamide gels, GP lb has an apparent molecular weight of 170,000 and cnsists of two disulfide-linked subunits, GP Iba (Mr = 135,000) and GP Ibβ (Mr = 25,000),whilst GP IX has an equivalent molecularweight under both nonreducing and reducing conditions (Mr = 22,000).The ±-chain ofGP lb has a central macroglycopeptide core (Mr =90,000) which is highly glycosylated. At each end of themacroglycopeptide region is a domainsensitive to proteolytic cleavage. Cleavage at the end proximal to the platelet membrane, e.g. by calpain, Serratia marcescens metalloprotease and trypsin, generates two fragments :a Mr =130,000 highly glycosylated fragment termed glycocalicin anda membrane-associated region consisting ofa Mr -25,000 fragment that remains disulfide-linkedto GP Ibβ and associated with GP IX. In resting platelets, the membrane-associated region spans the lipid bilayer linking the GP Ib-IX complex to the platelet endoskeleton via actin-binding protein. This membrane-associated region also contains the domain(s) recognized by quinine/quinidine drug-dependent antibodies. Cleavage at the plasma end of the macroglycopeptide, e.g. by human leukocyte elastase, generates a poorly glycosylated Mr = 45,000 fragment of GP Ibα (peptide tail region) and a heavily glycosylated Mr = 100,000 fragment that remains disulfide-linked to GP Ibg and associated with GP IX. Platelets lacking the N-terminal peptide tail region of GP Iba fail to agglutinate with ristocetin and vWF and show a delayed response to a-thrombin.Polyclonal and monoclonal antibodies against this region also inhibit both these platelet responses suggesting that the peptide tail region contains the binding sites for both α-thrombinand vWF. Rotary shadowingelectron microscopy of purified GP Ib-IX complex shows the structure to be highlyasymmetric with each complex existing asa flexible rod with a globular domain at each end. The overall length of the complexwas =60 nm.The smaller globular domain (peptidetail region) has a diameter of =9nm; the larger globular domain (membrane-associated region), a diameter of =16 nmWe have recently examined whetherthe human platelet GP Ib-IX complex is the receptor for the ristocetin-dependent binding of vWF by reconstitution with the purified components using a solid-phasebead assay. Our approach was to indirectlybind and orientate the GP Ib-IX complex onthe beads via a monoclonal antibody directed against the membrane-associated region of the complex (FMC 25, epitope on GP IX).Immunobeads were chosen as the insoluble matrix because they are uniform in size (=10μm in diameter), impermeable,specifically designed for the coupling of IgG, and because, like platelets, the beads have a net negative charge atneutral pH.Specific binding of 125I-labelled human vWF tothe GP Ib-IX complex-coated immunobeads was strictly ristocetin-dependent with maximal binding occurring atristocetin concentrations >1 mg/ml. Ristocetin-dependent specificbinding of 125I-labelled vWF was saturable.Scatchardanalysis revealed a single classof binding sites for vWF with purified GP Ib-IX complex.Monoclonal antibodies against the Mr = 45,000 peptide tail region ofGP lb which stronglyinhibitthe ristocetin-dependent binding ofvWF toplatelets also strongly inhibited the ristocetin-dependent binding of vWFto the GP Ib-IX coated beads. Monoclonalantibody against either themacroglycopeptide or membrane-associated regions of the GPIb-IX complex did not inhibit the ristocetin-dependent binding of vWF to platelets or to the GP Ib-IX complex-coated beads. Similar functional correlations were obtained with anti-vWFmonoclonal antibodies. The reconstitutiondata therefore confirm the functional roleof the GP Ib-IX complex as a major plateletvWF receptor. The region ofthe vWF molecule involved in binding to the GP Ib-IX complex has been localized toa Mr =50,000 domain towards theN-terminal end of the vWF subunit. The reconstitution assay should prove useful in the further definition of active peptides of vWF that bind tothe human platelet GP Ib-IX complex.
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Hessing, Martin, Joost C. M. Meijers, Jan A. van Mourik, and Bonno N. Bouma. "MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S AND C4b-BINDING PROTEIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644291.
Full textAbstract:
Protein S (PS) circulates in plasma both free and in reversible association with the complement component C4b-binding protein (C4bp). Only free PS is functional as a cofactor for activated protein C (APC). Cleavage of PS by thrombin at a site near the r-carboxyglutamic acid domain is associated with a loss of cofactor activity. This may be a control mechanism for the anticoagulant activity of APC. These observations led us to investigate the role of C4bp and thrombin in the regulation of PS. Complex formation between purified PS and C4bp was studied in plasma and in a system with purified components. 125I-labeled PS was first incubated with either C4bp or citrated plasma and then subjected to polyacrylamide gelelectrophoresis in the absence of SDS. The formation of the C4bp-PS complex in plasma and in the purified system was demonstrated by autoradiography. Crossed immuno-electrophoresis using an antiserum against PS was performed in the presence of 8 mM EDTA. Human citrated plasma showed two precipitin peaks. Free PS migrated rapidly in the first dimension, whereas the C4bp-PS complex was just anodal to the application slot. The addition of C4bp to either plasma or purified PS resulted in the disappearance of the free PS peak and an increase of the slower migrating peak. The effect of purified C4bp on the PS-cofactor function of APC was studied in citrated plasma. The prolongation of the APTT induced by the addition of APC could be inhibited by the addition of increasing amounts of C4bp. Monoclonal antibodies to PS and C4bp were prepared and characterized. The monoclonal antibodies to either PS or C4bp did not block the complex formation between and PS, as was demonstrated by dot blotting of C4bp with 125I-PS and agarose gelelectrophoresis followed by Western blotting. Three out of 7 monoclonal antibodies to PS did not detect PS after thrombin cleavage on an immunoblot after non-reduced SDS polyacrylamide gelelectrophoresis. These 3 antibodies gave a significant shortening of the prolonged APTT induced by the addition of APC to normal plasma, indicating that these monoclonals inhibited the cofactor function of PS. The other 4 monoclonals to PS that did detect PS after thrombin cleavage on an immunoblot, gave only a minor inhibition of the PS cofactor function.
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Koneti Rao, A., and Maria A. Kowalska. "ADP-INDUCED CYTOPLASMIC CALCIUM MOBILIZATION AND SHAPE CHANGE IN PLATELETS ARE MEDIATED BY DIFFERENT BINDING SITES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644466.
Full textAbstract:
Platelet stimulation with ADP results in a number of responses including increase in cytoplasmic ionized calcium concentration [Ca2+]i, shape change, aggregation, secretion, and inhibition of cAMP accumulation caused by PGI2.5'-Fluorosulphonylbenzoyladenosine (FSBA), which covalently labels ADP binding site on platelets, blocks platelet shape change but not inhibition of cyclic AMP levels by ADP, while p-chloromercuribenzenesulfonate (pCMBS), a non-penetrating thiol reagent, blocks ADP-induced inhibition of adenylate cyclase but not shape change. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i to determine whether it is linked to the binding site mediating shape change or that for inhibition of adenylate cyclase. In platelets loaded with Ca2+ indicators, quin 2 or fura 2, and in presence of adenosine deaminase (AD), FSBA (50-200 μM) induced a dose-dependent, rapid rise in [Ca2+]i. from basal levels of 70-90 nM to peak levels of 300-500 nM in the presence of 1 mM external Ca2+ providing direct evidence that FSBA is a platelet agonist. The [Ca2+ ]i. returned to near basal levels over 30 min. The effect of FSBA on [Ca2+]i. was inhibited by ZK 36,374 (40 nM), a stable PGI2 analog. AdP concentrations eliciting similar responses were about 10-fold less than those for FSBA. Platelet incubation with FSBA (50-100 μM) in the presence of AD for 30 min (to ensure optimal covalent labelling of the ADP binding sites) abolished shape change but jjid not inhibit ADP (5, 25 μM)-induced increase in [Ca2+]i. or block the inhibitory effect of ADP on cAMP accumulation in1platelets exposed to ZK 36,374 (50 nM) in.presence of theophylline (7 mM). Incubation with pCMBS (5-100 pM, 2 min) abolished the effect of ADP on [Ca2+]. and on the inhibition of cAMP levels; shape change was not 1 inhabited even at 1 mM. pCMBS (0.5-1 mM) inhibited the rise in [Ca2+ ]. by FSBA alone. These observations suggest that ADP-induced Ca mobilization is mediated by platelet binding sites which are distinct from those mediating shape change but probably the same as those modulating adenylate cyclase.
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Bourin, M. C., I. Bjôrk, M. C. Boffa, and U. Lindahl. "EFFECT OF RABBIT THROMBOMODULIN ON THE INHIBITION OF THROMBIN BY ANTITHROMBIN IN PRESENCE OF EXOGENOUS HEPARIN: ROLE OF THE ACIDIC DOMAIN OF THROMBOMODULIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643964.
Full textAbstract:
Thrombomodulin (TM) is an endothelial cell membrane protein chat acts as a cofactor for Protein C (PC) activation by thrombin (T). Rabbit TM also prevents fibrinogen clotting by T (direct anticoagulant activity) and accelerates T inhibition by antithrombin (AT-dependent anticoagulant activity). Rabbit TM was previously found to contain an acidic domain (presumably a heparin-like polysaccharide), separated from the PC cofactor site but required for the direct and AT-dependent anticoagulant activities. Although binding of TM to T modifies the specificity of T, it does not involve a modification of the catalytic site or a global conformational change of the enzyme.A non-acidic form of rabbit TM was obtained by limited proteolytic degradation of the acidic form. The non-acidic form retained only the PC cofactor activity. The acidic and non-acidic forms of rabbit TM were compared with regard to their effects on the inhibition of T by AT in presence of exogenous heparin (hep). The AT-heparin complex was preformed by incubation for 5 min at 37°C of AT (0.1 μM) with high-affinity heparin (HA-hep, 250 ng/ml) and then added to a solution of T (90 nM) or T preincubated with either the non-acidic form (250 nM) or the acidic form (160 nM) of TM. Residual T activity was determined after various times for up to 5 min. Controls were performed by substituting buffer for TM. It was found that T bound to the non-acidic form of TM was inhibited at the same rate as free T (90% T inhibited within 30 sec), whereas T bound to the acidic form of TM was inhibited at a much slower rate (40 to 50% T inhibited after 5 min). Similar results were obtained even when HA-hep was used in excess (up to 750 ng/ml). The data suggest that the acidic domain of rabbit TM is primarily responsible for the retardation of T-AT complex formation in presence of exogenous hep. It is proposed that the polyanionic (endogenous heparin-like) component of rabbit TM blocks a site on T required for binding of T to hep in the ternary T-Hep-AT complex.
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Larm, O., L. Adolfsson, I. Gouda, A. Malmberg, P. Olsson, and E. Scholander. "AN IMPROVED METHOD FOR COVALENT IMMOBILISATION OF HEPARIN BY END POINT ATTACHMENT." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643090.
Full textAbstract:
When creating a non-thrombogenic surface by immobilisation of heparin, most of the biological activity of heparin as it is expressed in blood should be retained after the coupling procedure. Consequently the chemical methods used in the immobilisation procedure shall be selected so that they do not, infavorably, affect the anti-thrombin (AT) binding site, the charge distribution and charge density of heparin.To ascertain that the AT-binding sequence is not involved in the coupling procedure, we have developed a method in which heparin is coupled by end point attachment (EPA). Heparin is partially degraded with nitrous acid and fragments with reactive aldehydo functions in the reducing terminal residues are formed. These are coupled to aminated surfaces by reductive amination. The primary amines have been introduced on the substrates in different ways, depending on their hydrophobic or hydrophilic characteristics.It has been possible to furnish such complex devices as hollow fibre oxygenators, tubing sets and cannulas with the heparin surface. Heparinized oxygenators have successfully been used in vivo and clinically without and/or with limited systemic heparinization.
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Koedam, Joost A., Rob J. Hamer, Nel H. Beeser-Visser, Etienne Jap Tjoen San, Kees Schippers, and Jan J. Sixma. "THE INTERACTION BETWEEN FACTOR VIII AND VON WILLEBRAND FACTOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644771.
Full textAbstract:
Factor VIII (FVIII) circulates in plasma as a non-covalent complex with von Willebrand factor (VWF), a large multimeric adhesive glycoprotein. VWF serves as a carrier for FVIII and is thought to stabilize FVIII. The interaction between the two proteins was studied by binding purified human 125I-FVIII to VWF which was coated on a solid matrix. Experiments employing isolated heavy and light chains of FVIII and monoclonal antibodies indicated that binding occurred through the carboxyterminal 80kDa light chain of factor VIII. Treatment of VWF-bound 125I-FVIII with thrombin resulted in the release of a light chain-derived 70kDa fragment and a heavy chain-derived 50kDa fragment. A 42kDa heavy chain-derived fragment was found in the fraction which remained bound to VWF. Treatment with factor Xa (FXa) resulted in the release of 63, 50, 45, and 42kDa fragments. No phospholipids were required for proteolysis of FVIII by either of these enzymes. In solution, the activation of FVIII by FXa, but not by thrombin, was inhibited by VWF. Neither activation, nor cleavage or release from VWF were observed when FVIII was incubated with factor IXa. Activation of FVIII was parallelled by its release from VWF. We conclude that the thrombin-activated form of FVIII consists of a complex between the 70kDa and 50kDa fragments. Inactivation of FVIII by activated protein C (APC) was inhibited when FVIII was complexed to VWF. This protective effect of VWF was abolished upon activation of FVIII and its subsequent release from VWF.In order to locate the binding site for FVIII on the VWF molecule, we digested VWF with Staphylococcal V8 protease (Sp). Digestion products were isolated with Mono Q ion-exchange chromatography and identified as Spl (39 kDa), SpII dimers (220 kDa) and Spill dimers (a triplet ranging from 210-280 kDa) by their molecular weight and chromatographic behaviour (J.-P. Girma et al.. Biochemistry 1986, 25:3156-3163). Purified VWF or digestion products were spotted on nitrocellulose paper, followed by blocking with an albumin solution. Binding of FVIII was studied by incubating the filters with 125I-FVIII, followed by autoradiography. Fifty ng of VWF was sufficient in order to detect FVIII binding. No binding was observed to partially reduced dimeric undigested VWF. Of the isolated digestion products, only the SpIII dimer was able to bind 125I-FVIII. After Western blotting of VWF-fragments from SDS-polyacrylamide gels, 125I-FVIII bound only to the bands which represented SpIII. Therefore, the domain on VWF responsible for the binding of FVIII seems to be located on its aminoterminal SpIII fragment. The integrity of internal disulfide bonds and dimerisation of VWF are required for FVIII binding.
Reports on the topic "Non-OR copper binding site"
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Hodges, Thomas K., and David Gidoni. Regulated Expression of Yeast FLP Recombinase in Plant Cells. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7574341.bard.
Full textAbstract:
Research activities in both our laboratories were directed toward development of control of the FLP/frt recombination system for plants. As described in the text of the research proposal, the US lab has been engaged in developing regulatory strategies such as tissue-specific promoters and the steroid-inducible activation of the FLP enzyme while the main research activities in Israel have been directed toward the development and testing of a copper-regulated expression of flp recombinase in tobacco (this is an example of a promoter activation by metal ions). The Israeli lab hat additionally completed experiments of previous studies regarding factors affecting the efficiency of recombinase activity using both a gain-of-function assay (excisional-activation of a gusA marker) and loss of function assay (excision of a rolC marker) in tobacco. Site-specific recombinase systems, in particular the FLP/frt and R/RS systems of yeast and the Cre/lox system of bacteriophage P1, have become an essential component of targeted genetic transformation procedures both in animal and plant organisms. To provide more flexibility in transgene excisions by the recombinase systems as well as gene targeting, and to widen possible applications, the development of controlled or regulated recombination systems is highly desirable and was therefore the subject of this research proposal. There are a few possible mechanisms to regulate expression of a recombinase system. They include: 1) control of the recombination system by having the target sites (e.g. frt) in one plant and the flp recombinase gene in another, and bringing the two together by cross fertilization. 2) regulation of promoter activities by external stimuli such as temperature, chemicals, metal ions, etc. 3) regulation of promoter activities by internal signals, i.e. cell- or tissue-specific, or developmental regulation. 4) regulation of enzyme activity by providing cofactors essential for biochemical reactions to take place such as steroid molecules in conjunction with a steroid ligand-binding protein (domains). During the course of this research our major emphasis have been focused toward studying the feasibility of hybrid seed production in Arabidopsis, using FLP/frt. Male-sterility was induced using the antisence of a pollen- and tapetum-specific gene, bcp1, isolated from Arabidopsis. The sterility inducing gene was flanked by frt sites. Upon cross pollination of flowers of male-sterile plants with pollen from FLP-containing plants, viable seeds were produced, and the progeny hybrid plants developed normally. The major achievement from this work is the first demonstration of using a site-specific recombinase to restore fertility in male-sterile plants (see attached paper, Luo et al., Plant J 2000; 23:423-430). The implication from this finding is that site-specific recombination systems can be applied in crop plants as a useful alternative method for hybrid seed production.
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Gurevitz, Michael, William A. Catterall, and Dalia Gordon. face of interaction of anti-insect selective toxins with receptor site-3 on voltage-gated sodium channels as a platform for design of novel selective insecticides. United States Department of Agriculture, December 2013. http://dx.doi.org/10.32747/2013.7699857.bard.
Full textAbstract:
Voltage-gated sodium channels (Navs) play a pivotal role in excitability and are a prime target of insecticides like pyrethroids. Yet, these insecticides are non-specific due to conservation of Navs in animals, raising risks to the environment and humans. Moreover, insecticide overuse leads to resistance buildup among insect pests, which increases misuse and risks. This sad reality demands novel, more selective, insect killers whose alternative use would avoid or reduce this pressure. As highly selective insect toxins exist in venomous animals, why not exploit this gift of nature and harness them in insect pest control? Many of these peptide toxins target Navs, and since their direct use via transformed crop plants or mediator microorganisms is problematic in public opinion, we focus on the elucidation of their receptor binding sites with the incentive of raising knowledge for design of toxin peptide mimetics. This approach is preferred nowadays by agro-industries in terms of future production expenses and public concern. However, characterization of a non-continuous epitope, that is the channel receptor binding site for such toxins, requires a suitable experimental system. We have established such a system within more than a decade and reached the stage where we employ a number of different insect-selective toxins for the identification of their receptor sites on Navs. Among these toxins we wish to focus on those that bind at receptor site-3 and inhibit Nav inactivation because: (1) We established efficient experimental systems for production and manipulation of site-3 toxins from scorpions and sea anemones. These peptides vary in size and structure but compete for site-3 on insect Navs. Moreover, these toxins exhibit synergism with pyrethroids and with other channel ligands; (2) We determined their bioactive surfaces towards insect and mammalian receptors (see list of publications); (3) We found that despite the similar mode of action on channel inactivation, the preference of the toxins for insect and mammalian channel subtypes varies greatly, which can direct us to structural features in the basis of selectivity; (4) We have identified by channel loop swapping and point mutagenesis extracellular segments of the Navinvolved with receptor site-3. On this basis and using channel scanning mutagenesis, neurotoxin binding, electrophysiological analyses, and structural data we offer: (i) To identify the residues that form receptor site-3 at insect and mammalian Navs; (ii) To identify by comparative analysis differences at site-3 that dictate selectivity toward various Navs; (iii) To exploit the known toxin structures and bioactive surfaces for modeling their docking at the insect and mammalian channel receptors. The results of this study will enable rational design of novel anti-insect peptide mimetics with minimized risks to human health and to the environment. We anticipate that the release of receptor site-3 molecular details would initiate a worldwide effort to design peptide mimetics for that site. This will establish new strategies in insect pest control using alternative insecticides and the combined use of compounds that interact allosterically leading to increased efficiency and reduced risks to humans or resistance buildup among insect pests.
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Gurevitz, Michael, Michael E. Adams, Boaz Shaanan, Oren Froy, Dalia Gordon, Daewoo Lee, and Yong Zhao. Interacting Domains of Anti-Insect Scorpion Toxins and their Sodium Channel Binding Sites: Structure, Cooperative Interactions with Agrochemicals, and Application. United States Department of Agriculture, December 2001. http://dx.doi.org/10.32747/2001.7585190.bard.
Full textAbstract:
Integrated pest management in modern crop protection may combine chemical and biological insecticides, particularly due to the risks to the environment and livestock arising from the massive use of non-selective chemicals. Thus, there is a need for safer alternatives, which target insects more specifically. Scorpions produce anti-insect selective polypeptide toxins that are biodegradable and non-toxic to warm-blooded animals. Therefore, integration of these substances into insect pest control strategies is of major importance. Moreover, clarification of the molecular basis of this selectivity may provide valuable information pertinent to their receptor sites and to the future design of peptidomimetic anti-insect specific substances. These toxins may also be important for reducing the current overuse of chemical insecticides if they produce a synergistic effect with conventional pesticides. Based on these considerations, our major objectives were: 1) To elucidate the three-dimensional structure and toxic-site of scorpion excitatory, "depressant, and anti-insect alpha toxins. 2) To obtain an initial view to the sodium channel recognition sites of the above toxins by generating peptide decoys through a phage display system. 3) To investigate the synergism between toxins and chemical insecticides. Our approach was to develop a suitable expression system for toxin production in a recombinant form and for elucidation of toxin bioactive sites via mutagenesis. In parallel, the mode of action and synergistic effects of scorpion insecticidal toxins with pyrethroids were studied at the sodium channel level using electrophysiological methods. Objective 1 was achieved for the alpha toxin, LqhaIT Zilberberg et al., 1996, 1997; Tugarinov et al., 1997; Froy et al., 2002), and the excitatory toxin, Bj-xtrIT (Oren et al., 1998; Froy et al., 1999; unpublished data). The bioactive surface of the depressant toxin, LqhIT2, has been clarified and a crystal of the toxin is now being analyzed (unpublished). Objective 2 was not successful thus far as no phages that recognize the toxins were obtained. We therefore initiated recently an alternative approach, which is introduction of mutations into recombinant channels and creation of channel chimeras. Objective 3 was undertaken at Riverside and the results demonstrated synergism between LqhaIT or AaIT and pyrethroids (Lee et al., 2002). Furthermore, negative cross-resistance between pyrethroids and scorpion toxins (LqhaIT and AaIT) was demonstrated at the molecular level. Although our study did not yield a product, it paves the way for future design of selective pesticides by capitalizing on the natural competence of scorpion toxins to distinguish between sodium channels of insects and vertebrates. We also show that future application of anti-insect toxins may enable to decrease the amounts of chemical pesticides due to their synergism.
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Pines, Mark, Arieh Bar, David A. Carrino, Arnold I. Caplan, and James A. Dennis. Extracellular Matrix Molecules of the Eggshell as Related to Eggshell Quality. United States Department of Agriculture, 1997. http://dx.doi.org/10.32747/1997.7575270.bard.
Full textAbstract:
The extracellular matrix of the mineralizing eggshell contains molecules hypothesized to be regulators biomineralization. To study eggshell matrix molecules, a bank of monoclonal antibodies was generated that bound demineralized eggshell matrix or localized to oviduct epithelium. Immunofluorescence staining revealed several staining patterns for antibodies that recognized secretory cells: staining for a majority of columnar lining cells, staining for a minor sub-set of columnar lining cells, intensified staining within epithelial crypts, and staining of the entire tubular gland. Western blotting with the antibody Epi2 on eggshell matrix showed binding to molecules with the apparent molecular weight of eggshell matrix dermatan sulfate proteoglycan (eggshell DSPG) (Carrino, et al., 1997). Immunoblots of cyanogen bromide-cleaved eggshell DSPG revealed broad band of reactivity that shifted to 25 kDa after chondroitinase digestion; indicating that the Epi2 binding site is located on a fragment which contains dermatan sulfate side chains. Immunogold labeling showed that Epi2 binds to secretory vesicles within the non-ciliated cells of the columnar epithelium, while the antibodies Tg1 and Tg2 bind to secretory vesicles of tubular gland cells. Immunogold labeling of demineralized shell matrix showed binding of Epi2, Tg1, and Tg2 to the matrix of the palisades layer, and showed little reactivity to other regions of the shell matrix. Quantification of the immunogold particles within the eggshell matrix revealed that antibodies Epi2 and Tg1 bind all calcified regions equally while antibody Tg2 has a greater affinity for the baseplate region of the calcium reserve assembly.
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Rafaeli, Ada, and Russell Jurenka. Molecular Characterization of PBAN G-protein Coupled Receptors in Moth Pest Species: Design of Antagonists. United States Department of Agriculture, December 2012. http://dx.doi.org/10.32747/2012.7593390.bard.
Full textAbstract:
The proposed research was directed at determining the activation/binding domains and gene regulation of the PBAN-R’s thereby providing information for the design and screening of potential PBAN-R-blockers and to indicate possible ways of preventing the process from proceeding to its completion. Our specific aims included: (1) The identification of the PBAN-R binding domain by a combination of: (a) in silico modeling studies for identifying specific amino-acid side chains that are likely to be involved in binding PBAN with the receptor and; (b) bioassays to verify the modeling studies using mutant receptors, cell lines and pheromone glands (at tissue and organism levels) against selected, designed compounds to confirm if compounds are agonists or antagonists. (2) The elucidation ofthemolecular regulationmechanisms of PBAN-R by:(a) age-dependence of gene expression; (b) the effect of hormones and; (c) PBAN-R characterization in male hair-pencil complexes. Background to the topic Insects have several closely related G protein-coupled receptors (GPCRs) belonging to the pyrokinin/PBAN family, one with the ligand pheromone biosynthesis activating neuropeptide or pyrokinin-2 and another with diapause hormone or pyrokinin-1 as a ligand. We were unable to identify the diapause hormone receptor from Helicoverpa zea despite considerable effort. A third, related receptor is activated by a product of the capa gene, periviscerokinins. The pyrokinin/PBAN family of GPCRs and their ligands has been identified in various insects, such as Drosophila, several moth species, mosquitoes, Triboliumcastaneum, Apis mellifera, Nasoniavitripennis, and Acyrthosiphon pisum. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates pheromone production in moths plus other functions indicating the pleiotropic nature of these ligands. Based on the alignment of annotated genomic sequences, the primary and secondary structures of the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors found in vertebrates. Major conclusions, solutions, achievements Evolutionary trace analysisof receptor extracellular domains exhibited several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding. Through site-directed point mutations, the 3rd extracellular domain of PBAN-R was shown to be critical for ligand selection. We identified three receptors that belong to the PBAN family of GPCRs and a partial sequence for the periviscerokinin receptor from the European corn borer, Ostrinianubilalis. Functional expression studies confirmed that only the C-variant of the PBAN-R is active. We identified a non-peptide agonist that will activate the PBAN-receptor from H. zea. We determined that there is transcriptional control of the PBAN-R in two moth species during the development of the pupa to adult, and we demonstrated that this transcriptional regulation is independent of juvenile hormone biosynthesis. This transcriptional control also occurs in male hair-pencil gland complexes of both moth species indicating a regulatory role for PBAN in males. Ultimate confirmation for PBAN's function in the male tissue was revealed through knockdown of the PBAN-R using RNAi-mediated gene-silencing. Implications, both scientific and agricultural The identification of a non-peptide agonist can be exploited in the future for the design of additional compounds that will activate the receptor and to elucidate the binding properties of this receptor. The increase in expression levels of the PBAN-R transcript was delineated to occur at a critical period of 5 hours post-eclosion and its regulation can now be studied. The mysterious role of PBAN in the males was elucidated by using a combination of physiological, biochemical and molecular genetics techniques.
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Dubcovsky, Jorge, Tzion Fahima, and Ann Blechl. Molecular characterization and deployment of the high-temperature adult plant stripe rust resistance gene Yr36 from wheat. United States Department of Agriculture, November 2013. http://dx.doi.org/10.32747/2013.7699860.bard.
Full textAbstract:
Stripe rust, caused by Puccinia striiformis f. sp. tritici is one of the most destructive fungal diseases of wheat. Virulent races that appeared within the last decade caused drastic cuts in yields. The incorporation of genetic resistance against this pathogen is the most cost-effective and environmentally friendly solution to this problem. However, race specific seedling resistance genes provide only a temporary solution because fungal populations rapidly evolve to overcome this type of resistance. In contrast, high temperature adult plant (HTAP) resistance genes provide a broad spectrum resistance that is partial and more durable. The cloning of the first wheat HTAP stripe rust resistance gene Yr36 (Science 2009, 323:1357), funded by our previous (2007-2010) BARD grant, provided us for the first time with an entry point for understanding the mechanism of broad spectrum resistance. Two paralogous copies of this gene are tightly linked at the Yr36 locus (WKS1 and WKS2). The main objectives of the current study were to characterize the Yr36 (WKS) resistance mechanism and to identify and characterize alternative WKSgenes in wheat and wild relatives. We report here that the protein coded by Yr36, designated WKS1, that has a novel architecture with a functional kinase and a lipid binding START domain, is localized to chloroplast. Our results suggest that the presence of the START domain may affect the kinase activity. We have found that the WKS1 was over-expressed on leaf necrosis in wheat transgenic plants. When the isolated WKS1.1 splice variant transcript was transformed into susceptible wheat it conferred resistance to stripe rust, but the truncated variant WKS1.2 did not confer resistance. WKS1.1 and WKS1.2 showed different lipid binding profiling. WKS1.1 enters the chloroplast membrane, while WKS1.2 is only attached outside of the chloroplast membrane. The ascorbate peroxidase (APX) activity of the recombinant protein of TmtAPXwas found to be reduced by WKS1.1 protein in vitro. The WKS1.1 mature protein in the chloroplast is able to phosphorylate TmtAPXprotein in vivo. WKS1.1 induced cell death by suppressing APX activity and reducing the ability of the cell to detoxify reactive oxygen. The decrease of APX activity reduces the ability of the plant to detoxify the reactive H2O2 and is the possible mechanism underlying the accelerated cell death observed in the transgenic plants overexpressing WKS1.1 and in the regions surrounding a stripe rust infection in the wheat plants carrying the natural WKS1.1 gene. WKS2 is a nonfunctional paralog of WKS1 in wild emmer wheat, probably due to a retrotransposon insertion close to the alternative splicing site. In some other wild relatives of wheat, such as Aegilops comosa, there is only one copy of this gene, highly similar to WKS2, which is lucking the retrotransposon insertion. WKS2 gene present in wheat and WKS2-Ae from A. showed a different pattern of alternative splice variants, regardless of the presence of the retrotransposon insertion. Susceptible Bobwhite transformed with WKS2-Ae (without retrotansposon insertion in intron10), which derived from Aegilops comosaconferred resistance to stripe rust in wheat. The expression of WKS2-Ae in transgenic plants is up-regulated by temperature and pathogen infection. Combination of WKS1 and WKS2-Ae shows improved stripe rust resistance in WKS1×WKS2-Ae F1 hybrid plants. The obtained results show that WKS1 protein is accelerating programmed cell death observed in the regions surrounding a stripe rust infection in the wheat plants carrying the natural or transgenic WKS1 gene. Furthermore, characterization of the epistatic interactions of Yr36 and Yr18 demonstrated that these two genes have additive effects and can therefore be combined to increase partial resistance to this devastating pathogen of wheat. These achievements may have a broad impact on wheat breeding efforts attempting to protect wheat yields against one of the most devastating wheat pathogen.
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Ohad, Itzhak, and Himadri Pakrasi. Role of Cytochrome B559 in Photoinhibition. United States Department of Agriculture, December 1995. http://dx.doi.org/10.32747/1995.7613031.bard.
Full textAbstract:
The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.