Academic literature on the topic 'Enzyme activation'
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Journal articles on the topic "Enzyme activation"
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Wollenberger, Ulla, and Frieder W. Scheller. "Enzyme activation for activator and enzyme activity measurement☆." Biosensors and Bioelectronics 8, no. 6 (1993): 291–97. http://dx.doi.org/10.1016/0956-5663(93)85009-d.
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Wang, Fang, Yuchen Liu, Chang Du, and Renjun Gao. "Current Strategies for Real-Time Enzyme Activation." Biomolecules 12, no. 5 (April 19, 2022): 599. http://dx.doi.org/10.3390/biom12050599.
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Enzyme activation is a powerful means of achieving biotransformation function, aiming to intensify the reaction processes with a higher yield of product in a short time, and can be exploited for diverse applications. However, conventional activation strategies such as genetic engineering and chemical modification are generally irreversible for enzyme activity, and they also have many limitations, including complex processes and unpredictable results. Recently, near-infrared (NIR), alternating magnetic field (AMF), microwave and ultrasound irradiation, as real-time and precise activation strategies for enzyme analysis, can address many limitations due to their deep penetrability, sustainability, low invasiveness, and sustainability and have been applied in many fields, such as biomedical and industrial applications and chemical synthesis. These spatiotemporal and controllable activation strategies can transfer light, electromagnetic, or ultrasound energy to enzymes, leading to favorable conformational changes and improving the thermal stability, stereoselectivity, and kinetics of enzymes. Furthermore, the different mechanisms of activation strategies have determined the type of applicable enzymes and manipulated protocol designs that either immobilize enzymes on nanomaterials responsive to light or magnetic fields or directly influence enzymatic properties. To employ these effects to finely and efficiently activate enzyme activity, the physicochemical features of nanomaterials and parameters, including the frequency and intensity of activation methods, must be optimized. Therefore, this review offers a comprehensive overview related to emerging technologies for achieving real-time enzyme activation and summarizes their characteristics and advanced applications.
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Hamilton-Miller, J. M. T., and Q. Li. "Enzyme-Catalyzed Antimicrobial Activation." Antimicrobial Agents and Chemotherapy 46, no. 11 (November 1, 2002): 3692. http://dx.doi.org/10.1128/aac.46.11.3692.2002.
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Hadfield, Andrea T. "Electron-Induced Enzyme Activation." Structure 14, no. 1 (January 2006): 1–2. http://dx.doi.org/10.1016/j.str.2005.12.002.
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Bott, R., G. Ganshaw, M. Soltis, P. Kuhn, and M. Knapp. "Snapshots of Enzyme Activation." Acta Crystallographica Section A Foundations of Crystallography 56, s1 (August 25, 2000): s247. http://dx.doi.org/10.1107/s0108767300025319.
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Shisler, Krista A., Rachel U. Hutcheson, Masaki Horitani, Kaitlin S. Duschene, Adam V. Crain, Amanda S. Byer, Eric M. Shepard, et al. "Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme." Journal of the American Chemical Society 139, no. 34 (August 22, 2017): 11803–13. http://dx.doi.org/10.1021/jacs.7b04883.
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Cassels, R., R. Fears, and R. A. Smith. "The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro." Biochemical Journal 247, no. 2 (October 15, 1987): 395–400. http://dx.doi.org/10.1042/bj2470395.
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The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.
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Arcus, Vickery L., and Adrian J. Mulholland. "Temperature, Dynamics, and Enzyme-Catalyzed Reaction Rates." Annual Review of Biophysics 49, no. 1 (May 6, 2020): 163–80. http://dx.doi.org/10.1146/annurev-biophys-121219-081520.
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We review the adaptations of enzyme activity to different temperatures. Psychrophilic (cold-adapted) enzymes show significantly different activation parameters (lower activation enthalpies and entropies) from their mesophilic counterparts. Furthermore, there is increasing evidence that the temperature dependence of many enzyme-catalyzed reactions is more complex than is widely believed. Many enzymes show curvature in plots of activity versus temperature that is not accounted for by denaturation or unfolding. This is explained by macromolecular rate theory: A negative activation heat capacity for the rate-limiting chemical step leads directly to predictions of temperature optima; both entropy and enthalpy are temperature dependent. Fluctuations in the transition state ensemble are reduced compared to the ground state. We show how investigations combining experiment with molecular simulation are revealing fundamental details of enzyme thermoadaptation that are relevant for understanding aspects of enzyme evolution. Simulations can calculate relevant thermodynamic properties (such as activation enthalpies, entropies, and heat capacities) and reveal the molecular mechanisms underlying experimentally observed behavior.
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Vater, C. A., H. Nagase, and E. D. Harris. "Proactivator-dependent activation of procollagenase induced by treatment with EGTA." Biochemical Journal 237, no. 3 (August 1, 1986): 853–58. http://dx.doi.org/10.1042/bj2370853.
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A new mechanism for activation of the proactivator of procollagenase [Vater, Nagase & Harris (1983) J. Biol. Chem. 258, 9374-9382] has been found. Collagenolytic and other proteolytic enzyme activities in the medium of cultured rabbit synovial fibroblasts were found to be activated by a new mechanism: short-term incubation at 37 degrees C performed in the presence of EGTA followed by replacement of Ca2+ during enzyme assay. The crucial event in procollagenase activation is the production of a functional activator enzyme. Activation of procollagenase in the culture medium did not occur when proactivator was removed by immunoprecipitation. Proteolytic activity of proactivator was fully activated, whereas procollagenase alone could not be activated by the same sequence. EGTA treatment of the culture medium at 0 degrees C did not result in enzyme activation if Ca2+ was replaced before incubation at 37 degrees C. Certain other bivalent metal ions (e.g. Sn2+, Cd2+, Zn2+ and Mn2+) could substitute for Ca2+ to stabilize the proactivator as a zymogen and therefore prevent the appearance of proteolytic activity in culture medium. Isolation of proactivator and procollagenase from EGTA-treated radiolabelled culture medium by immunoprecipitation and subsequent analyses by fluorography revealed that a time-dependent proteolysis of both molecules occurred after replacement of Ca2+ and incubation at 37 degrees C. However, comparison of enzyme activity with fluorographic analyses showed that the maximal activation of both enzymes was achieved before any detectable decrease in Mr. The results suggest that the activation of proactivator and the subsequent activation of procollagenase may be initiated by conformational changes in structure of the proactivator molecule produced by removal of stabilizing bivalent metal ions.
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KHOLODENKO, Boris N., and Guy C. BROWN. "Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?" Biochemical Journal 314, no. 3 (March 15, 1996): 753–60. http://dx.doi.org/10.1042/bj3140753.
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It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties.
Dissertations / Theses on the topic "Enzyme activation"
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Eddeb, Fadel. "Stabilisation and activation of horseradish peroxidase." Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294853.
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Aubatin, Aude. "Modulation de la réponse immune par IL4I1 : rôle dans les évènements précoces d’activation lymphocytaire T." Thesis, Paris Est, 2016. http://www.theses.fr/2016PESC0069.
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Modulation de la réponse lymphocytaire T par IL4I1 - RESUME : L’enzyme Interleukin-four Induced Gene 1 (IL4I1), qui dégrade la phénylalanine, est exprimée par des cellules présentatrices d’antigène (CPA) en réponse à des stimuli pro-inflammatoires. Elle affecte la prolifération et les fonctions lymphocytaires T et pourrait donc participer au rétrocontrôle des réponses immunitaires. Cependant, les mécanismes d’action d’IL4I1 restent encore mal connus. Mon projet de thèse a comporté deux axes. Dans un premier axe, j’ai participé à la caractérisation du rôle d’IL4I1 dans la différenciation des lymphocytes T CD4+ naïfs en lymphocytes T régulateurs et T auxiliaires effecteurs. Dans mon deuxième axe, qui représente la majeure partie de mon travail, j’ai analysé l’action d’IL4I1 sur les évènements précoces de l’activation T. J’ai observé qu’IL4I1 inhibe la phosphorylation de ZAP70 dès les premières minutes d’activation. Ce défaut se propage aux trois voies de signalisation principales : la voie calcique, la voie des MAP kinases et la voie NFκB. Elle retentit secondairement sur la capacité à former des synapses avec les CPA, ainsi que sur l’expression de différents marqueurs membranaires d’activation. L’activité enzymatique d'IL4I1 n’est pas responsable du défaut d’activation observé. L’étude des synapses CPA-T a montré une polarisation des vésicules de sécrétion contenant IL4I1 en direction de la cellule T ainsi qu’occasionnellement leur présence au contact du lymphocyte T. Des expériences complémentaires confirment la fixation d’IL4I1 sur les lymphocytes T. Ces données suggèrent un nouveau mécanisme d’action d’IL4I1 dépendant de sa liaison à un récepteur membranaire de la cellule T. - Mots clefs : Interleukin-4 induced gene 1 (IL4I1), enzyme immunosuppressive, signalisation du lymphocyte TModulation of the T cell response by IL4I1 - SUMMARY: The enzyme Interleukin-four Induced Gene 1 (IL41I), which degrades phenylalanine, is expressed by antigen presenting cells (APC) in response to pro-inflammatory stimuli. IL4I1 modifies the proliferation and function of T lymphocytes, and may participate in the negative feedback of the immune response. Its mechanism of action remains poorly understood.My thesis project included two tasks. In the first task, I participated in the characterisation of the role of IL4I1 in naive CD4+ T cell differentiation into regulatory and helper T lymphocytes. In the second task, and the main part of my thesis, I have studied the effect of IL4I1 on early T cell activation. I observed that ZAP70 phosphorylation was rapidly decreased after TCR stimulation. This alteration was transmitted to the three main downstream signalling pathways: calcium fluxes, the MAP kinase pathway, and the NFκB pathway. The enzymatic activity of IL4I1 was not responsible for the observed decreased activation. Analysis of the APC-T cell synapse showed the polarised secretion of IL4I1 toward the T cell. Labelling of IL4I1 was sometimes detected directly on T lymphocytes. Complementary experiments indicate that IL4I1 binds to T lymphocytes. These data suggest a new mechanism of action of IL4I1 dependent on its ability to bind a membrane receptor on T lymphocytes. - Key-words: Interleukin-4 induced gene 1 (IL4I1), Immunosuppressive Enzyme, T lymphocyte signalling
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Quayle, Katherine Amanda. "Mechanisms of regulation of acetyl-CoA carboxylase." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/30657.
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One of the major physiological responses to insulin secretion is the activation of lipogenesis in target tissues (principally fat and liver). As acetyl-CoA carboxylase (ACC) is the rate limiting enzyme in fatty acid synthesis, the mechanisms involved in the short term regulation of this enzyme represent a pertinent model system for determining elements involved in amplification of the signals produced in response to stimulation of cells with lipogenic and counter regulatory hormones. The regulation of mammalian ACC by hormones is a complex phenomenon involving interplay between allosteric and covalent mechanisms. While the effects of adrenaline and glucagon are well characterised, the mechanism of regulation by insulin has still to be defined and formed the focus for the work presented in this thesis.
To study the role of phosphorylation in the response of ACC to insulin, the site-specific phosphorylation of the enzyme observed following exposure of intact cells to insulin has been reproduced in vitro. These studies for the first time describe the conditions required to achieve distribution of [³²P] in vitro among sites of acetyl-CoA carboxylase, very similar to that seen after hormone treatment of intact cells and employing endogenous polyamine-sensitive kinase(s). No corresponding increase in catalytic activity was detected following phosphorylation, in vitro, of insulin directed phosphorylation sites on purified rat liver acetyl-CoA carboxylase in these studies. Subsequently, ACC was phosphorylated by an exogenous protein kinase from maturation activated sea-star oocytes which led to high stoichiometric incorporation of ³²P into the unique site (I-site) phosphorylated in intact cells in
response to insulin (0.3 mol phosphate / mol 240,000 kD subunit). Again no change in ACC activity was observed following I-site phosphorylation. The peptide containing the I-site was separated from other tryptic phosphopeptides by reverse phase HPLC and then sequenced. Phosphorylation of serine 1186 was determined to be the major phosphorylation site of ACC in response to insulin. The amino acid sequence corresponding to the peptide containing Ser 1186 is located in the putative "hinge" region of ACQ which is some 300 amino acids towards the C-terminal of the biotin binding site (Lys-784).
Subsequent re-evaluation of the kinetic properties of acetyl-CoA carboxylase during purification has led to the identification of a fraction containing low Mr inhibitor(s) and an apparently novel protein activator present in rat liver. Affinity purified rat liver acetyl-CoA carboxylase can be activated 2-3 fold at physiological citrate concentrations (0.1-0.5mM) by the addition of the heat and pro tease-sensitive cytosolic protein. The ACC activator has been extensively purified (though not yet to homogeneity) from a 100,000 g supernatant fractions from rat liver extract, by a combination of ammonium sulphate precipitation, ion-exchange chromatography and gel filtration. From these results we concluded that the activator is a protein and the native molecular weight in solution is estimated to be approximately 75 kDaltons.
A popular hypothesis regarding the short term regulation of ACC involves a phosphorylation-dephosphorylation mechanism resulting in inhibition and activation respectively. A number of experiments have been carried out in order to test the hypothesis that the activator preparation may contain protein phosphatase activity directed towards ACC. The results strongly suggest that under the assay conditions
described for the expression of activation of catalytic activity of ACC, there is little or no apparent dephosphorylation. Indeed, the most purified preparations of ACC activator do not contain any detectable phosphatase activity towards the model substrates histone III-S and casein. The activation of ACC occurs rapidly, in a time dependent manner (within 20 min at 37°C) and involves protein-protein interaction which is antagonized by avidin. The interactions between ACC, avidin and activator protein suggest that the activator not only induces conformational change at the active site of ACC but may also bind in such a way as to be displaced (perhaps directly) by avidin. From the data presented it is concluded that this acetyl-CoA carboxylase activator protein represents a novel factor which may be involved in the short term regulation of ACC activity.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Lee, Charles Kai-Wu. "Eurythermalism of a deep-sea symbiosis system from an enzymological aspect." The University of Waikato, 2007. http://hdl.handle.net/10289/2588.
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The recently proposed and experimentally validated Equilibrium Model provides the most detailed description of temperature's effect on enzyme catalytic activity to date. By introducing an equilibrium between Eact, the active form of enzyme, and Einact, a reversibly inactivated form of enzyme, the Equilibrium Model explains apparent enzyme activity loss at high temperatures that cannot be accounted for by irreversible thermal denaturation. The Equilibrium Model describes enzyme behavior in the presence of substrates and under assay conditions; thus its associated parameters, deltaHeq and Teq, may have physiological significance. The Equilibrium Model parameters have been determined for twenty-one enzymes of diverse origins. The results demonstrated the wide applicability of the Equilibrium Model to enzymes of different types and temperature affinity. The study has also established deltaHeq as the first quantitative measure of enzyme eurythermalism and demonstrated the relationship between Teq and optimal growth temperature of organisms. The Equilibrium Model is therefore a useful tool for studying enzyme temperature adaptation and its role in adaptations to thermophily and eurythermalism. Moreover, it potentially enables a description of the originating environment from the properties of the enzymes. The Equilibrium Model has been employed to characterize enzymes isolated from bacterial episymbionts of Alvinella pompejana. A. pompejana inhabits one of the most extreme environments known to science and has been proposed as an extremely eurythermal organism. A metagenomic study of the A. pompejana episymbionts has unveiled new information related to the adaptive and metabolic properties of the bacterial consortium; the availability of metagenomic sequences has also enabled targeted retrieval and heterologous expression of A. pompejana episymbiont genes. By inspecting enzymes derived from the unique episymbiotic microbial consortium intimately associated with A. pompejana, the study has shed light on temperature adaptations in this unique symbiotic relationship. The findings suggested that eurythermal enzymes are one of the mechanisms used by the microbial consortium to achieve its adaptations. By combining metagenomic and enzymological studies, the research described in this thesis has lead to insights on the eurythermalism of a complex microbial system from an enzymological aspect. The findings have enhanced our knowledge on how life adapts to extreme environments, and the validation of the Equilibrium Model as a tool for studying enzyme temperature adaptation paves the way for future studies.
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Trinconi, Cristiana de Melo. "Investigação sobre a atividade de Ceramida Sintase em Leishmania amazonensis." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-13022012-091618/.
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A leishmaniose é uma doença parasitária de ampla distribuição e de difícil tratamento. Recentemente foi identificada a atividade leishmanicida de tamoxifeno, levando à proposta de sua utilização como alternativa para o tratamento de leishmaniose. Neste trabalho, propusemo-nos a caracterizar a atividade de ceramida sintase (CerS) em L. amazonensis e testar a interferência do tamoxifeno nesta enzima. Identificamos, no genoma de L. amazonensis, uma ORF que codifica uma proteína similar à CerS de Saccharomyces cerevisiae, com seis domínios transmembrana e um motivo Lag1 característico. A caracterização da atividade enzimática in vitro mostrou que a enzima reconhece esfingosina/esfinganina e palmitoil/miristoil CoA como precursores. A enzima não é sensível à fumonisina B2 ou a tamoxifeno. Isto indica que esta droga não atua através da inibição da CerS. A caracterização completa desta enzima fornecerá dados valiosos sobre o metabolismo de esfingolipídeos nestes protozoários.Leishmaniasis is a widely distributed parasitic disease with difficult treatment. The leishmanicidal activity of tamoxifen was recently identified, leading to its proposal as an alternative treatment for leishmaniasis. In this work, we aimed at characterizing the activity of ceramide synthase (CerS) in L. amazonensis and testing whether this enzymatic activity in inhibited by tamoxifen. We have identified, in the L. amazonensis genome, an ORF which encodes a protein similar to the Saccharomyces cerevisiae CerS, with six transmembrane domains and a characteristic Lag1 motif. The characterization of the in vitro enzymatic activity showed that the enzyme recognizes sphingosine/sphinganine as well as palmitoyl/miristoyl CoA as precursors. This enzyme is not sensitive to fumonisin B2 or tamoxifen. This indicates that this drug does not act through the inibition of CerS. The complete characterization of this enzyme will provide valuable information about the sphingolipid methabolism of these protozoa.
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Eatock, Susan A. (Susan Amelia) Carleton University Dissertation Chemistry. "Activation and reconstitution studies of the enzyme L-Phenylalanine Hydroxylase." Ottawa, 1989.
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Valdivia, Ciro Pablo Kopp. "Pruebas de elaboracion de leche de soya (Glycine max (L.) Merril) y derivados proyecto de viabilidad industrial /." Diss., Cochabamba, 1998. http://contentdm.lib.byu.edu/u?/Benson,4195.
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Valdivia, Ciro Pablo Kopp. "Pruebas de elaboracion de leche de soya (Glycine max (L.) Merril) y derivados proyecto de viabilidad industrial /." La Paz, 1997. http://www.lib.byu.edu/valdivia.
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Celik, Haydar. "Enzyme-catalyzed Reductive Activation Of Anticancer Drugs Idarubicin And Mitomycin C." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/3/12609247/index.pdf.
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Idarubicin (IDA) and mitomycin C (MC) are clinically effective quinone-containing anticancer agents used in the treatment of several human cancers. Quinone-containing anticancer drugs have the potential to undergo bioreduction by oxidoreductases to reactive species, and thereby exert their cytotoxic effects. In the present study, we investigated, for the first time, the potential of IDA, in comparison to MC, to undergo reductive activation by NADPH-cytochrome P450 reductase (P450R), NADH-cytochrome b5 reductase (b5R) and P450R-cytochrome P4502B4 (CYP2B4) system by performing both in vitro plasmid DNA damage experiments and enzyme assays. In addition, we examined the potential protective effects of some antioxidants against DNA-damaging effects of IDA and MC resulting from their reductive activation. To achieve these goals, we obtained P450R from sheep lung, beef liver and PB-treated rabbit liver microsomes, b5R from beef liver microsomes and CYP2B4 from PB-treated rabbit liver microsomes in highly purified forms.
The plasmid DNA damage experiments demonstrated that P450R is capable of effectively reducing IDA to DNA-damaging species. The effective protections provided by antioxidant enzymes, SOD and catalase, as well as scavengers of hydroxyl radical, DMSO and thiourea, revealed that the mechanism of DNA damage by IDA involves the generation of ROS by redox cycling of IDA with P450R under aerobic conditions. The extent of DNA damages by both IDA and MC were found to increase with increasing concentrations of the drug or the enzyme as well as with increasing incubation time. IDA was found to have a greater ability to induce DNA damage at high drug concentrations than MC. The plasmid DNA experiments using b5R, on the other hand, showed that, unlike P450R, b5R was not able to reduce IDA to DNA-damaging reactive species. It was also found that in the presence of b5R and cofactor NADH, MC barely induced DNA strand breaks. All the purified P450Rs reduced IDA at about two-fold higher rate than that of MC as shown by the measurement of drug-induced cofactor consumption. This indicates that IDA may be a more potent cytotoxic drug than MC in terms of the generation of reactive metabolites. The results obtained from enzyme assays confirmed the finding obtained from plasmid DNA experiments that while MC is a very poor substrate for b5R, IDA is not a suitable substrate for this enzyme unlike P450R. The reconstitution experiments carried out under both aerobic and anaerobic conditions using various amounts of CYP2B4, P450R and lipid DLPC revealed that reconstituted CYP2B4 produced about 1.5-fold and 1.4-fold rate enhancements in IDA and MC reduction catalyzed by P450R alone, respectively. The present results also showed that among the tested dietary antioxidants, quercetin, rutin, naringenin, resveratrol and trolox, only quercetin was found to be highly potent in preventing DNA damage by IDA.
These results may have some practical implications concerning the potential use of P450R as therapeutic agent on their own in cancer treatment strategies. Selective targeting of tumor cells with purified P450R by newly developed delivery systems such as using polymers, liposomes or antibodies may produce greater reductive activation of bioreductive drugs in tumor cells. Consequently, this strategy has a high potential to increase the efficacy and selectivity of cancer chemotherapy.
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Lai, Chung-Jeng. "Fumarate Activation and Kinetic Solvent Isotope Effects as Probes of the NAD-Malic Enzyme Reaction." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc278864/.
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The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.
Books on the topic "Enzyme activation"
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H, Stenmark, ed. Phosphoinositides in subcellular targeting and enzyme activation. Berlin: Springer, 2004.
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Stenmark, Harald, ed. Phosphoinositides in Subcellular Targeting and Enzyme Activation. Berlin, Heidelberg: Springer Berlin Heidelberg, 2004. http://dx.doi.org/10.1007/978-3-642-18805-3.
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Alan, Wiseman, ed. Enzyme induction, mutagen activation, and carcinogen testing in yeast. Chichester, West Sussex, England: E. Horwood, 1987.
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Mordechai, Liscovitch, ed. Signal-activated phospholipases. Austin: R.G. Landes Co., 1994.
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H, Schmid-Schönbein, Wurzinger L. J, Zimmermann R. E, and Commission of the European Communities. Committee on Medical and Public Health Research., eds. Enzyme activation in blood-perfused artificial organs: Proceedings of an interdisciplinary meeting. Boston: Nijhoff, 1985.
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Light, P. E. An investigation into factors controlling activation of the enzyme protein Kinase C in the regulation of neurotransmitter release at the frog neuromuscular junction. Birmingham: University of Birmingham, 1990.
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Chen, Chang-Hwei. Activation and Detoxification Enzymes. New York, NY: Springer New York, 2012. http://dx.doi.org/10.1007/978-1-4614-1049-2.
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Chen, Chang-Hwei. Activation and Detoxification Enzymes. Cham: Springer Nature Switzerland, 2024. http://dx.doi.org/10.1007/978-3-031-55287-8.
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Zdeněk, Deyl, ed. HPLC in enzymatic analysis. 2nd ed. New York: Wiley, 1998.
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1923-, Lorand Laszlo, and Mann Kenneth G, eds. Proteolytic enzymes in coagulation, fibrinolysis, and complement activation. San Diego: Academic Press, 1993.
Find full textBook chapters on the topic "Enzyme activation"
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Yang, Yanhui, Yu Chen, Herve Aloysius, Daigo Inoyama, and Longqin Hu. "ENZYMES AND TARGETED ACTIVATION OF PRODRUGS." In Enzyme Technologies, 163–235. Hoboken, NJ: John Wiley & Sons, Inc, 2013. http://dx.doi.org/10.1002/9781118739907.ch5.
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Chen, Chang-Hwei. "Diets Rich in Enzyme Modulators." In Activation and Detoxification Enzymes, 103–11. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4614-1049-2_11.
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Chen, Chang-Hwei. "Diversified Classes of Enzyme Modulators." In Activation and Detoxification Enzymes, 155–65. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4614-1049-2_16.
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Chen, Chang-Hwei. "Diversified Classes of Enzyme Modulators." In Activation and Detoxification Enzymes, 189–202. Cham: Springer Nature Switzerland, 2024. http://dx.doi.org/10.1007/978-3-031-55287-8_16.
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Chen, Chang-Hwei. "Metabolic Enzyme Induction for Health Benefits." In Activation and Detoxification Enzymes, 215–27. Cham: Springer Nature Switzerland, 2024. http://dx.doi.org/10.1007/978-3-031-55287-8_18.
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Scheller, Frieder, Ulla Wollenberger, Florian Schubert, Dorothea Pfeiffer, Alexander Makower, and C. McNeil. "Multienzyme Biosensors — Coupled Enzyme Reactions and Enzyme Activation." In Uses of Immobilized Biological Compounds, 171–79. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-011-1932-0_17.
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Workman, Paul, and Michael I. Walton. "Enzyme-Directed Bioreductive Drug Development." In Selective Activation of Drugs by Redox Processes, 173–91. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4615-3768-7_16.
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Figarella, C., D. Basso, and O. Guy-Crotte. "Lysosomal Enzyme Activation of Digestive Enzymes During Chronic Pancreatitis?" In Chronic Pancreatitis, 134–39. Berlin, Heidelberg: Springer Berlin Heidelberg, 1990. http://dx.doi.org/10.1007/978-3-642-75319-0_15.
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Baici, Antonio. "Multiple Interactions: Essential Activation and Liberation." In Kinetics of Enzyme-Modifier Interactions, 357–66. Vienna: Springer Vienna, 2015. http://dx.doi.org/10.1007/978-3-7091-1402-5_7.
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Mawson, Raymond, Mala Gamage, Netsanet Shiferaw Terefe, and Kai Knoerzer. "Ultrasound in Enzyme Activation and Inactivation." In Food Engineering Series, 369–404. New York, NY: Springer New York, 2010. http://dx.doi.org/10.1007/978-1-4419-7472-3_14.
Full textConference papers on the topic "Enzyme activation"
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Wallace, Robert W., E. Ann Tallant, and Lynn M. Brumley. "POSSIBLE ROLE FOR THE CA2+-DEPENDENT PROTEASE (CALPAIN I) AS AN IRREVERSIBLE ACTIVATOR OF CA2+/CALMODULIN-MEDIATED REACTIONS IN THE HUMAN PLATELET." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644528.
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Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and 125I-CaM. Ten proteins of 245, 225. 175, 150, 90. 82(2), 60 and 41(2) kilodaltons (kDa) bind 125I-CaM in a Ca2+-dependent manner; the binding is blocked by both trifluoperazine and nonradiolabeled CaM. The 225 and 90 kDa proteins are labeled by antisera against myosin light chain kinase (MLCK); the 60 kDa and one of the 82 kDa proteins have been identified as the CaM-dependent phosphatase (calcineurin) and caldesmon. The other proteins are presumed to be other Ca2+/CaM regulated enzymes and proteins which may be important in platelet function. Most of the CaM-binding proteins are degraded upon addition of Ca2+ to a platelet homogenate; the degradation may be blocked by either EGTA, leupeptin or N-ethylmaleimide which suggests that the degradation is due to a Ca2+-dependent protease. Activation of intact platelets under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca2+) also results in limited proteolysis of CaM-binding proteins including those labeled with anti sera against MLCK and the phosphatase. In vitro studies utilizing purified phosphatase and calpain I indicate that the phosphatase is irreversibly activated upon Ca2+-dependent proteolysis. The proteolytically-activated enzyme is insensitive to either Ca2+ or Ca2+/CaM; in addition, its activity in the absence of Ca2+ is even greater than the activity of the unproteolyzed enzyme in the presence of Ca2+ and CaM. Proteolytic stimulation of the phosphatase is accompanied by degradation of the 60 kDa subunit of the enzyme (subunit A) to 56, 52 and 45 kDa fragments, sequentially; proteolysis results in the loss of CaM binding to the enzyme. These results suggest that the Ca2+-dependent protease may have a physiological role in platelet activation as an irreversible activator of Ca2+/ CaM-dependent reactions. Supported by NIH grant HL29766.
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Noerager, Brett D., Xin Xu, Svetlana Okafor, J. E. Blalock, and Patricia L. Jackson. "Acrolein Treatment Of Human Neutrophils Affects Enzyme Release And Activation." In American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a2550.
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Selak, M. A., M. Chignard, and J. B. Smith. "CHARACTERIZATION OF A NEUTROPHIL CPYMOTRYPSIN-LIKE ENZYME THAT ACTIVATES PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643157.
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Communication between neutrophils and platelets was previously investigated by measuring platelet aggregation, serotonin release and changes in cytosolic free calcium subsequent to specific stimulation of neutrophils by fMet-Leu-Phe (FMLP) in a suspension of both cell types. The addition of the chemotactic peptide was shown to elicit secondary platelet activation as a consequence of primary stimulation of neutrophils. Cell-free supernatants from FMLP-stimulated neutrophils were capable of inducing platelet activation thus demonstrating that a factor released bv neutrophils was responsible for the observed platelet responses. After eliminating classical platelet agonists as the acitive agent, it was shown that an enzyme termed neutroohilin induced platelet calcium mobilization, secretion and aggregation. The current studies were conducted to characterize the mediator released bv neutrophils. Neutrophilin bound bo cation exchange resins but failed to bind to anion exchangers. The biological activity associated with neutroohilin was unaffected by leupeptin, only very weakly diminished by N-bosyl-Lvs-chloromethvl ketone and was strongly inhibited by N-tosvl-Phe-chloromethvl ketone, aloha-l-antitrvpsin, soybean trypsin inhibitor and Z-Glv-Leu-Phe-chloromethvl ketone. Neutroohilin was released from stimulated neutrophils only after cytochalasin B treatment, as was beta-glucuronidase, suggesting that both enzymes are located in azurophilic granules. Neutroohilin-induced platelet activation was inhibited bv antiserum to human catheosin G in a dose-deoendent manner but was unaffected by antiserum to human elastase or alpha-fetoprotein. The inhibitor sensitivity, immunological cross-reactivity, ionic properties and probable subcellular localization indicate that neutrophilin is a cationic chymotrvosin-like enzyme related, if not identical to, catheosin G. Neutroohilin-induced platelet activation could explain different pathological events in which platelets and neutroohils are known to be involved.
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Maschio, A. Del, E. Corvazier, F. Maillet, M. Kazatchkine, and J. Maclouf. "PLATELET-DEPENDENT ACTIVATION AND AMPLIFICATION OF THE POLYMORPHONUCLEAR LEUKOCYTES LYSOSOMAL ENZYME RELEASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644858.
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The degranulatlon of human PMNs by opsonlsed zymosan (OpZ) was studied In the presence or In the absence of platelet alone or after stimulation by thrombin. Evidence Is presented that the presence of platelets Increased the extent of the liberation of lysozyme from PMNs stimulated by OpZ with a maximal intensity when they were stimulated by thrombin. The extent of the amplification was higher when the PMNs trigger was lower (i.e. 0.5 x 108 particles/ml as compared to 3.0 x 108 particles). This effect was dependent on the platelet concentration (from 10-80 platelets/PMN). Platelets stimulated by thrombin could alsoactivate the resting PMNs with a maximum obtained ata thrombin concentration of 0.1 U/ml, corresponding to the maximal release by these cells of products stored In their granules. However, the substitution ofplatelet suspensions by the released products found In their supernatant after stimulation by thrombin, resulted In a comparable stimulation only at platelet concentrations above the ones for coincubation experiments. These findings suggest that the presence of platelets themselves or In combination with their released products are responsible for this amplification. The use of zymosan alone or coated with IgG, C3b1, C3b or OpZ did not reveal any specificity of the Inducer for this amplification suggesting that platelets and/or platelet products acted by enhancing acommon step of PMNs activation Independent of the stimulus carried by the particles. Additionally, It could be noted that the maximal effect of the amplification by platelets occurred when the level of stimulation of the PMNs alone was the weakest.
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Del Maschio, A., M. Albors, F. Bucchi, M. Tomasiak, V. Bertele, C. Cerletti, and G. de Gaetano. "HUMAN POLYMORPHONUCLEAR LEUKOCYTE ACTIVATION INDUCED BY PLATELET ACTIVATING FACTOR (PAF)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643482.
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Human polymorphonuclear leukocytes (PMNs) loaded with the photoprotein Aequorin, were exposed to PAF in the presence of extracellular Ca2+ (1 mM). PMNs aggregation measured In the “Platelet Ionized Calcium Aggregometer” (P.I.C.A.) was dependent on the concentration of the stimulus. Ca2+ cytoplasmatic increase was monitored in parallel at concentrations of PAF which did not modify cellular integrity (10-7-10-5M). The intracellular Ca2+ flux (up to 19±3 µM) triggered by PAF was also concentration-dependent. In order to establish the role played by this intracellular messenger, we studied some cellular responses possibly related to Ca2+ mobilization: enzymatic release, oxygen radicals production, and arachidonic acid metabolism. PAF induced release of both lysozyme , and β-glucuronldase (15% to 20% of the total enzyme content at the maximal concentration). However PAF (10-712-10“Vl) stimulated the production of only small amounts of oxygen radicals as compared to Phorbol Myristate Acetate (PMA). Leukotriene B4 (LTB4), the main arachidonic acid metabolite in PMNs and the products of its catabolism (20-OH and 20-C00H LTBO were assayed by two different technics (HPLC and RIA) in the same cellular suspensions. PAF (10-4 M)-stimulated PMNs (0.5-2xl07 cells/ml) did not produce any detectable amount of these arachidonic acid metabolites. In contrast, calcium ionophore A 23187 (2 μM)-stimulated PMNs (in the same range of cellular concentration) produce up to 170 ng/ml of LTB4. In conclusion, cytoplasmatic Ca2+ increase in PAF-stimulated PMNs was not accompanied either by oxygen radicals production or by activation of arachidonic acid metabolism catalyzed by 5-1 ipoxygenase.
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Black, Kvar, Mingzhou Zhou, Pinaki Sarder, Maryna Kuchuk, Amal Y. Al-Yasiri, Sean P. Gunsten, Kexian Liang, et al. "Dual-radiolabeled nanoparticle probes for depth-independent in vivo imaging of enzyme activation." In Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications X, edited by Samuel Achilefu and Ramesh Raghavachari. SPIE, 2018. http://dx.doi.org/10.1117/12.2301033.
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Mannervik, Bengt, and Birgitta I. Sjödin. "Abstract LB-B23: Prodrug activation by designer enzyme targeted to CD123-expressing tumors." In Abstracts: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA. American Association for Cancer Research, 2018. http://dx.doi.org/10.1158/1535-7163.targ-17-lb-b23.
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Collins, Christian, and Christopher J. Ackerson. "Remote enzyme activation using gold coated magnetite as antennae for radio frequency fields." In Colloidal Nanoparticles for Biomedical Applications XIII, edited by Xing-Jie Liang, Wolfgang J. Parak, and Marek Osiński. SPIE, 2018. http://dx.doi.org/10.1117/12.2290478.
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Menasni, S., W. Hornebeck, L. Robert, and Y. Legrand. "ELASTASE TYPE ACTIVITY OF ENDOTHELIAL CELLS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643360.
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Elastin degrading enzymes have been reported in the vessel wall and both fibroblasts and smooth muscle cells have been shown to produce elastase type enzymes in culture. Data is presented here showing that porcine aortic endothelial cells produce enzyme activities hydrolyzing elastin and synthetic substrates I Sue Ala Ala Ala nitroanilide, SAPNAI considered specific for elastase. Enzyme activity against the SAPNA but not against H-elastin was found to be associated with the cells after triton lysis .This activity was not secreted into the culture medium . The elastolytic activity has been partially characterized in relation to the kinetic of hydrolysis, pH optimum and susceptibility to different inhibitors. These studies revealed the presence of at least two enzymes: a metalo-protease with a pH optimum of 7.5 which accounts for approx. 80% of the total activity, and a serine protease with pH optimum of 8.0 which accounts for the remaining 20% . When the conditioned culture medium was studied, virtually no proteolytic activity could be detected even after activation with an organomercurial agent. However fractionation of the culture medium by gel filtration on HPLC resulted in elastolytic activity both against H-elastin and SAPNA. Proteolytic activity against casein could also be revealed after separation on SDS-PAGE. It is likely that these separation techniques remove an inhibitor also produced by the endothelial cells and allow the expression of proteolytic activity. That the elastolytic activity and the caseinolytic activity revealed by HPLC and PAGE respectively represent the activity of the same enzyme hase not yet been determined, and its relationship to the Stromelysin described by Herron et al(J. Biol. Chem. , 1986, 261. 2810-2813) in rabbit brain capillary endothelial cells is being investigated.
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Molina, Luis, y. Vedia, and Eduardo G. Lapetina. "ENZYMES THAT DEPHOSPHORYLATE INOSITOL PHOSPHATES IN HUMAN PLATELETS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644522.
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Inositol trisphosphate (IP3) is now recognized as a second messenger molecule that mobilizes Ca2+ from intracellular stores to the cytosol. The persistence of the action of IP3 depends on the specific phosphatase that converts IP3 to inositol bisphosphate (IP2). The activation of IP3 phosphatase is important in terminating the Ca2+ signal in stimulated cells. In platelets it has previously been shown that this enzyme is regulated by protein kinase C since it is stimulated by phorbol esters and 1,2-diacylglycerol (Molina y Vedia, L., and Lapetina, E.G. J. Biol Chem. 261, 10493-10495, 1986) and the cytosolic platelet enzyme is phosphorylated by brain protein kinase C, resulting in a 4-fold increase in IP3 phosphatase activity (Connolly, T. M., Lawing, W.J., Jr., and Majerus, P.W., Cell, 46, 951-958, 1986). We have studied the subcellular distribution of the phosphatases that hydrolyze IP3, IP2 and inositol monophosphate (IP) in human platelets. Three subcellular fractions were obtained from human platelets lysed by freezing and thawing: a cytosolic fraction, a membrane fraction and a mixed particulate fraction containing granules, mitochondria and membranes. These fractions have been characterized by specific marker enzymes. The highest specific activity of IP3 -phosphatase is associated with the membrane fraction and accounts for about 10-15% of the total activity. The mixed particulate fraction has 35-40% of the activity while about 50% is cytosolic. The Km of the membrane fraction enzyme is 100 μM. This enzyme is extracted by 1M NaCl and hydrodynamic studies revealed a molecular weight of 50 kDa. The NaCl extracted-enzyme has been further purified by hydrophobic and gel filtration chromatographies. This activity does not hydrolyses IP but hydrolyse IP2 at a lower rate. The enzyme that hydrolyses IP to inositol is confined to the cytosolic fraction, has a Km of 130 μM, is inhibited by Li+, and hydrodynamic studies show an apparent molecular weight of 91 kDa.
Reports on the topic "Enzyme activation"
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Wang, Min, and Garry Beuttner. The Enzyme MnSOD Suppresses Malignant Breast Cell Growth by Preventing HIF-1 Activation. Fort Belvoir, VA: Defense Technical Information Center, May 2003. http://dx.doi.org/10.21236/ada418673.
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Prusky, Dov, Noel Keen, and Rolf Christoffersen. Involvement of Epicatechin in the Regulation of Natural Resistance of Avocado Fruit against Postharvest Pathogens. United States Department of Agriculture, January 1997. http://dx.doi.org/10.32747/1997.7613028.bard.
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In this project it was found that the activation of the mechanism of resistance in avocado fruits to Colletotrichum gloeosporioides depends on the increase of the level of the preformed antifungal diene. This increase is regulated by the synthesis of the flavonoid epicatechin present in the fruit peel. Epicatechin is an inhibitor of the enzyme lipoxygenase whose activity catalyze the breakdown of the antifungal diene. Increase in epicatechin concentration inhibits the breakdown of the antifungal compound and since the compound is continuously synthesized, both combined processes result in the increase of the antifungal level. Biotic and abiotic elicitors affecting the mechanism of resistance, all activate the synthesis of epicatechin. As abiotic elicitors were tested wounding, ethylene and CO2 treatments. As biotic elicitors were tested challenge inoculation with C. gloeosporioides, Colletotrichum magna (a non pathogen of avocado) and also non pathogenic strain of C. magna. In all the cases activation of the key enzymes of the phenylpropanoic pathway is followed by an enhance in the level of epicatechin and the antifungal diene. In order to determine the level of regulation by the different elicitors of the mechanism, the genes encoding for key enzymes of the phenylpropanoic pathway were cloned and it was found that the different elicitors regulate the expression of those genes at a translational level. Modulation of the mechanism of resistance could also be done by activation of lipoxygenase gene expression. For this purpose lipoxygenase from avocado was cloned and its over-expression, under the effect of methyl jasmonate, determined.
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Locy, Robert D., Hillel Fromm, Joe H. Cherry, and Narendra K. Singh. Regulation of Arabidopsis Glutamate Decarboxylase in Response to Heat Stress: Modulation of Enzyme Activity and Gene Expression. United States Department of Agriculture, January 2001. http://dx.doi.org/10.32747/2001.7575288.bard.
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Most plants accumulate the nonprotein amino acid, g-aminobutyric acid (GABA), in response to heat stress. GABA is made from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), an enzyme that has been shown by the Israeli PI to be a calmodulin (CaM) binding protein whose activity is regulated in vitro by calcium and CaM. In Arabidopsis there are at least 5 GAD genes, two isoforms of GAD, GAD1 and GAD2, are known to be expressed, both of which appear to be calmodulin-binding proteins. The role of GABA accumulation in stress tolerance remains unclear, and thus the objectives of the proposed work are intended to clarify the possible roles of GABA in stress tolerance by studying the factors which regulate the activity of GAD in vivo. Our intent was to demonstrate the factors that mediate the expression of GAD activity by analyzing the promoters of the GAD1 and GAD2 genes, to determine the role of stress induced calcium signaling in the regulation of GAD activity, to investigate the role of phosphorylation of the CaM-binding domain in the regulation of GAD activity, and to investigate whether ABA signaling could be involved in GAD regulation via the following set of original Project Objectives: 1. Construction of chimeric GAD1 and GAD2 promoter/reporter gene fusions and their utilization for determining cell-specific expression of GAD genes in Arabidopsis. 2. Utilizing transgenic plants harboring chimeric GAD1 promoter-luciferase constructs for isolating mutants in genes controlling GAD1 gene activation in response to heat shock. 3. Assess the role of Ca2+/CaM in the regulation of GAD activity in vivo in Arabidopsis. 4. Study the possible phosphorylation of GAD as a means of regulation of GAD activity. 5. Utilize ABA mutants of Arabidopsis to assess the involvement of this phytohormone in GAD activation by stress stimuli. The major conclusions of Objective 1 was that GAD1 was strongly expressed in the elongating region of the root, while GAD2 was mainly expressed along the phloem in both roots and shoots. In addition, GAD activity was found not to be transcriptionally regulated in response to heat stress. Subsequently, The Israeli side obtained a GAD1 knockout mutation, and in light of the objective 1 results it was determined that characterization of this knockout mutation would contribute more to the project than the proposed Objective 2. The major conclusion of Objective 3 is that heat-stress-induced changes in GAD activity can be explained by heat-stress-induced changes in cytosolic calcium levels. No evidence that GAD activity was transcriptionally or translationally regulated or that protein phosphorylation was involved in GAD regulation (objective 4) was obtained. Previously published data by others showing that in wheat roots ABA regulated GABA accumulation proved not to be the case in Arabidopsis (Objective 5). Consequently, we put the remaining effort in the project into the selection of mutants related to temperature adaptation and GABA utilization and attempting to characterize events resulting from GABA accumulation. A set of 3 heat sensitive mutants that appear to have GABA related mutations have been isolated and partially characterized, and a study linking GABA accumulation to growth stimulation and altered nitrate assimilation were conducted. By providing a better understanding of how GAD activity was and was not regulated in vivo, we have ruled out the use of certain genes for genetically engineering thermotolerance, and suggested other areas of endeavor related to the thrust of the project that may be more likely approaches to genetically engineering thermotolerance.
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Lurie, Susan, John Labavitch, Ruth Ben-Arie, and Ken Shackel. Woolliness in Peaches and Nectarines. United States Department of Agriculture, 1995. http://dx.doi.org/10.32747/1995.7570557.bard.
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The overall goal of the research was to understand the processes involved in the development of woolliness in peaches and nectarines. Four specific hypotheses were proposed and in the course of the research evidence was gathered t support two of them and to not support two others. The hypotheses and a summary of the evidence are outlined below. 1. That woolliness arises from an imbalance between the activities of the cell wall pectin degrading enzymes. Using 'Flavortop' nectarines and 'Hermoza' peaches as model systems, storage regimes were manipulated to induce or prevent woolliness. The expression (mRNA abundance), protein content (Western blotting), and activity of polygalacturonase (PG) and pectin esterase (PE) were followed. Expression of the enzymes was not different, but activity and the ratio between PG and PE activities were quite different in fruits developing woolliness or ripening normally. This was also examined by looking at the substrate, the pectin moiety of the cell wall, and i woolly fruit there were more high molecular weight pectins with regions of non-methylated galacturonic acid residues. Taking an in vitro approach it was found a) that PE activity was stable at 0oC while PG activity decreased; b) incubating the calcium pectate fraction of the cell wall with PE extracted from peaches caused the polymers to form a gel characteristic of the visual woolly symptoms in peaches. 2. That continued cell wall synthesis occurs during storage and contributes to structural changes i cell walls and improper dissolution and softening after storage. We tried to adapt our technique of adding 13C-glucose to fruit discs, which was used successfully to follow cell wall synthesis during tomato ripening. However, the difference in sugar content between the two fruits (4% in tomato and 12% in peach) meant that the 13C-glucose was much more diluted within the general metabolite pool. We were unable to see any cell wall synthesis which meant that either the dilution factor was too great, or that synthesis was not occurring. 3. That controlled atmosphere (CA) prevents woolliness by lowering all enzyme activities. CA was found to greatly reduce mRNA abundance of the cell wall enzymes compared to regular air storage. However, their synthesis and activity recovered during ripening after CA storage and did not after regular air storage. Therefore, CA prevented the inhibition of enzyme activation found in regular air storage. 4. That changes in cell wall turgor and membrane function are important events in the development of woolliness. Using a micro pressure probe, turgor was measured in cells of individual 'O'Henry' and 'CalRed' peaches which were woolly or healthy. The relationship between firmness and turgor was the same in both fruit conditions. These data indicate that the development and expression of woolliness are not associated with differences in membrane function, at least with regard to the factors that determine cell turgor pressure. In addition, during the period of the grant additional areas were explored. Encoglucanase, and enzyme metabolizing hemicellulose, was found to be highly expressed air stored, but not in unstored or CA stored fruit. Activity gels showed higher activity in air stored fruit as well. This is the first indication that other components of the cell wall may be involved in woolliness. The role of ethylene in woolliness development was also investigated at it was found a) that woolly fruits had decreased ability to produce ethylene, b) storing fruits in the presence of ethylene delayed the appearance of woolliness. This latter finding has implication for an inexpensive strategy for storing peaches and nectarines.
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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.
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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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Sandermann, Heinrich, Duncan Jr., and Thomas M. Lipid-Dependent Membrane Enzymes. Kinetic Modelling of the Activation of Protein Kinase C by Phosphatidylserine. Fort Belvoir, VA: Defense Technical Information Center, January 1991. http://dx.doi.org/10.21236/ada302987.
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Prusky, Dov, Noel T. Keen, and Stanley Freeman. Elicitation of Preformed Antifungal Compounds by Non-Pathogenic Fungus Mutants and their Use for the Prevention of Postharvest Decay in Avocado Fruits. United States Department of Agriculture, January 1996. http://dx.doi.org/10.32747/1996.7570573.bard.
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C. gloeosporioides attacks unripe avocado fruits in the orchard. Germinated spores produce appressoria that germinate and breach the cuticle, but the resultant subcuticular hyphae become quiescent and do not develop further until fruit is harvested and ripens. Resistance of unripe avocado to attach by C. gloeosporioides is correlated with the presence of fungitoxic concentrations of the preformed antifungal compound, 1-acetoxy-2-hydroxy-4-oxoheneicosa-12, 15 diene in the pericarp of unripe fruits. The objective of this proposal was to study the signal transduction process by which elicitors induce resistance in avocado. It was found that abiotic elicitors, infection of avocado fruit with C. gloeosporioides or treatment of avocado cell suspension with cell-wall elicitor induced a significant production of reactive oxygen species (ROS). Ripe and unripe fruit tissue differ with regard to the ROS production. The unripe, resistant fruit are physiologically able to react and to produce high levels of ROS and increased activity of H+ATPase that can enhance the phenylpropanoid pathway ad regulate the levels of the antifungal compound-diene, inhibit fungal development, resulting in its quiescence. Interestingly, it was also found that growth regulators like cytokinin could do activation of the mechanism of resistance. Postharvest treatments of cytokinins strongly activated the phenylpropanoid pathway and induce resistance. We have developed non-pathogenic strains of C. gloeosporioides by Random Enzyme Mediated Integration and selected a hygromycin resistance, non-pathogenic strain Cg-142 out of 3500 transformants. This non-pathogenic isolate activates H+ATPase and induces resistance against Colletotrichum attack. As a basis for studying the importance of PL in pathogenicity, we have carried out heterologous expression of pel from C. gloeosporioides in the non-pathogenic C. magna and determine the significant increase in pathogenicity of the non-pathogenic strain. Based on these results we can state that pectate lyase is an important pathogenicity factor of C. gloeosporioides and found that fungal pathogenicity is affected not by pel but by PL secretion. Our results suggest that PH regulates the secretion of pectate lyase, and support its importance as a pathogenicity factor during the attack of avocado fruit by C. gloeosporioides . This implicates that if these findings are of universal importance in fungi, control of disease development could be done by regulation of secretion of pathogenicity factors.
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Freeman, Stanley, and Russell J. Rodriguez. The Interaction Between Nonpathogenic Mutants of Colletotrichum and Fusarium, and the Plant Host Defense System. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7573069.bard.
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The intent of this proposal was to study the interaction between nonpathogenic mutants of Colletotrichum magna and Fusarium oxysporum, and the cucurbit host defense system. We had shown previously that a nonpathogenic endophytic mutant path- 1 of C. magna, caused no visible disease symptoms but protected watermelon seedlings from disease caused by the wildtype isolate and F. o. niveum. Objectives were: 1) Determine the microscopic, biochemical and molecular genetic interaction between "protected" (path- 1 colonized) cucurbit hosts and wildtype isolates of C. magna; 2) Isolate non-pathogenic mutants of F.o. melonis and test feasibility for protecting plants against fungal diseases. We found that path-1 caused no visible disease symptoms in cucurbit seedlings but conferred disease resistance against pathogenic isolates of C. magna, C. orbiculare, and F. oxysporum. Disease resistance conferred by path-1 correlated to a decrease in the time of activation of host defense systems after exposure of path-1 colonized plants to virulent pathogens. This was determined by monitoring the biochemical activity of PAL and peroxidase, and the deposition of lignin. It appears that path-1-conferred disease resistance is a multigenic phenomenon which should be more difficult for pathogen to overcome than single gene conferred resistance. Based on the benefits conferred by path-1, we have defined this mutant as expressing a mutualistic lifestyle. REMI (restriction enzyme-mediated integration) nonpathogenic mutants were also isolated using pHA1.3 plasmid linearized with Hind III and transformed into wildtype C. magna. The integrated vector and flanking genomic DNA sequences in REMI mutant R1 was re-isolated and cloned resulting in a product of approximately 11 kb designated pGMR1. Transformations of wildtype C. magna with pGMR1 resulted in the same non-pathogenic phenotype. A nonpathogenic mutant of F.o. melonis (pathogenic to melon) was isolated that colonized melon plants but elicited no disease symptoms in seedlings and conferred 25 - 50% disease protection against the virulent wildtype isolate. Subsequently, nonpathogenic mutant isolates of F.o. niveum (pathogenic to watermelon) were also isolated. Their protection capacity against the respective wildtype parent is currently under investigation. This research has provided information toward a better understanding of host-parasite interactions; specifically, endophytes, pathogens and their hosts. It will also allow us to assess the potential for utilizing nonpathogenic mutants as biological control agents against fungal pathogens and isolating molecular genetic factors of pathogenicity in Fusarium.
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Rafaeli, Ada, Wendell Roelofs, and Anat Zada Byers. Identification and gene regulation of the desaturase enzymes involved in sex-pheromone biosynthesis of pest moths infesting grain. United States Department of Agriculture, March 2008. http://dx.doi.org/10.32747/2008.7613880.bard.
Full textAbstract:
The original objectives of the approved proposal included: 1. Establishment of the biosynthetic pathways for pheromone production using labeled precursors and GC-MS. 2. The elucidation of a circadian regulation of key enzymes in the biosynthetic pathway. 3. The identification, characterization and confirmation of functional expression of the delta-desaturases. 4. The identification of gene regulatory processes involved in the expression of the key enzymes in the biosynthetic pathway. Background to the topic: Moths constitute one of the major groups of pest insects in agriculture and their reproductive behavior is dependent on chemical communication. Sex-pheromone blends are utilized by a variety of moth species to attract conspecific mates. The sex pheromones used are commonly composed of blends of aliphatic molecules that vary in chain length, geometry, degree and position of double bonds and functional groups. They are formed by various actions of specific delta-desaturases to which chain shortening, elongation, reduction, acetylation, and oxidation of a common fatty acyl precursor is coupled. In most of the moth species sex-pheromone biosynthesis is under circadian control by the neurohormone, PBAN (pheromone-biosynthesis-activating neuropeptide). The development of specific and safe insect control strategies utilizing pheromone systems depends on a clear knowledge of the molecular mechanisms involved. In this proposal we aimed at identifying and characterizing specific desaturases involved in the biosynthetic pathway of two moth pest-speciesof stored products, P. interpunctella and S. cerealella, and to elucidate the regulation of the enzymes involved in pheromone biosynthesis. Due to technical difficulties the second stored product pest was excluded from the study at an early phase of the research project. Major conclusions: Within the framework of the planned objectives we confirmed the pheromone biosynthetic pathway of P. interpunctella and H. armigera by using labeled precursor molecules. In addition, in conjunction with various inhibitors we determined the PBAN-stimulated rate-limiting step for these biosynthetic pathways. We thereby present conclusive evidence that the enzyme Acetyl Coenzyme A Carboxylase is activated as a result of PBAN stimulation. We also found that P. interpunctella produce the main pheromone component Z9, E12 Tetradecenyl acetate through the action of a D11 desaturase working on the 16:Acid precursor. This is evidenced by the high amount of incorporation of ²H-labeled 16:Acid into pheromone when compared to the incorporation of ²H-labeled 14:Acid. However, in contrast to reports on other moth species, P. interpunctella is also capable of utilizing the 14:Acid precursor, although to a much lesser extent than the 16:Acid precursor. Despite the discovery of nine different desaturase gene transcripts in this species, from the present study it is evident that although PCR detected all nine gene transcripts, specific to female pheromone glands, only two are highly expressed whereas the other 7 are expressed at levels of at least 10⁵ fold lower showing very low abundance. These two genes correspond to D11-like desaturases strengthening the hypothesis that the main biosynthetic pathway involves a D11 desaturase.
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Prusky, Dov, Noel Keen, and John Browse. Modulation of the synthesis of the main preformed antifungal compound as abasis for the prevention of postharvest disease of C. gloeosporioides in avocado fruits. United States Department of Agriculture, December 2001. http://dx.doi.org/10.32747/2001.7575273.bard.
Full textAbstract:
The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned , The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned D9 and D12 desaturase, a protein kinase and a elongase that their transcriptional activation is significantly enhanced during the enhanced synthesis of the antifungal diene. Although we are far away from a complete elucidation of the synthesis of the antifungal compound we have stepped forward determining some of the key steps that might be involved in its synthesis.