Actomyosin ATPase Activity Research Articles

The green tea polyphenol (−)-epigallocatechin-3-gallate inhibits magnesium binding to the C-domain of cardiac troponin C

Cardiac muscle contraction is activated via the single Ca(2+)-binding site (site II) in the N-domain of troponin C (cTnC). The two Ca(2+)/Mg(2+) binding sites in the C-domain of cTnC (sites III and IV) have been considered to play a purely structural role in anchoring cTnC to the thin filament. However, several recent discoveries suggest a possible role of this domain in contractile regulation. The green tea polyphenol (-)-epigallocatechin 3-gallate (EGCg), which binds specifically to the C-domain of cTnC, reduces cardiac myofilament Ca(2+) sensitivity along with maximum force and acto-myosin ATPase activity. We have determined the effect of EGCg on Ca(2+) and Mg(2+) binding to the C-domain of cTnC. In the absence of Mg(2+) there was no significant effect of EGCg on the Ca(2+)-cTnC affinity. Surprisingly, in the presence of Mg(2+) EGCg caused an increase in Ca(2+) affinity for sites III and IV of cTnC. However, in the absence of Ca(2+) the addition of EGCg caused a significant reduction in Mg(2+)-cTnC affinity. This reduction is presumably responsible for the increase in Ca(2+)-cTnC affinity produced by EGCg in the presence of Mg(2+). We propose that the inhibitory effect of EGCg on myofilament Ca(2+) activation may be related to an enhanced Ca(2+)-Mg(2+)exchange at sites III and IV of cTnC, which might reduce the myosin crossbridge dependent component of thin filament activation.

S-Nitrosylation Decreases Ca2+ Sensitivity and Actomyosin ATPase Activity of Contractile Proteins in Cardiac Myofibrils

Increases in muscle nitric-oxide (NO) production can be buffered by reaction with intracellular glutathione, forming S-nitrosoglutathione (GSNO). GSNO has been shown to S-nitrosylate Cys thiols of cardiac contractile proteins in vivo and in vitro, but effects on maximal force, thin-filament Ca2+ sensitivity and actomyosin ATPase activity are unknown. Here, we analyzed the targets of S-nitrosylation in mouse cardiac contractile proteins, and examined the effects of these modifications on function in myocytes and skinned cardiac myofibrils. S-Nitrosylation and denitrosylation were detected using resin-assisted capture (SNO-RAC) and targets for S-nitrosylation were identified by quantitative LC-MS/MS. Isolated cardiomyocytes treated with S-nitrosocysteine (CysNO, 500μM, 10min) showed an increase in total protein-SNO, followed by progressive denitrosylation (30-60min with CysNO). At 10min, CysNO dose-dependently increased S-nitrosylation of specific Cys thiols in myosin heavy chain, actin, TnC, TnI, myosin-binding protein C and other muscle proteins. Myofibril thin-filament Ca2+ sensitivity decreased (P 0.05). Loss of Ca2+ sensitivity was partially reversed by the denitrosation agent, ascorbate. Relaxation kinetics of skinned fibers, as measured by flash photolysis, were also significantly reduced by100μM GSNO (k1,15.33 to 11.68/s; k2 2.33 to 0.87/s; fit to double exponential). Maximal myofibrillar ATPase activity (pCa 5.0) was also dose-dependently inhibited (8, 15, 30%) by 50, 100 and 500μM GSNO, an effect that was reversed by ascorbate. The findings suggest that S-nitrosylation of regulatory Cys thiol(s) can reversibly modulate cardiac muscle contraction and may be able to protect the heart by reducing myosin ATPase demand without affecting the maximal force of contraction.

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca(2+) sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

Peptide mapping of polymorphic myosin heavy chain isoforms in different muscle types of some freshwater teleosts

A modified SDS-PAGE system has been employed to resolve polymorphic myosin heavy chain (MyHC) isoforms in different muscle types of three freshwater teleosts displaying different modes of respiration, adaptive features and life styles. Investigated species include accessory air-breather Channa punctata along with exclusive aquatic breather major carps Labeo rohita and Catla catla. All the selected species show specificity and expressivity of at least three MyHC isoforms, one each in red, head and pectoral muscles. Chymotryptic peptide maps unambiguously support substructural individuality of each MyHC isoforms with the type-specific dispersal of chymotryptic cleavage sites. Specific Ca(2+)- and Mg(2+)-ATPase activities of natural actomyosin (NAM) of lateral line red muscle of C. punctata were low and less sensitive to pH, but sensitive to KCl concentrations between 0.05 and 0.15M. In comparison, the specific enzymatic activities of NAM of red muscle from the carps (L. rohita and C. catla) were substantially high with prominent peaks at pH 7.5 and near insensitivity to 0.05-0.15M KCl, while C. punctata had shown a different response at these molarities. Thus, the data favor a correlation between breathing modes and life style and the differences in pH or ionic strength sensitivities of ATPases. Unique profiles of peptide maps and the dispersal patterns of hydrophobic residues (cleavage sites of chymotrypsin) in MyHC of different muscle types further reflect individuality of their evolutionary histories.

High prevalence of Arginine to Glutamine Substitution at 98, 141 and 162 positions in Troponin I (TNNI3) associated with hypertrophic cardiomyopathy among Indians

BackgroundTroponin I (TNNI3) is the inhibitory subunit of the thin filament regulatory complex Troponin, which confers calcium-sensitivity to striated muscle actomyosin ATPase activity. Mutations (2-7%) in this gene had been reported in hypertrophic cardiomyopathy patients (HCM). However, the frequencies of mutations and associated clinical presentation have not been established in cardiomyopathy patients of Indian origin, hence we have undertaken this study.MethodsWe have sequenced all the exons, including the exon-intron boundaries of TNNI3 gene in 101 hypertrophic cardiomyopathy patients (HCM), along with 160 healthy controls, inhabited in the same geographical region of southern India.ResultsOur study revealed a total of 16 mutations. Interestingly, we have observed Arginine to Glutamine (R to Q) mutation at 3 positions 98, 141 and 162, exclusively in HCM patients with family history of sudden cardiac death. The novel R98Q was observed in a severe hypertrophic obstructive cardiomyopathy patient (HOCM). The R141Q mutation was observed in two familial cases of severe asymmetric septal hypertrophy (ASH++). The R162Q mutation was observed in a ASH++ patient with mean septal thickness of 29 mm, and have also consists of allelic heterogeneity by means of having one more synonymous (E179E) mutation at g.4797: G → A: in the same exon 7, which replaces a very frequent codon (GAG: 85%) with a rare codon (GAA: 14%). Screening for R162Q mutation in all the available family members revealed its presence in 9 individuals, including 7 with allelic heterogeneity (R162Q and E179E) of which 4 were severely affected. We also found 2 novel SNPs, (g.2653; G → A and g.4003 C → T) exclusively in HCM, and in silico analysis of these SNPs have predicted to cause defect in recognition/binding sites for proteins responsible for proper splicing.ConclusionOur study has provided valuable information regarding the prevalence of TNNI3 mutations in Indian HCM patients and its risk assessment, these will help in genetic counseling and to adopt appropriate treatment strategies.

Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.

Impact of Carbon Nanotubes on ATPase Activity and Superprecipitation Reaction of Natural Actomyosin

Impact of Carbon Nanotubes on ATPase Activity and Superprecipitation Reaction of Natural Actomyosin

Physiological Effects of FHC-Causing K104E Mutation in the Myosin Regulatory Light Chain

We have studied the physiological effects of the Lysine 104 to Glutamic Acid (K104E) mutation in the ventricular myosin regulatory light chain (RLC), shown to cause familial hypertrophic cardiomyopathy (FHC). In vitro and in vivo experiments were performed using transgenic (Tg) mouse cardiac muscle preparations carrying the K104E-RLC mutation. We observed a slight but significant decrease in maximal force (ΔFmax≅8kN/m2) and an increase in the Ca2+-sensitivity (ΔpCa50≅0.1) of isometric contraction in glycerinated skinned muscle fibers from Tg-K104E compared to Tg-WT mice. No mutant related changes in rigor binding of transgenic K104E mouse myosin to pyrene-actin were observed. Likewise, no changes in actomyosin or myofibrillar ATPase activities were monitored. Histological examination of Masson's trichrome stained Tg-hearts showed signs of fibrosis in 8 mo-old Tg-K104E mice compared to age matched Tg-WT. These observed changes in myocyte organization in Tg-K104E mice could be due to ∼4-fold decrease in the myosin heavy chain-RLC interaction determined by the binding of bacterially expressed K104E mutant to RLC-depleted porcine myosin. Echocardiography examination of senescent Tg-K104E mice confirmed a hypetrophic phenotype and showed a significantly enlarged interventricular septum and LVPWd (left ventricular posterior wall in diasole), ∼1.6-fold increase in LV mass and significantly decreased LVIDs (LV inner diameter in systole) compared to Tg-WT mice. However, EF (ejection fraction) was higher in Tg-K104E mice (73±8%) compared to 61±7% observed in Tg-WT mice. In addition, Doppler E velocity was also 1.3-fold higher in Tg-K104E mice compared to Tg-WT. These results confirm a mutation induced hypertrophic phenotype in Tg-K104E mice, similar to the patients carrying this FHC-mutation. No drastic changes at the level of actomyosin interaction or in cardiac function assessed in Tg-K104E mice suggest that the mutation might be associated with good prognosis. Supported by NIH-HL071778 (DSC).

Ca2+ Sensitization of Myocardial Force and Actomyosin ATPase by (D-Ala2, Met5) Enkephalinamide

The presence of proenkephalin mRNA and proenkephalin peptides in cardiac muscle cells suggests the local production of enkephalins in the myocardium. Yet, the effects of these peptides on the function of the contractile proteins are unknown. The effects of (D-Ala2, Met5) enkephalinamide (DALA) on the activity of the actin stimulated Ca, Mg-myosin ATPase in myofibrils and on the contractility and the activity of the related actomyosin ATPase of chemically skinned muscle fibres from pig myocardium were studied. In this article, it is shown that the myofibrillar actomyosin ATPase as well as the contractility and the actomyosin ATPase in skinned fibres are sensitized to Ca2+ ions by DALA. 10(-11) -10(-6) mol/l DALA decrease the effective concentration of Ca2+ stimulating the myofibrillar ATPase activity by 50% (EC50) from 4.0.10(-5) to 1.5.10(-5) mol/l (p < 0.05). The magnesium dependent myosin ATPase activity at low Ca2+ concentration (10(-9) mol/l) is increased. The EC50 values of Ca2+ for both force development and the related actomyosin ATPase activity of skinned fibres are decreased by DALA (10(-11) -10(-5) mol/l) from 2.5.10(-6) to 2.0.10(-6) mol/l (contractions; p < 0.01) and from 2.0.10(-6) to 1.3.10(-6) mol/l (ATPase activity; p < 0.01). The tension cost (ATPase/tension) of the fibres is unchanged by DALA. In conclusion, the results demonstrate a Ca2+ sensitization of the contractile proteins by low concentrations of DALA, indicating a direct regulatory involvement of enkephalins in the regulation of myocardial contractility. These results correspond with the positive inotropic effects of enkephalins in isolated heart muscle cells.

Abstract P088: The Rate of Inorganic Phosphate Release in Contracting Rat Trabeculae Is Independent of Calcium After Stretch

We aim to reveal the molecular mechanism underlying the force response to stretch in the heart by examining the dependence of the rate of inorganic phosphate (P i ) release in response to stretch on the degree of calcium activation. Permeabilized trabeculae of rat heart are activated by photolytic release of ATP, at 20°C, in the presence of saturating (32µM), or half-saturating (1µM), calcium concentrations, at an initial sarcomere length of 1.9 or 2.1 µm. For trabeculae with an initial sarcomere length of 1.9 µm, the trabeculae are stretched to 2.1 µm during the isometric plateau, held at a constant length and returned to their original length. The rate of P i release is determined by the fluorescence change associated with phosphate binding to a fluorescently-labelled phosphate binding protein diffused into the preparation. At an initial sarcomere length of 1.9µm, the isometric force at full activation was 51 kN.m −2 compared to 26kN.m −2 at half-maximal activation. During the isometric phase, P i release is at a steady rate of 8.2 and 4.8 s −1 (assuming a myosin head concentration of 120µM) at 32 and 1µM calcium, respectively. During stretch, the rate of P i release decreases markedly at both activation levels, to1.0 and 1.4s −1 respectively. Activation from an initial sarcomere length of 2.1µm produced an isometric force of 65.5 kN.m −2 with a steady P i release rate of 8.1 s −1 at maximal activation, and an isometric force of 49.2 kN.m −2 with a steady P i release rate of 4.8 s −1 at half-maximal activation. The rates of P i release during stretch and after the end of the stretch at low activation are equal to that at high activation indicating stretch-induced activation which occurs for stretches applied prior to activation, and during activation. The results demonstrate that stretch has a dramatic effect causing an immediate reduction in the rate of P i release. Stretch contributes to activation of the trabecula's actomyosin ATPase in a calcium-independent manner, although the effect is more marked at low activation levels. Thus a direct effect of stretch on thin filament activation contributes to the Frank-Starling law.

Changes in isoform composition, structure, and functional properties of titin from mongolian gerbil (Meriones unguiculatus) cardiac muscle after space flight

Changes in isoform composition, secondary structure, and titin phosphorylation in Mongolian gerbil (Meriones unguiculatus) cardiac muscle were studied after 12-day-long space flight onboard the Russian spacecraft Foton-M3. The effect of titin on the actin-activated myosin ATPase activity at pCa 7.5 and 4.6 was also studied. Almost twofold increase in titin long N2BA isoform content relative to that of short N2B isoform was found on electrophoregrams of cardiac muscle left ventricle of the flight group gerbils. Differences in secondary structure of titin isolated from cardiac muscle of control and flight groups of gerbils were found. An increase in phosphorylation (1.30-1.35-fold) of titin of cardiac muscle of the flight group gerbils was found. A decrease in activating effect of titin of cardiac muscle of the flight group gerbils on actomyosin ATPase activity in vitro was also found. The observed changes are discussed in the context of M. unguiculatus cardiac muscle adaptation to conditions of weightlessness.

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.

Recent perspectives into biochemistry of decavanadate

The number of papers about decavanadate has doubled in the past decade. In the present review, new insights into decavanadate biochemistry, cell biology, and antidiabetic and antitumor activities are described. Decameric vanadate species (V(10)) clearly differs from monomeric vanadate (V(1)), and affects differently calcium pumps, and structure and function of myosin and actin. Only decavanadate inhibits calcium accumulation by calcium pump ATPase, and strongly inhibits actomyosin ATPase activity (IC(50) = 1.4 μmol/L, V(10)), whereas no such effects are detected with V(1) up to 150 μmol/L; prevents actin polymerization (IC(50) of 68 μmol/L, whereas no effects detected with up to 2 mmol/L V(1)); and interacts with actin in a way that induces cysteine oxidation and vanadate reduction to vanadyl. Moreover, in vivo decavanadate toxicity studies have revealed that acute exposure to polyoxovanadate induces different changes in antioxidant enzymes and oxidative stress parameters, in comparison with vanadate. In vitro studies have clearly demonstrated that mitochondrial oxygen consumption is strongly affected by decavanadate (IC(50), 0.1 μmol/L); perhaps the most relevant biological effect. Finally, decavanadate (100 μmol/L) increases rat adipocyte glucose accumulation more potently than several vanadium complexes. Preliminary studies suggest that decavanadate does not have similar effects in human adipocytes. Although decavanadate can be a useful biochemical tool, further studies must be carried out before it can be confirmed that decavanadate and its complexes can be used as anticancer or antidiabetic agents.

Increased Ca2+ sensitivity of myofibrillar tension in ischaemic vs dilated cardiomyopathy

1. There is evidence that different aetiologies of heart failure, especially ischaemic vs dilated cardiomyopathy (ICM and DCM, respectively), may influence the prognosis of patients with this disease. Patients with ICM have a worse prognosis than those with DCM; the mechanisms underlying this difference have not yet been clarified. The aim of the present study was to investigate whether there are changes in myofibrillar function depending on the aetiology of human heart failure. 2. Ca(2+) -dependent tension (DT) and actomyosin ATPase activity (MYO) in Triton X-skinned fibre preparations of the left ventricular myocardium from patients with heart failure due to ICM (n=5) and DCM (n=5) were measured. Tension-dependent ATP consumption was calculated by the ratio of DT and MYO ('tension cost'). Non-failing myocardium (NF) from donor hearts, which could not be transplanted because of technical reasons, was evaluated as a control. 3. Although DT was reduced, the myofibrillar Ca(2+) sensitivity of DT and MYO, as well as tension cost, were increased in preparations from ICM and DCM myocardium compared with NF. The Ca(2+) sensitivity of DT and MYO was significantly increased in ICM compared with DCM preparations, resulting in more economic cross-bridge cycling in ICM than in DCM. 4. In conclusion, ICM is associated with an increased Ca(2+) sensitivity of myofibrillar tension and ATPase activity accompanied by decreased tension cost compared with DCM. Thus, the worse prognosis associated with ICM does not seem to be due to differences in myofibrillar function.

HGAL Directly Interacts with Both Myosin and Actin and Increases the Binding of Myosin to Actin

AbstractAbstract 3097HGAL is a recently identified germinal center (GC)–specific gene whose expression by tumor cells correlates with a favorable prognosis in patients with diffuse large B-cell and classical Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and decreasing lymphoma cell migration and dissemination. The latter effect may contribute to the less aggressive clinical behavior of HGAL-expressing lymphomas. Actin filaments, usually in association with myosin, are responsible for many types of cell movements. The interaction between myosin and actin is responsible for muscle contraction, migration of nonmuscle cells and also plays a role in cell division. The movement of cells appears to be driven directly by actin polymerization and by actin-myosin interactions. In our previous work (Lu et al, Blood 2007), we demonstrated by co-immunoprecipitation and co-localization studies that HGAL interacts with myosin II and actin. However, the mechanistic consequences of this interaction are unknown. Because the interaction of actin with myosin is the key factor in cell motility and HGAL negatively regulates lymphocyte migration, we hypothesized that HGAL may decelerate cell migration by affecting the binding of myosin to actin. To confirm the direct and not interdependent interaction between myosin-HGAL and actin-HGAL, we performed in vitro co-sedimentation experiments with recombinant HGAL protein and purified actin and myosin, respectively. These studies demonstrated direct and independent binding of HGAL to both myosin and actin. We next demonstrated that the N terminal portion of the HGAL protein (aa 1–118) can bind the head region of myosin II (S1) that contains the actin and ATP binding sites, and to the rod part of the myosin molecule that is responsible for filament formation. While no effect of the HGAL on the actomyosin ATPase activity was observed, it reduced the rate of actin polymerization from G-actin to F-actin. Further binding studies using pyrene-labeled F-actin showed that HGAL increases the binding of myosin to F-actin. Taken together, our results indicate that HGAL is a cellular regulator of actin assembly that can also regulate the actin-myosin interaction. This in turn may explain the role of HGAL as a favorable prognosis marker for patients with diffuse large B-cell and classical Hodgkin lymphomas. Disclosures:No relevant conflicts of interest to declare.

C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration

Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca2+-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

Thin filaments of bivalve smooth muscle may contain a calponin-like protein

A novel 40-kDa calponin-like protein (CaP) was detected in thin filaments from catch muscles of the mussel Crenomytilus grayanus. The content of CaP in thin filaments depends on isolation conditions and varies from complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the CaP content in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22°C and optimal values of pH and ionic strength of the extraction solution, all CaP co-sediments with thin filaments. At 2°C it does not interact with thin filaments and remains in the supernatant. Like vertebrate smooth muscle calponin (33 kDa), the mussel CaP is thermostable, inhibits the Mg2+-ATPase activity of actomyosin, and can be phosphorylated, which is performed by endogenous (co-isolated) kinases in a Ca2+-independent manner. Thus, the C. grayanus CaP is a new member of the family of calponins, the function of which in muscle and nonmuscle cells is still obscure. We suggest that CaP is involved in Ca2+-independent regulation of smooth muscle contraction.

CHANGES IN SALT EXTRACTABLE PROTEIN AND CA2+‐ATPASE ACTIVITY OF MINCE FROM SILVER CARP (HYPOPHTHALMICHTHYS MOLLITRIX) DURING FROZEN STORAGE: A KINETIC STUDY

ABSTRACT Changes in salt extractable protein (SEP) and Ca2 + ‐ATPase activity of actomyosin from washed and unwashed silver carp stored at −10 and −20C were investigated. The resultant decrease of SEP and Ca2 + ‐ATPase activity mainly occurred in the first 5 and 7 weeks, and the changes obeyed first‐order model. The kinetic models of SEP and Ca2 + ‐ATPase changes were established during frozen storage. The rate constant k of decline in SEP and Ca2+‐ATPase activity of samples stored at −10C were higher than that of samples stored at −20C; the D value of samples stored at −10C were lower than that of samples stored at −20C. Compared with washed samples, unwashed samples had a higher rate constant k and activation energies E, and a lower D value. Activation energies E of SEP were higher than E of Ca2+‐ATPase activity in all samples.PRACTICAL APPLICATIONSSilver carp is one of the main freshwater fish species in China. Washing is an important step for fish processing and temperature is an important factor on fish protein storage. The changes of the kinetic models of SEP and Ca2+‐ATPase during frozen storage can explain the effect of temperature and time on silver carp mince quality. These kinetic models can be used to predict the silver carp protein functional changes during frozen storage, and help to provide better methods to effectively utilize silver carp protein resource.

BIOCHEMICAL AND PHYSICOCHEMICAL PROPERTIES OF ACTOMYOSIN AND MYOFIBRILS FROM FROZEN STORED FLOUNDER (PARALICHTHYS PATAGONICUS) FILLETS

ABSTRACT The biochemical and physicochemical properties of myofibrils and actomyosin (AM) from frozen stored fillets of flounder were investigated. Solubility of proteins increased after freezing and decreased during frozen storage. No significant (P < 0.05) changes were detected in both Mg2+ (Ca2+) and Mg2+ Ethylene glycol tetraacetic acid (EGTA) Adenosinetriphosphatase (ATPase) activities of myofibrils due to freezing and frozen storage. Freezing produced a significant (P < 0.05) increase in Mg2+ Ca2+ and Mg2+(EGTA) and Mg2+ATPase activities of AM. The (Ca2+) ATPase activity remained unchanged. All the ATPases activities of AM decreased during frozen storage. The changes in the enzymatic activities of AM during storage were accompanied by a decrease in the reduced viscosity of the protein. No signals of proteolysis were detected in the profiles of SDS-Polyacrilamyde Gel Electrophoresis 10% gels of both myofibrils and AM. A significant decrease (P < 0.05) in the relative percentage of myosin and myosin/actin ratio was observed at the end of storage. These results suggest that denaturation–aggregation of myosin occurs at the end of storage in frozen stored fillets. PRACTICAL APPLICATIONS Flounder (Paralichthys patagonicus) is an important species of fish in South American waters. In recent years, catches in Argentina greatly increased. The fishing is commonly called “fine fishing” because of the high quality of the meat and its high price in the market. It is processed and exported as frozen interfoliated fillets and consumed as fresh fish in the internal market. Only a few reports on changes in biochemical and physicochemical properties of both AM and myofibrils from iced flounder are available. However, there is a lack of information on the behavior of myofibrillar proteins from flounder (P. patagonicus) during freezing and frozen storage. This information could be very important to establish technological actions to obtain optimal quality in frozen flounder.

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

In this study we explore the mechanisms by which a double mutation (E59D/D75Y) in cardiac troponin C (CTnC) associated with dilated cardiomyopathy reduces the Ca(2+)-activated maximal tension of cardiac muscle. Studying the single mutants (i.e. E59D or D75Y) indicates that D75Y, but not E59D, causes a reduction in the calcium affinity of CTnC in troponin complex, regulated thin filaments (RTF), and the Ca(2+) sensitivity of contraction and ATPase in cardiac muscle preparations. However, both D75Y and E59D are required to reduce the actomyosin ATPase activity and maximal force in muscle fibers, indicating that E59D enhances the effects of D75Y. Part of the reduction in force/ATPase may be due to a defect in the interactions between CTnC and cardiac troponin T, which are known to be necessary for ATPase activation. An additional mechanism for the reduction in force/ATPase comes from measurements of the binding stoichiometry of myosin subfragment-1 (S-1) to the RTF. Using wild type RTFs, 4.8 mol S-1 was bound per mol filament (seven actins), whereas with E59D/D75Y RTFs, the number of binding sites was reduced by approximately 23% to 3.7. Altogether, these results suggest that the reduction in force and ATPase activation is possibly due to a thin filament conformation that promotes fewer accessible S-1-binding sites. In the absence of any family segregation data, the functional results presented here support the concept that this is likely a dilated cardiomyopathy-causing mutation.