Dissertations / Theses on the topic 'Sarcoplasmic proteins'

The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membraneous network that, through the cyclical release and re-uptake of Ca²⁺ into and from, respectively, the cytoplasmic space, regulates myofilament shortening and, therefore, muscle contraction. SR derived from the terminal cisternae (HSR) demonstrates the property of Ca²⁺-induced Ca²⁺ release. Upon attainment of a threshold intralumenal Ca²⁺ load, application of a small pulse of extralumenal Ca²⁺ stimulates the release of a pool of intralumenal Ca²⁺ via the ligand gated Ca²⁺ permeable pore of the Ca²⁺ release channel/ryanodine receptor complex. It was hypothesised that intralumenal Ca²⁺ regulates the opening of the release channel. HSR vesicles were purified from skeletal and cardiac muscle by a novel technique. Structural characterisation of these membranes demonstrated an enrichment of harvested fractions in the Ca²⁺ release channel and the intralumenal Ca²⁺ binding protein, calsequestrin. In radiometric studies, skeletal HSR vesicles were shown to bind ryanodine with high capacity at both low and high affinity sites, with 2 fold stimulation of Ca²⁺ accumulation by the polyorganic cation Ca²⁺ channel blocker, ruthenium red. HSR vesicles passively loaded Ca²⁺. Passive loading of HSR vesicles with Ca²⁺ was found to be non-linearly dependent upon the concentration of Ca²⁺ within the loading medium. This suggested the presence of 2 intralumenal Ca²⁺ binding sites with different affinities for Ca²⁺. A spectroscopic dual-wavelength assay of Ca²⁺ release was developed that took advantage of peculiar spectral properties of the metallochromic sensitive dye Antipyrylazo III. In the presence of mM MgATP and mM Mg2+ the initial fast phase of HSR Ca²⁺ was well resolved. Evidence was presented that initial rapid uptake was associated with high affinity binding to an intralumenal compartment. Ca²⁺ -induced Caz+ release was shown to occur with a threshold loading of intralumenal Ca²⁺. The intralumenal Ca²⁺ threshold for Ca²⁺-induced Ca²⁺ release was decreased in the presence of ryanodine. Ryanodine induced Ca²⁺ release was also dependent upon the amount of intralumenal Ca²⁺. Ryanodine was also shown to inhibit sustained Ca²⁺-induced Ca²⁺ release by apparent inhibition of the binding of Ca²⁺ to intralumenal sites. These results suggested that junctional state transitions of the Ca²⁺ channel and calsequestrin were interdependent. Purified mM and mM Ca²⁺ activated neutral protease isoforms selectively cleaved the Ca²⁺ channel into 410 and 150kDa peptides with limited proteolysis. This was demonstrated in both HSR vesicles and the purified Ca²⁺ release channel. A novel 88kDa protein was also shown to be fragmented by both CANP isoforms. The identity of this prominent HSR associated protein remains obscure. CANP fragmentation of HSR protein elevated passive and active 4^Ca²⁺ loading in vesicles. This indicated that selective structural modification of the cytoplasmic portion of the release channel modified the comformational states of a intralumenal Ca²⁺ binding compartment in HSR vesicles. In spectroscopic studies, CANP proteolysis of HSR proteins increased the sensitivity to Ca²⁺ and ryanodine-induced Ca²⁺ release through decreases in the required intralumenal Ca²⁺ threshold for release. These functional alterations coincided with apparent single site cleavage of the release channel. Further proteolysis of the initial 410 and 150kDa peptides was without further significant effect upon function. Based upon the hypothesis that primary sequences rich in proline (P), glutamate (E), aspartate (D), serine (S) and threonine (T) (PEST regions) are recognition sites for CANP binding to substrates, a search for PEST regions within the Ca²⁺ channel was undertaken. It was tentatively proposed that two PEST regions near the N-terminal of the Caz release channel may represent sites close to the CANP cleavage site. The results of this work were discussed in relation to a possible role of Ca²⁺-induced Ca²⁺ release in regulating the patterning of Ca²⁺ cytosolic transients. The frequency and amplitude of cytosolic Ca²⁺ transients appear to be important in regulating protein expression. The requirement of intralumenal Ca²⁺-induced Ca²⁺ release may be a means by which the cyclical uptake and release of Ca²⁺ during muscle relaxation and contraction can be coordinated. This coordination may define the patterning of cytosolic Ca²⁺ transients. The increased sensitivity to Ca²⁺-induced Ca²⁺ release by HSR after CANP treatment may represent a means by which the patterning of cytosolic Ca²⁺ transients can be altered to effect changes in protein synthesis.
Graduate and Postdoctoral Studies
Graduate

This study was carried out to determine the characteristics of common carp surimi. In Australia, common carp (Cyprinus carpio) is an environmental pest, strongly coloured (dark-muscle fish), large (2-3 kg), low cost (AUD 2.5/kg) and not highly valued as it is every where else. Surimi could add value to carp, but the colour would have to be modified as surimi manufacturers prefer white coloured flesh. So, firstly the efficiency of Hydrogen peroxide (H2O2; 1-3% v/v) solution at alkaline side of pH (7.0-11.5) on whitening of light fillets of common carp was examined. The whiteness (L*-3b*) of surimi produced from treated (3% H2O2, pH 8.2) common carp light fillets was significantly (p less than 0.05) greater than that of threadfin bream surimi and was not significantly different to that of Alaska pollock. Based on a temperature sweep test, a similar pattern in G of tested surimi was observed which started at ca. 47?C and was completed at ca. 73-74?C. However, thread fin bream kamaboko showed better texture profile characteristics (hardness and gel strength) than that of the other kamaboko tested. To improve the quality of common carp surimi and kamaboko, alternative methods were applied such as modified conventional method (MCM), alkaline-aided method (AAM) and pH modified method (PMM) and the resultant surimi and kamaboko were compared with those produced by the traditional method (TM). In MCM each washing cycle was followed by a centrifugation step for a more effective dewatering and removal of sarcoplasmic proteins (Sp-P). Kamaboko prepared from MCM was whiter and had significantly (p less than 0.05) improved textural characteristics (hardness and gel strength) than that from TM, AAM and PMM. Furthermore, SEM of surimi and kamaboko showed higher number of polygonal structure/mm2 in the gel matrix of MCM kamaboko, as a result of more cross-linking of the myofibrillar proteins, than that recorded for TM, AAM and PMM samples tested. Finally, this study examined the effect of adding common carp sarcoplasmic proteins (Sp-P) on the gel characteristics of threadfin bream surimi and kamaboko. Based on the temperature sweep test, the depths of the valley in the G thermograph of the gels decreased as the concentration of added Sp-P increased from 5% to 35%. Storage modulus (G) of the gels showed greater elasticity in the samples with added Sp-P compared with the control samples without added Sp-P. Furthermore, the breaking force and breaking distance and consequently gel strength of the resultant kamaboko were improved, significantly (p less than 0.05) with added Sp-P. Thus, added Sp-P did not interfere with the gelling of myofibrillar proteins during sol-gel transition phase and was associated with textural quality enhancement for the resultant kamaboko. However, the addition of freeze-dried Sp-P from the dark muscle of the carp decreased the whiteness of the resultant surimi. Furthermore, the gel strength could not be associated with either the number of polygonal structures/mm2 or the area of the polygonal structures.

Tryptic digestion of SRV labelled with N-cyclohexyl-N'-(4-dimethylamino--naphthyl) carbodiimide (NCD-4), was shown to expose Ca^2+-protectable NCD-4-labelled amino acid residues, manifested by a time dependent quench (38%) of the initial fluorescence of the preparation. This finding is discussed in terms of a 3D model of the (Ca2+ + Mg2+)-ATPase. The inhibition of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by the diphenolic antioxidants: 4,4'-isopropylidene diphenol, 4-hydroxy, 4'-methoxy isopropylidene diphenol, 4,4'dimethoxy isopropylidene diphenol and bis (2-hydroxy-3,5-methyl phenyl) methane, was studied, using the fluorescence response to bound NCD-4. They were shown to modulate conformational transitions of the ATPase subsequent to Ca2+ binding. It is suggested that the diphenolic antioxidants may have a target/binding site on the ATPase polypeptide via which they compete with Ca2+ ion binding and translocation. The stoichiometry of incorporation into the ATPase of tritated NCD-4 was studied. The ATPase was incubated with [3H]-NCD-4 for 4h. in the presence and in the absence of Ca2+ ions and then treated with trypsin for 35 mins. The four fragments: A(Mr= 50KD), B(Mr= 45KD), A1(Mr= 30KD) and A2(Mr= 20KD) were purified by preparative SDS-PAGE. The stoichiometry of incorporation of the radioactive label into fragments A1, A2 and the A2 subfragments SI (Mr= 13KD) and SII(Mr= 8.5KD) from CNBr cleavage, was measured by liquid scintillation counting of the gel eluates. In the absence of Ca2+, fragment A2 was found to incorporate 2.3 nmol [^3H]-NCD-4 per nmol of protein more than the equivalent preparation in the presence of Ca^2+. Subfragment S_I was shown to incorporate roughly equal amounts of label in both cases. S_II, however, in the absence of Ca^2+ was found to incorporate 1.15-2.3 nmol [^3H]-NCD-4 more than the equivalent preparation in the presence of Ca^2+. These differences were attributed to inhibitor binding at the Ca^2+-binding sites of the enzyme. However, discrepancies in the absolute incorporation values when experiments were compared, were related to loss of bound label due to nucleophilic attack. Four isomers of NCD-4 were prepared and evaluated in terms of inhibitory capacity towards the (Ca^2+ + Mg^2+)-ATPase. Preliminary evidence suggested that even related (isomeric) carbodimides can label different sites on the ATPase polypeptide. The synthesis and the evaluation of (N-cyclohexyl, N'-dimethylamino phenylazophenyl) carbodiimide (ACD) as a chromophoric probe for the Ca^2+ sites of the ATPase, is also described: this compound emerged as a relatively poor inhibitor of the enzyme, despite showing favourable chromophoric properties.

Abstract Skeletal muscles are composed of long, multinucleated cells called myofibers, which are highly differentiated cells and therefore unique in structure. In the present study the organization of the endocytic and exocytic pathways in isolated rat skeletal myofibers was defined with confocal and electron microscopic methods. In isolated myofibers the I band areas were shown to be active in endocytosis. The sorting endosomes were distributed in a cross-striated fashion while the recycling and late endosomal compartments were located to perinuclear areas and interfibrillar spaces, where they followed the course of microtubules. Protein trafficking in the different stages of muscle cell differentation was also analyzed. The studies with L6 myoblasts and myotubes showed that during myogenesis varying fractions of different viral glycoproteins were sorted from the endoplasmic reticulum (ER) into a specific compartment that did not recycle with the Golgi apparatus. This compartment is suggested to be the sarcoplasmic reticulum (SR). The studies with living muscle cells showed further changes in vesicle trafficking taking place during myogenesis. With GFP-tagged tsO45G protein, transport containers were detected in 20% of the infected myofibers, while all infected L6 myoblasts or myotubes showed intense movement of corresponding structures. We also detected significant differences between the pre-and post-Golgi traffickings in myofibers. When the distribution of the ER in adult myofibers was studied, the confocal microscopic data showed that the labeling patterns of the rough endoplasmic reticulum (RER) and the SR markers were different. Blocking of different cargo proteins in the RER revealed two discrete distribution patterns, neither of them identical with the SR. The collected electron microscopic data supported the idea that in mature myofibers there are two separate RER compartments. We suggest that the RER compartment capable of export function located around the myonuclei and on the Z lines, while the non-exporting RER compartment localized to terminal cisternae and probably took care of the synthesis of the SR proteins.

Fatigue and skeletal muscle weakness are problems associated with congestive heart failure. Research does not support the theory that the affected cardiac function is responsible for the fatigue. During skeletal muscle fatigue, calcium handling is altered. Thus, the fatigue associated with congestive heart failure could be attributed to altered calcium handling. The main proteins involved in calcium release are the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The main proteins involved in calcium uptake are the fast and slow isoforms of sarco(endo)plasmic reticulum calcium ATPase (SERCA 1 and SERCA 2 respectively). Calsequestrin (Csq) and calmodulin (CaM) play regulatory roles in calcium handling. Changes in the levels of these proteins could explain alterations in calcium handling and subsequent muscle function. The purpose of this study was to use a genetic model of heart failure, the SHHF rat, to examine the levels of regulatory calcium handling proteins to determine if changes in the amounts of RyR, DHPR, SERCA1, SERCA2, Csq and CaM are altered in congestive heart failure. A significant decrease was found in the amounts of RyR, DHPR, and SERCA 1 of the SHHF gastrocnemius and diaphragm samples in comparison to the control. There was no significant difference found in the amounts of CaM or SERCA 2 between the two groups. Csq was not found to be statistically different between the two groups of the gastrocnemius samples. However, there was an increase in Csq in the SHHF diaphragm samples in comparison to the control. In conclusion, the calcium handling proteins are affected in the genetic model of heart failure. These changes could explain previous reports of altered calcium handling within the skeletal muscles of congestive heart failure animals.
Master of Science

Abstract Skeletal muscle cells (myofibers) are huge multinucleated cells responsible for muscle contraction and hence for the everyday movements of the joints. The structure of these voluminous cells differs greatly from that of the mononucleated cells – the characteristic features of the myofibers include dozens of peripherally located nuclei, tightly packed contractile apparatus and a sophisticatedly organized endomembrane system. The basic physiology involving myofibers is quite well known, but scarce data exist on the membrane biology of the myofibers. The purpose of this study was to examine the localization of mRNA and the site of protein synthesis in the myofibers. The characterization of the membrane dynamics in muscle cells was also performed. In this study we utilized a primary cell culture model obtained from the rat flexor digitorum brevis (FDB) muscle. Also frozen sections from the rat extensor digitorum longus muscle were used. The precursor cells of the myofibers – myoblasts and myotubes – were also utilized in some experiments. Furthermore, methods of immunohistochemistry and molecular biology were applied extensively in this study. We found that in FDB myofibers the mRNA lies just under the plasma membrane. Protein synthesis seemed to be concentrated in the vicinity of nuclei locating beneath the plasma membrane but also in interfibrillar dot-like structures. Protein products moved hundreds of micrometers away from the nuclei of origin. Moreover, there were no barriers for protein movement into the core regions of the myofibers. Movement of proteins was found to be rapid in the cytosol and in the endomembrane system, too. Interestingly, when examining exocytic trafficking we observed that ER-to-Golgi trafficking significantly differed from that of mononucleated cells. Finally, myofibers were found to be able to generate lipid bodies under stress conditions. The dynamics of lipid bodies seemed to deviate from the dynamics found in other cells types. Nowadays not much muscle research with primary myofibers is done worldwide, and therefore dilemmas involving myofibers such as insulin resistance and myotoxicity of statins are mostly unresolved. The knowledge gained from this study may be used in the future to solve clinical problems related to the cell biology of the myofibers
Tiivistelmä Luurankolihassolut eli myofiiberit ovat jättimäisiä monitumaisia soluja, jotka vastaavat lihassupistuksen aikaansaamisesta ja siten mahdollistavat jokapäiväisen liikkumisemme. Näiden suurten solujen rakenne poikkeaa selkeästi yksitumaisten solujen rakenteesta: myofiiberien tunnusomaisia piirteitä ovat kymmenet solun reunoille sijoittuneet tumat, tiiviisti pakkautunut supistumiskoneisto ja monimutkaisesti järjestynyt solukalvostojärjestelmä. Vaikka myofiiberien perusfysiologia tunnetaankin hyvin, niin tiedetään itse myofiiberien kalvostobiologiasta sangen vähän. Kokonaisuutena tämän tutkimuksen tarkoituksena oli tarkastella mRNA:n ja proteiinisynteesin sijaintia myofiibereissä. Lisäksi selvitimme lihassolujen kalvostodynamiikkaa. Tässä tutkimuksessa käytimme rotan flexor digitorum brevis (FDB) -lihaksesta saatua primääristä soluviljelymallia. Lisäksi hyödynsimme rotan extensor digitorum longus -lihaksesta hankittuja jääleikkeitä. Joissakin kokeissa käytimme myös myofiiberien esiastesoluja (myoblasteja ja myotuubeja). Immunohistokemian ja molekyylibiologian menetelmiä sovellettiin tutkimuksessa laajasti. Havaitsimme, että FDB –myofiibereissä mRNA sijaitsee aivan solukalvon alla. Proteiinisynteesi vaikutti olevan keskittynyt solukalvon alla sijaitsevien tumien ympärille, mutta myös solusisäisiin pistemäisiin rakenteisiin. Proteiinituotteet ylsivät satojen mikrometrien päähän alkuperäisestä tumastaan. Lisäksi proteiineille ei ilmennyt leviämisestettä myofiiberin sisäosiin. Leviämisen havaittiin olevan nopeaa sekä solulimassa että solulimakalvostoissa. Tutkiessamme solun eritystoimintaa huomasimme, että kuljetus ER:stä Golgin laitteeseen eroaa huomattavasti yksitumaisten solujen vastaavasta kuljetuksesta. Lopuksi havaitsimme myofiiberien pystyvän muodostamaan rasvapisaroita rasitusolosuhteissa. Rasvapisaroiden käyttäytyminen näytti myös poikkeavan siitä, mitä muissa soluissa on havaittu. Nykyään lihastutkimusta primäärisoluilla ei juuri tehdä maailmalla, minkä vuoksi myofiibereihin liittyvät lääketieteelliset pulmat kuten insuliiniresistenssi ja statiinien lihashaitat ovat suurelta osin ratkaisematta. Tästä tutkimuksesta saatuja tuloksia voitaneen jatkossa käyttää myofiiberien solubiologiaan liittyvien kliinisten ongelmien selvittämiseen

One advantage of higher life-forms over less developed organisms is their ability to respond to signals from their environment with motion. This requires highly specialized contractile cells and a whole locomotion apparatus. In vertebrates, the cells responsible for movement are the skeletal muscle cells. They receive signals from the autonomic nervous system in the form of an action potential, and they contract in an appropriate manner. Calcium is a vital intracellular passenger whose role in muscular function is to initiate contraction. It is released via specific channel proteins from an internal Ca++ store, the sarcoplasmic reticulum, and triggers muscular contraction, the actual interplay of actin and myosin filaments. The step that is still not fully understood is the coupling process between arrival of an action potential and the subsequent contraction, called excitation-contraction coupling. Several theories have been proposed to explain this process. Some years ago, our laboratory introduced the hypothesis that an oxidation-reduction reaction of critical sulfhydryls associated with the Ca+t channel protein are involved in the regulation of channel gating. In an effort to understand more about the Ca++ channel gating mechanism at the molecular level, this thesis focuses on the interaction between o-phthalaldehyde, a reagent which specifically forms an isoindole derivative with the amino acids cysteine and lysine, and the Ca++ release channel complex. In this thesis, the planar lipid bilayer technique was used to study the Ca++ release channel protein from skeletal muscle sarcoplasmic reticulum at the single channel level. Utilizing this experimental technique, the direct interaction between OP A and the channel was investigated. In this study, it was shown that the interaction of o-phthalaldehyde with the channel increases the channel's open probability as well as its mean open time. Furthermore, the covalent nature of o-phthalaldehyde binding to the calcium release channel complex is shown and its inhibiting effects on chloride channels are demonstrated.

Heart failure represents an important cause of death in Western Countries. The pathophysiology of heart failure is mainly associated with abnormalities in intracellular calcium control. We previously showed that Egr-1 negatively regulates expression of sodium-calcium exchanger (NCX) in vivo and in vitro. Here we tested the hypothesis that Egr-1 regulates expression of calcium storage proteins in the sarco-endoplasmic reticulum (SER), calsequestrin (CSQ) and/or ER, calreticulin (CRT) directly or indirectly via Egr-1:NFAT (nuclear factor of activated T-cells) formation. Secondarily, we hypothesized that this will reduce calcium mobilization. We found that undifferentiated 1293F cells, overexpressing Egr-1, have reduced CSQ compared to control H9c2 cells. We demonstrated that Egr-1 negatively regulates CSQ but not CRT expression. The Egr-1 mediated decrease in CSQ is linked to decreased calcium availability. Repression is by a novel NAB-independent (NGFI-A binding protein) activity localized to a.a. region 1-307. We conclude that Egr-1-mediated reductions in calcium storage protein expression alter calcium availability for cardiac contraction/relaxation.

The aim of this study was to investigate the mode of action of pavetamine on rat cardiomyocytes. Pavetamine is the causative agent of gousiekte (“quick-disease”), a disease of ruminants characterized by acute heart failure following ingestion of certain rubiaceous plants. Two in vitro rat cardiomyocyte models were utilized in this study, namely the rat embryonic cardiac cell line, H9c2, and primary neonatal rat cardiomyocytes. Cytotoxicity of pavetamine was evaluated in H9c2 cells using the MTT and LDH release assays. The eventual cell death of H9c2 cells was due to necrosis, with LDH release into the culture medium after exposure to pavetamine for 72 h. Pavetamine did not induce apoptosis, as the typical features of apoptosis were not observed. Electron microscopy was employed to study ultrastructural alterations caused by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormalities after 48 h exposure of the cells to pavetamine. Abundant secondary lysosomes with electron dense material were present in treated cells. Numerous vacuoles were also present in treated cells, indicative of autophagy. During this exposure time, the nuclei appeared normal, with no chromatin condensation as would be expected for apoptosis. Abnormalities in the morphology of the nuclei were only evident after 72 h exposure. The nuclei became fragmented and plasma membrane blebbing occurred. The mitochondrial membrane potential was investigated with a fluorescent probe, which demonstrated that pavetamine caused significant hyperpolarization of the mitochondrial membrane, in contrast to the depolarization caused by apoptotic inducers. Pavetamine did not cause opening of the mitochondrial permeability transition pore, because cyclosporine A, which is an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly. Fluorescent probes were used to investigate subcellular changes induced by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormal features compared to the control cells, which is consistent with the electron microscopy studies. The lysosomes of treated cells were more abundant and enlarged. The activity of cytosolic hexosaminidase was nearly three times higher in the treated cells than in the control cells, which suggested increased lysosomal membrane permeability. The activity of acid phosphatase was also increased in comparison to the control cells. In addition, the organization of the cytoskeletal F-actin of treated cells was severely affected by pavetamine. Rat neonatal cardiomyocytes were labelled with antibodies to detect the three major contractile proteins (titin, actin and myosin) and cytoskeletal proteins (F-actin, desmin and β-tubulin). Cells treated with pavetamine had degraded myosin and titin, with altered morphology of sarcomeric actin. Vacuoles appeared in the β-tubulin network, but the appearance of desmin was normal. F-actin was severely disrupted in cardiomyocytes treated with pavetamine and was degraded or even absent in treated cells. Ultrastructurally, the sarcomeres of rat neonatal cardiomyocytes exposed to pavetamine were disorganized and disengaged from the Z-lines, which can also be observed in the hearts of ruminants that have died of gousiekte. It is concluded that the pathological alteration to the major contractile and cytoskeleton proteins caused by pavetamine could explain the cardiac dysfunction that characterizes gousiekte. F-actin is involved in protein synthesis and therefore can play a role in the inhibition of protein synthesis in the myocardium of ruminants suffering from gousiekte. Apart from inhibition of protein synthesis in the heart, there is also increased degradation of cardiac proteins in an animal with gousiekte. The mitochondrial damage will lead to an energy deficiency and possibly to generation of reactive oxygen species. The sarcoplasmic reticula are involved in protein synthesis and any damage to them will affect protein synthesis, folding and post-translational modifications. This will activate the unfolded protein response (UPR) and sarcoplasmic reticula-associated protein degradation (ERAD). If the oxidizing environment of the sarcoplasmic reticula is disturbed, it will activate the ubiquitin-proteasome pathway (UPP) to clear aggregated and misfolded proteins. Lastly, the mitochondria, sarcoplasmic reticula and F-actin are involved in calcium homeostasis. Any damage to these organelles will have a profound influence on calcium flux in the heart and will further contribute to the contractile dysfunction that characterizes gousiekte.
Thesis (PhD)--University of Pretoria, 2010.
Paraclinical Sciences
unrestricted

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Biomateriais, Reabilitação e Biomecânica)
In 2014, fish captures and aquaculture supplied ca. 167 million tonnes of fish, of which about 87% were used for human food and 10% was destined for fishmeal and fish oil. The remain 3% is waste that can be used as raw material for direct feeding in aquaculture. Fish sarcoplasmic proteins (FSP) are soluble in water. Large quantities of those proteins are discarded as part of the waste water resulting from fish surimi preparation. FSP constitutes around 25-30% of the total fish muscle protein. FSP comprise heme proteins and enzymes, which are associated with the energy-producing metabolism of the muscle. Herein, FSP from codfish (Gadus morhua) were isolated, resulting in FSP extracts. Both FSP extracts and membranes were physicochemically characterized and their cytocompatibility with human lung fibroblasts (MRC-5 cell line) was also evaluated. By SDS-PAGE, it was possible to define the composition of FSP extracts. From the differential scanning calorimetry (DSC) thermograms, it was possible to define that FSP denature at 44.12 ± 2.34˚C. By circular dichroism (CD) spectroscopy, it was possible to defined that the secondary structure of FSP is mainly composed by α-helix structures. For concentrations lower than 10 mg/mL, no cytotoxicity was observed over 72h of culture. Further on, the FSP extracts were processed into FSP membranes by the spin coating. FSP membranes shown an uniform surface when analyzed by scanning electron microscopy. By CD spectrum, it was verified an increase in the amount of α-helix structures. The FSP membranes were more stable than the FSP extracts, since the denaturation temperature was higher as determined by DSC. FSP membranes were hydrophobic, with a surface zeta potential of -33.4 mV, showing distinctive mechanical properties, with a stiffness of 16.57 ± 3.95 MPa and a yield strength of 23.85 ± 5.97 MPa. Human fibroblasts cultured in direct contact with FSP membranes demonstrate their cytocompatibility until 48h. Based on these results, FSP can be considered a potential biomaterial recovered from the waste water of fish, constituting a pool of enzymes that has potential interest for wound healing applications.
Em 2014, a captura de peixes e a aquacultura forneceram aproximadamente 167 milhões de toneladas de peixe, das quais 87% foram para consumo humano e 10% destinado a farinhas e óleos de peixes. Os restantes 3% são considerados desperdício, que podem ser utilizados como alimento direto na aquacultura. As proteínas sarcoplasmáticas de peixe são solúveis em água, pelo que grandes quantidades são descartadas juntamente com a água desperdiçada durante a preparação das delícias do mar. As proteínas sarcoplasmáticas constituem cerca de 25-30% das proteínas totais do músculo. Estas proteínas são compostas por proteínas heme e enzimas, que estão associadas com a produção de energia no músculo. Assim, as proteínas sarcoplasmáticas do bacalhau (Gadus morhua) foram isoladas, dando origem a extratos de proteínas sarcoplasmáticas. Tanto os extratos como as membranas foram caracterizados física e quimicamente e a sua citocompatibilidade foi avaliada com fibroblastos do pulmão humano (linha celular MRC-5). Pelo SDS-GE foi possível definir a composição dos extratos das proteínas sarcoplasmáticas. Pelo termograma da calorimetria diferencial de varrimento (DSC) foi possível definir que os extratos desnaturam a 44.12 ± 2.34˚C. Pela dicroísmo circular (CD) verificou-se que a estrutura secundária dos extratos era composta maioritariamente por estruturas α-hélices. Os extratos não apresentaram citotoxicidade para concentrações inferiores a 10 mg/mL durante 72h de cultura. Neste sentido, os extratos foram processados em membranas por spin coating. As membranas compostas por proteínas sarcoplasmáticas foram analisadas através de microscopia eletrónica de varrimento, verificando-se que a superfície era uniforme. Pela análise do espectro de CD, notou-se um aumento na quantidade de estruturas α-hélices. As membranas foram mais estáveis que os extratos, uma vez que temperatura de desnaturação foi superior, como determinado pelo DSC. As membranas foram hidrofóbicas, com uma carga de superfície de -33.4 mV. Por outro lado, apresentaram propriedades mecânicas distintivas, com uma rigidez de 16.57 ± 3.95 MPa e uma tensão de cedência de 23.85 ± 5.97 MPa. Os fibroblastos de pulmão humano cultivados em contacto direto com as membranas demostraram citocompatibilidade até 48h. Com base nestes resultados, as proteínas sarcoplasmáticas de peixe, constituídas maioritariamente por enzimas, podem ser consideradas um potencial biomaterial recuperado das águas residuais dos peixes, com um potencial de interesse para cicatrização de feridas.

Protein solubility of Pacific whiting muscle with isoelectric point at pH 5.5 was significantly affected by pH. The highest breaking force was measured from fish proteins treated at pH 11, while high deformation values were obtained at pH 2 and 11. Texture of gels made using the conventional method were quite inferior to gels made using fish proteins treated at pH 2 or 11, while color of conventional gels was significantly better than the other treatments. SDS-PAGE revealed that fish proteins were highly denatured during acid or alkali treatment. High cathepsin B-like activity was detected from acid-aided fish proteins. Strong cathepsin L-like activity was found in fish proteins treated at pH 10.5, which corresponded with the lower breaking force and deformation obtained from those samples. Disulfide bonds contributed to high texture value in fish proteins treated at pH 11. Physicochemical characteristics of sarcoplasmic proteins (SP) from rockfish and their interaction with Alaska pollock surimi (myofibrillar proteins) were investigated. Solubility of SP was significantly suppressed at acidic pH (2-4) plus high salt concentration (0.5 M NaCl). This was also supported by SDS-PAGE results (extensively degraded SP). DSC results revealed SP gave three endothermic transitions. The least amount ofproteins was lost when treated at pH 2 or 3 followed by precipitation at pH 5.5. SP did not enhance the gelation properties of myofibrillar proteins, but positively contributed to gelation with myofibrillar proteins when compared to sucrose. Myofibrillar proteins were primary components contributing to heat-induced gelation. Salt effect on acid- or alkali-treated surimi gel was investigated. Good gels were obtained without salt using acid- and alkali-treated fish proteins. Their texture properties decreased as NaCl content increased, unlike conventional surimi gels. Consequently, NaCl did not solubilize myofibrillar proteins once the fish proteins were treated by acid or alkali. Solubility was apparently not a key factor for the texture properties of acid- or alkali-treated surimi. Transglutaminase-mediated setting reaction was partially inactivated during acid or alkaline treatment. Acid-treated surimi gel gave the best color properties.
Graduation date: 2003

碩士
國立臺灣海洋大學
食品科學系
92
Abstract The objectives of this research was to study the function of physiological active peptides from neritic squid (Loligo edulis Hoyle) sarcoplasmic protein hydrolysate. The neritic squid sarcoplasmic protein was hydrolyzed with papain, bromelin, protease Type XIV, protease Type XXIII and autolysis. The antioxidant activities of protein hydrolysates were measured by four methods inculding the Trolox equivalent antioxidant capacity, scavenging effect of DPPH(α, α-diphanyl-β- picrylhydrazyl) radical, reducing power and chelating Cu2+ effect. As results, the hydrolysate, treated with 0.03% papain 12h, showed the best antioxidativity. The papain hydrolysates were separated by using Amicon, Sephadex G-25 and DEAE Sephadex A-50 chromatography. All the steps were monitored antioxidativity. Using the four antioxidant methods, the papain hydrolysate (MW<5kDa) was found having the best antioxidativity. It was further separated by Sephadex G-25 gel filtration chromatography, and five major fractions: A (MW>1355Da), B, C, D (MW 1355~204Da) and E (MW<204Da) were obtained. The fractions (C and E) showed the best TEAC antioxidant capacity, the fraction D showed the best scavenging effect of DPPH radical and reducing power, the fractions (B and E) showed the best chelating Cu2+ effect. After DEAE Sephadex A-50 and Sephadex G-25 chromatography, the MW of fraction D was about 800Da. According to amino acid analysis, the major amino acid of fraction D was Gly (63.8%); then His (14.6%) and Tyr (14%).

The sarcoplasmic reticulum (SR) is one of the major regulators of the cytosolic Ca2+ concentration in cardiac ventricular muscle cells. In the myocardium, relaxation results from a decrease in cytoplasmic free Ca2+ levels mediated through an efflux of Ca2+ from the cell via the sarcolemmal Na+/Ca2+- exchanger and by the sequestration of 0a2+ by the network SR membranes through the actions of a calcium pump (a Mg2+dependent, Ca2+/K+activated adenosine triphosphatase; Ca2+/K+-ATPase). During an action potential, the Ca2+ stored in the SR is released to the cytoplasm via a Ca2+release channel present in the junctional SR and Ca2+ also enters the cell through voltage-controlled Ca2+ channels in the sarcolemmal membrane. These two processes result in an increase in cytoplasmic Ca2+ concentration which leads to contraction of the myocardium. SR membrane function is regulated in part by the phosphorylation of proteins present in this membrane. Subsequent to 3-adrenergic stimulation of the heart by catecholamines, levels of cAMP are increased leading to the activation of cAMP-dependent protein kinase (PK A). In isolated cardiac SR membrane vesicles and perfused hearts, phosphorylation of an indigenous SR protein, phospholamban, is mediated by PK A. Phosphorylated phospholamban acts as a modulator of the Ca2+/K+-ATPase to stimulate active Ca2+ uptake by increasing the affinity of this enzyme for Ca2+. This stimulation of Ca2+-uptake is the main mechanism by which catecholamines accelerate relaxation in the heart. When phospholamban is in the dephosphorylated state, a cytoplasmic portion of the molecule interacts near the phosphorylation site of the Ca2+-pump to inhibit Ca2+-transport. [More abstract follows]

碩士
國立臺灣海洋大學
食品科學系
92
This research was to study the antioxidant ability of hydrolysates extracted from hard clam (Meretrix lusoria) sarcoplasmic protein. First, the extracted sarcoplasmic protein was hydrolyzed by autolysis and adding of papain, bromelain, protease Type XXIII (Aspergillus oryzae) and Type XIV (Streptomyces griseus) at 37oC with different time. The scavenging effect of DPPH radical, reducing power, chelating Fe2+ effect and Trolox equivalent antioxidant capacity (TEAC) was employed to monitored the antioxidativity of hydrolysates. As result, the hydrolysate that treated with 0.02% bromelain 12hrs had the best antioxidant ability (scavenging DPPH & reducing power). The hydrolysates that treated with protease Type XXIII 24hrs had the best ability of the scavenging DPPH. The hydrolysate that treated with protease Type XXIII 6 hrs had best chelating Fe2+ effect. The hydrolysate that treated with protease Type XIV 24hrs had best TEAC. Using Amicon ultrafiltration, the fraction (MW<5KDa) had better antioxidativity. Then after Sephadex G-25 chromatography, the fraction D (800Da) had the best antioxidativity. After DEAE Sephadex A-50 chromatography of fraction D (G-25), the adsorbed fraction (Fraction II) having better reducing power & Trolox equivalent antioxidant capacity, and unadsorbed fraction (Fraction I) having better chelating Fe2+ effect were found. According to the amino acid analysis, the Fraction II contained P-Ser, Arg, Lys etc., the Fraction I contained Gly, Ser, Tyr, His etc. After protease (pepsin, trypsin, chymotrypsin, carboxypeptidase A) treatment, the antioxidativity of Fraction I, II were decreased.

The overall objective of this thesis was to examine mechanisms involved in the acute regulation of sarcoplasmic reticulum (SR) Ca2+-handling properties by second messenger signaling pathways in skeletal and cardiac muscle. The aim of the first study (Chapter Two) was to characterize changes in the kinetic properties of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA) proteins in cardiac and skeletal muscles in response to b-adrenergic, Ca2+-dependent calmodulin kinase II (CaMKII) and protein kinase C (PKC) signaling. The aim of the second study (Chapter Three) was to determine if insulin signaling could acutely regulate SERCA kinetic properties in cardiac and skeletal muscle. The aim of the final study (Chapter Four) was to determine if alterations in plasma glucose, epinephrine and insulin concentrations during exercise are able to influence SR Ca2+-handling properties in contracting human skeletal muscle. Data collected in Chapter Two and Chapter Three were obtained using tissue prepared from a group of 28 male Sprague-Dawley rats (9 weeks of age; mass = 280 ± 4 g: X ± S.E). Crude muscle homogenates (11:1 dilution) were prepared from selected hind limb muscles (soleus, SOL; extensor digitorum longus, EDL; the red portion of gastrocnemius, RG; and the white portion of gastrocnemius, WG) and the left ventricle (LV). Enriched SR membrane fractions, prepared from WG and LV, were also analyzed. A spectrophotometric assay was used to measure kinetic properties of SERCA, namely, maximal SERCA activity (Vmax), and Ca2+-sensitivity was characterized by both the Ca50, which is defined as the free Ca2+-concentration needed to elicit 50% Vmax, and the Hill coefficient (nH), which is defined as the relationship between SERCA activity and Ca2+f for 10 to 90% Vmax. The observations made in Chapter Two indicated that b-adrenergic signaling, activated by epinephrine, increased (P<0.05) Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50), without altering Vmax in LV and SOL but had no effect (P<0.05) on EDL, RG, or WG. Further analysis using a combination of cAMP, the PKA activator forskolin, and/or the PKA inhibitor KT5270 indicated that the reduced Ca50 in LV was activated by cAMP- and PKA-signaling mechanisms. However, although the reduced Ca50 in SOL was cAMP-dependent, it was not influenced by a PKA-dependent mechanism. In contrast to the effects of b-adrenergic signaling, CaMKII activation increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 and increased nh, without altering SERCA Vmax in LV but was without effect in any of the skeletal muscles examined. The PKC activator PMA significantly reduced SERCA Ca2+-sensitivity, by inducing a right-shift in Ca50 and decreased nH in the LV and all skeletal muscles examined. PKC activation also reduced Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG and WG), but did not alter Vmax in LV or SOL. The results of Chapter Three indicated that insulin signaling increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50) and an increased nH, without altering SERCA Vmax in crude muscle homogenates prepared from LV, SOL, EDL, RG, and WG. An increase in SERCA Ca2+-sensitivity was also observed in enriched SERCA1a and SERCA2a vesicles when an activated form of the insulin receptor (A-INS-R) was included during biochemical analyses. Co-immunoprecipitation experiments were conducted and indicated that IRS-1 and IRS-2 proteins bind SERCA1a and SERCA2a in an insulin-dependent manner. However, the binding of IRS proteins with SERCA does not appear to alter the structural integrity of the SERCA Ca2+-binding site since no changes in NCD-4 fluorescence were observed in response to insulin or A-INS-R. Moreover, the increase in SERCA Ca2+-sensitivity due to insulin signaling was not associated with changes in the phosphorylation status of phospholamban (PLN) since Ser16 or Thr17 phosphorylation was not altered by insulin or A-INS-R in LV tissue. The data described in Chapter Four was collected from 15 untrained human participants (peak O2 consumption, VO2peak= 3.45 ± 0.17 L/min) who completed a standardized cycle test (~60% VO2peak) on two occasions during which they were provided either an artificially sweetened placebo (PLAC) or a 6% glucose (GLUC) beverage (~1.00 g CHO per kg body mass). Muscle biopsies were collected from the vastus lateralis at rest, after 30 min and 90 min of exercise and at fatigue in both conditions to allow assessment of metabolic and SR data. Glucose supplementation increased exercise ride time by ~19% (137 ± 7 min) compared to PLAC (115 ± 6 min). This performance increase was associated with elevated plasma glucose and insulin concentrations and reduced catecholamine concentrations during GLUC compared to PLAC. Prolonged exercise reduced (p<0.05) SR Ca2+-uptake, Vmax, Phase 1 and Phase 2 Ca2+-release rates during both PLAC and GLUC. However, no differences in SR Ca2+-handling properties were observed between conditions when direct comparisons were made at matched time points between PLAC and GLUC. In summary, the results of the first study (Chapter Two) indicate that b-adrenergic and CaMKII signaling increases SERCA Ca2+-sensitivity in the LV and SOL; while PKC signaling reduces SERCA Ca2+-sensitivity in all tissues. PKC activation also reduces Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG, and WG) but has no effect on Vmax in the LV and SOL. The results of the second study (Chapter Three) indicate that insulin signaling acutely increases the Ca2+-sensitivity of SERCA1a and SERCA2a in all tissues examined, without altering the Vmax. Based on our observations, it appears that the increase in SERCA Ca2+-sensitivity may be regulated, in part, through the interaction of IRS proteins with SERCA1a and SERCA2a. The results of the final study (Chapter Four) indicate that alterations in plasma glucose, epinephrine and insulin concentrations associated with glucose supplementation during exercise, do not alter the time course or magnitude of reductions in SERCA or Ca2+-release channel (CRC) function in working human skeletal muscle. Although glucose supplementation did increase exercise ride time to fatigue in this study, our data does not reveal an association with SR Ca2+-cycling measured in vitro. It is possible that the strength of exercise signal overrides the hormonal influences observed in resting muscles. Additionally, these data do not rule out the possibility that glucose supplementation may influence E-C coupling processes or SR Ca2+-cycling properties in vivo.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.