Actomyosin MgATPase Activity Research Articles

Age, Sex and Overall Health, Measured As Frailty, Modify Myofilament Proteins in Hearts From Naturally Aging Mice

We investigated effects of age, sex and frailty on contractions, calcium transients and myofilament proteins to determine if maladaptive changes associated with aging were sex-specific and modified by frailty. Ventricular myocytes and myofilaments were isolated from middle-aged (~12 mos) and older (~24 mos) mice. Frailty was assessed with a non-invasive frailty index. Calcium transients declined and slowed with age in both sexes, but contractions were largely unaffected. Actomyosin Mg-ATPase activity increased with age in females but not males; this could maintain contractions with smaller calcium transients in females. Phosphorylation of myosin-binding protein C (MyBP-C), desmin, tropomyosin and myosin light chain-1 (MLC-1) increased with age in males, but only MyBP-C and troponin-T increased in females. Enhanced phosphorylation of MyBP-C and MLC-1 could preserve contractions in aging. Interestingly, the age-related decline in Hill coefficients (r = −0.816; p = 0.002) and increase in phosphorylation of desmin (r = 0.735; p = 0.010), tropomyosin (r = 0.779; p = 0.005) and MLC-1 (r = 0.817; p = 0.022) were graded by the level of frailty in males but not females. In these ways, cardiac remodeling at cellular and subcellular levels is graded by overall health in aging males. Such changes may contribute to heart diseases in frail older males, whereas females may be resistant to these effects of frailty.

Slit2 Protects Hearts Against Ischemia-Reperfusion Injury by Inhibiting Inflammatory Responses and Maintaining Myofilament Contractile Properties.

BackgroundThe secreted glycoprotein Slit2, previously known as an axon guidance cue, has recently been found to protect tissues in pathological conditions; however, it is unknown whether Slit2 functions in cardiac ischemia–reperfusion (IR) injury.MethodsLangendorff-perfused isolated hearts from Slit2-overexpressing (Slit2-Tg) mice and C57BL/6J mice (background strain) were subjected to 20 min of global ischemia followed by 40 min of reperfusion. We compared Slit2-Tg with C57BL/6J mice in terms of left ventricular function and infarct size of post-IR hearts along with tissue histological and biochemical assessments (mRNA and protein expression, phosphorylation status, and myofilament contractile properties).ResultsSlit2 played cardioprotective roles in maintaining contractile function and reducing infarct size in post-IR hearts. IR increased the expression of the Slit2 receptor Robo4 and the membrane receptor Slamf7, but these increases were suppressed by Slit2 overexpression post IR. This suppression was associated with inhibition of the nuclear translocation of NFκB p65 and reductions in IL-1β and IL-18 release into perfusates. Furthermore, Slit2 overexpression attenuated the increases in myofilament-associated PKCs and phosphorylation of cTnI at Ser43 in the post-IR myocardium. The myofilament calcium sensitivity and actomyosin MgATPase activity were preserved in the post-IR Slit2 myocardium.ConclusionOur work demonstrates that Slit2 inhibits inflammatory responses and maintains myofilament contractile properties, thus contributing, at least in part, to the prevention of structural and functional damage during IR.

Abstract 906: Age-Dependent Modifications in Cardiac Myofilament Proteins are Graded by Overall Health, Measured as Frailty, in Aging Male and Female C57BL/6 Mice

Previously, we showed that the age-dependent decline in ventricular function in male mice is proportional to overall health, as quantified with a frailty index (FI) tool. This tool is based on the accumulation of deficits in health across many systems, but not the cardiovascular system per se . Here we investigated whether frailty grades age-dependent modifications in myofilaments in young and aged mice of both sexes. Myofilaments were isolated from ventricles of adult (12-13 mos; n=5 males, 5 females) and aged (22-23 mos; n=6 males, 5 females) mice. FI scores were obtained from all mice. Actomyosin Mg-ATPase activity as a function of increasing calcium concentration and phosphorylation of major myofilament proteins were quantified and compared between groups. Results showed that myofilament calcium sensitivity (EC 50 values) was similar regardless of age or sex and was not affected by FI score. Maximal actomyosin Mg-ATPase activity increased with age in females, but this was not linked to frailty. Hill coefficients declined with age in males only and were lower than females in the older group (p<0.05). However when all mice were examined, Hill coefficients declined as frailty increased (r=0.68, p=0.001). Total phosphorylation levels of myosin-binding protein C (MyBP-C), desmin, tropomyosin and essential myosin light chain (ELC) increased with age, but only in males. Interestingly, phosphorylation of MyBP-C (r=0.49; p=0.03), desmin (r=0.65; p=0.003), tropomyosin (r=0.62; p=0.004) and ELC (r=0.71; p=0.001) were highly correlated with overall health (FI scores). By contrast there was no link between frailty and phosphorylation of actin, troponin T and troponin I. These findings suggest that poor overall health, quantified in an FI, predicts changes in the myofilaments across the life course in both sexes. This may contribute to changes in cardiac contractile function in frail older adults.

Smooth muscle-like Ca2+-regulation of actin\u2013myosin interaction in adult jellyfish striated muscle

Cnidaria is an animal phylum, whose members probably have the most ancestral musculature. We prepared and characterized, for the first time to our knowledge, native actomyosin from the striated myoepithelium of the adult moon jelly Aurelia sp. The actomyosin contained myosin, paramyosin-like protein, Ser/Thr-kinase, actin, and two isoforms of tropomyosin, but not troponin, which is known to activate contraction dependent on intracellular Ca2+ signaling in almost all striated muscles of bilaterians. Notably, the myosin comprised striated muscle-type heavy chain and smooth muscle-type regulatory light chains. In the presence of Ca2+, the Mg-ATPase activity of actomyosin was stimulated and Ser21 of the regulatory light chain was concomitantly phosphorylated by the addition of calmodulin and myosin light chain kinase prepared from chicken smooth muscle. Collectively, these results suggest that, similar to smooth muscle, the contraction of jellyfish striated muscle is regulated by Ca2+-dependent phosphorylation of the myosin light chain.

Acute exposure to progesterone attenuates cardiac contraction by modifying myofilament calcium sensitivity in the female mouse heart.

We investigated myocardial effects of acute application of progesterone. In females, but not males, progesterone attenuates and slows cardiomyocyte contraction with no effect on calcium transients. Progesterone also reduces myofilament calcium sensitivity in female hearts. This may adversely affect heart function, especially when serum progesterone levels are high in pregnancy.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/acute-progesterone-modifies-cardiac-contraction/.

Regulation of Nonmuscle Myosin II by Tropomyosin Isoforms

Tropomyosin (Tm) regulates actin myosin interactions in eukaryotic cells ranging from yeast to mammalian muscle and nonmuscle cells. Tropomyosin is a α-helical coiled-coil actin binding protein that associates end-to-end to form continuous strands along both sides of the actin filament. Tropomyosins can inhibit or activate actomyosin MgATPase activity and motility depending on the myosin and Tm isoforms. In this study, we have attempted to determine whether activation or inhibition is specified by the Tm or the myosin isoform. We carried out in vitro motility assays with four myosin isoforms (skeletal muscle myosin, and nonmuscle IIA, IIB and IIC HMMs) in the presence of five Tm isoforms (skeletal muscle αTm, and nonmuscle isoforms, Tm2, Tm5a, Tm5NM1 and Tm4) and skeletal muscle actin. With skeletal muscle myosin, actin-Tm filament velocities were inhibited by αTm (∼60%) but activated by Tm5a and Tm5NM1 (30-60%) relative to actin alone, whereas Tm2 and Tm4 had little or no effect. In the case of nonmuscle IIA and IIC HMMs, all nonmuscle Tms activated filament velocities, whereas αTm had no effect, relative to actin alone. None of the Tm isoforms affected filament velocities with nonmuscle IIB HMM. Therefore, the primary determinant of the effect of Tm on actin filament velocities on myosin is the myosin isoform. The actin-activated MgATPase activities of IIA, IIB and IIC HMMs were measured to determine the mechanism of activation by a nonmuscle Tm, Tm5NM1. Tm5NM1 increased the Vmax of both IIA (26%), and IIC (19%), with a much smaller effect on IIB (12%) compared to actin alone thus supporting the motility data. Tm5NM1 also decreased the KATPase of IIA and IIB. Therefore, Tm5NM1 activates the MgATPase activity of IIA and IIB by increasing the Vmax and decreasing KATPase. Supported by NIH.

Rapid Changes in Cardiac Myofilament Function following the Acute Activation of Estrogen Receptor-Alpha

Estrogens have well-recognized and complex cardiovascular effects, including altering myocardial contractility through changes in myofilament function. The presence of multiple estrogen receptor (ER) isoforms in the heart may explain some discrepant findings about the cardiac effects of estrogens. Most studies examining the impact of estrogens on the heart have focused on chronic changes in estrogen levels, and have not investigated rapid, non-genomic pathways. The first objective of this study was to determine how acute activation of ERα impacts cardiac myofilaments. Nongenomic myocardial estrogen signaling is associated with the activation of a variety of signaling pathways. p38 MAPK has been implicated in acute ER signaling in the heart, and is known to affect myofilament function. Thus, the second objective of this study was to determine if acute ERα activation mediates its myofilament effects through p38 MAPK recruitment. Hearts from female C57Bl/6 mice were perfused with the ERα agonist PPT and myofilaments isolated. Activation of ERα depressed actomyosin MgATPase activity and decreased myofilament calcium sensitivity. Inhibition of p38 MAPK attenuated the myofilament effects of ERα activation. ERα stimulation did not affect global myofilament protein phosphorylation, but troponin I phosphorylation at the putative PKA phosphorylation sites was decreased. Changes in myofilament activation did not translate into alterations in whole heart function. The present study provides evidence supporting rapid, non-genomic changes in cardiac myofilament function following acute ERα stimulation mediated by the p38 MAPK pathway.

Cardiac Actin Capping Protein Reduction and Protein Kinase C Inhibition Maintain Myofilament Function During Cardioplegic Arrest

Background: Heart transplantation is associated with cold, cardioplegic arrest that impairs myocardial function. Protein Kinase C (PKC) suppression of myofilaments may contribute to this dysfunction. CapZ-deficient cardiac myofilaments are unresponsive to PKC. We hypothesized that myofilaments from CapZ-deficient transgenic hearts are resistant to cardioplegic dysfunction and that PKC inhibition improves function. Methods: Heart function was assessed using a Langendorff apparatus. Myofilaments isolated from murine hearts were assessed with an actomyosin MgATPase assay and protein phosphorylation gels. PKC activation was examined by immunoblotting. Results: Wildtype hearts showed impaired function after cardioplegic arrest. CapZ-deficient transgenic mouse hearts performed significantly better after 1 h cardioplegia than wildtype hearts, but not after 4 h cardioplegic arrest. Wildtype myofilaments had depressed activation at 1 and 4 h cardioplegic arrest, as demonstrated by reduced actomyosin MgATPase activity. CapZ-deficient myofilaments showed no reduced actomyosin MgATPase activity at either time. Troponin I (TnI) phosphorylation increased by approximately 20% at 1 and 4 h in wildtype mice. Myosin binding protein C (MyBP-C),and troponin T (TnT) phosphorylation increased by less than 10% at 1 h, and tended to rise at 4 h. Myofilament protein phosphorylation was largely unchanged in CapZ-deficient hearts at 1 h, but MyBP-C tended to be dephosphorylated at 4 h cardioplegic arrest. Myofilament-associated PKC-α, -βII, -δ, and -ε increased at 1 and 4 h cardioplegia in wildtype hearts, whereas only PKC-α increased in transgenic myofilaments at 1 h. PKC inhibition abolished the cardioplegic-dependent changes in actomyosin MgATPase activity and TnI phosphorylation of wildtype myofilaments. Conclusions: We demonstrate a direct link between PKC activation and myofilament dysfunction associated with cold, cardioplegic arrest. Moreover, we show for the first time a cardioprotective benefit of decreased cardiac CapZ.

A101. Uncovering the mechanisms of CapZ control of myofilament activation

Cardiac Z-discs act as intracellular communications centres, anchoring and controlling molecular messengers. We have shown that reductions in the Z-disc protein CapZ alter myofilament activation and regulation by several intracellular signalling pathways. While these protein messengers are unable to exert control over myofilament function, they are still able to alter the phosphorylation status of several myofilament proteins. We hypothesized that reductions in CapZ protein render myofilaments insensitive to changes in protein phosphorylation, thereby disrupting contractile regulation. To test this hypothesis we treated cardiac myofilaments with type 1α protein phosphatase (PP1α) before or after reducing myofilament CapZ protein levels. By extracting CapZ after PP1α treatment we ensure that the proper amino acids are targeted and that any alterations in PP1α-dependent control are not due to the targeting of different amino acids or insufficient protein dephosphorylation. Treatment of cardiac myofilaments with exogenous PP1α significantly increased maximum Ca2+-dependent actomyosin MgATPase activity and Ca2+ sensitivity. Extraction of 20% CapZ prior to PP1α treatment significantly attenuated these changes, while CapZ extraction after PP1α treatment did not impact the effects of PP1α on myofilament activation. These results demonstrate that reduced CapZ protein levels inhibit myofilament regulation by the intracellular messenger PP1α. Moreover, our findings indicate that cardiac myofilaments are not insensitive to the covalent modification of their components when CapZ levels are decreased, which contradicts our hypothetical mechanism.

Functional importance of Ca 2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca 2+-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca 2+ due to substitutions of the amino acid residues responsible for Ca 2+ liganding. To evaluate the functional importance of the Ca 2+-deficient N-terminal lobe in the Ca 2+-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC RA, and TnC SA, respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC AA). According to equilibrium dialysis using 45Ca 2+, TnC RA, and TnC SA bound stoichiometrically 3 mol Ca 2+/mol and 1 mol Ca 2+/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280–290 nm upon Ca 2+ binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6 M urea, if Ca 2+ was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca 2+-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC SA conferred as high a Ca 2+ sensitivity to Mg-ATPase activity of rabbit actomyosin–akazara scallop tropomyosin as did the troponin containing TnC AA, whereas the troponin containing TnC RA conferred virtually no Ca 2+ sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca 2+.

In vivo α-adrenergic responses and troponin I phosphorylation: anesthesia interactions

The mechanisms by which alpha-adrenergic stimulation of the heart in vivo can cause contractile dysfunction are not well understood. We hypothesized that alpha-adrenergic-mediated contractile dysfunction is mediated through protein kinase C phosphorylation of troponin I, which in in vitro experiments has been shown to reduce actomyosin Mg-ATPase activity. We studied pressure-volume loops in transgenic mice expressing mutant troponin I lacking protein kinase C phosphorylation sites and hypothesized altered responses to phenylephrine. As anesthesia agents can produce markedly different effects on contractility, we studied two agents: avertin and alpha-chloralose-urethane. With alpha-chloralose-urethane, at baseline, there were no contractile abnormalities in the troponin I mutants. Phenylephrine produced a 50% reduction in end-systolic elastance in wild-type controls, although a 9% increase in troponin I mutants (P <0.05). Avertin was associated with reduced contractility compared with alpha-chloralose-urethane. Avertin anesthesia, at baseline, produced a reduction in end-systolic elastance by 31% in the troponin I mutants compared with wild-type (P <0.05), and this resulted in further marked systolic and diastolic dysfunction with phenylephrine in the troponin I mutants. Dobutamine produced no significant difference in the contractile phenotype of the transgenic mice with either anesthetic regimen. In conclusion, these data (alpha-chloralose-urethane) demonstrate that alpha-adrenergic-mediated force reduction is mediated through troponin I protein kinase C phosphorylation. beta-Adrenergic responses are not mediated through this pathway. Altering the myofilament force-calcium relationship may result in in vivo increased sensitivity to negative inotropy. Thus choice of a negative inotropic anesthetic agent (avertin) with phenylephrine can lead to profound contractile dysfunction.

N-terminal modification and its effect on the biochemical characteristics of Akazara scallop tropomyosins expressed in Escherichia coli.

Akazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala- (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, nf-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar alpha-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca(2+)-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca(2+)-regulation together with troponin.

Identification of a Functionally Critical Protein Kinase C Phosphorylation Residue of Cardiac Troponin T

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.

Actin capping protein: an essential element in protein kinase signaling to the myofilaments.

Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC-Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca2+-dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca2+ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-epsilon after PHE or ET exposure of WT preparations. In contrast, myofilament-associated PKC-epsilon was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilament-associated PKC-beta were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca2+ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-beta.

Two distinct actin-binding sites of smooth muscle calponin.

Amino acid residues 145-163 of calponin have been proposed as a putative actin-binding site [Mezgueldi, M., Mendre, C., Calas, B., Kassab, R. & Fattoum, A. (1995) J. Biol. Chem. 270, 8867-8876]. Our previous work demonstrated that a fragment of calponin, which corresponded to the first repeated region of calponin and contained the preferred site of phosphorylation by protein kinase C [Nakamura, F., Mino, T., Yamamoto, J., Naka, M. & Tanaka, T. (1993) J. Biol. Chem. 268, 6194-6201] enhanced the Ca2+-induced contraction of permeabilized smooth muscle [Itoh, T., Suzuki, A., Watanabe, Y., Mino, T., Naka, M. & Tanaka, T. (1995) J. Biol. Chem. 270, 20400-20403]. In the present study, we compared the interactions with actin of a synthetic peptide (Lys172-His187) that encompassed the first repeated region with those of three other synthetic peptides. Lys172-His187 inhibited the binding of calponin to F-actin in a concentration-dependent manner but not the binding of caldesmon. Gly141-Gly160, including the above-mentioned putative actin-binding site, also competed with intact calponin to the same extent as Lys172-His187. Inhibition of actomyosin MgATPase activity was observed only with Gly141-Gly160. Lys172-His187 and other tested peptides had no effect. However, Gly141-Gly160 and Lys172-His187 reduced the fluorescence intensity of pyrene-labeled F-actin with approximately equal potency. Moreover, Lys172-His187 was able to reverse the inhibition of actomyosin MgATPase activity by calponin. Lys172-His187 was phosphorylated stoichiometrically by protein kinase C and phosphorylation of this peptide decreased its actin-binding activity. These observations suggest the direct involvement of two distinct actin-binding sites, with different regulatory functions, in the interactions of calponin with actin.

Adenovirus-mediated transfer of the smooth muscle cell calponin gene inhibits proliferation of smooth muscle cells and fibroblasts

Smooth muscle cell calponin ( h1 or basic isoform) is an actin-binding protein that inhibits actomyosin MgATPase activity and is abundantly expressed in differentiated smooth muscle. Western blots showed bovine tracheal (BT) smooth muscle cells in culture expressed only 2±1% ( n=8) of the amount of calponin in tissues, while NIH-3T3 fibroblasts expressed none. We tested the hypothesis that introduction of calponin to cultured BT and 3T3 cells would inhibit cytoskeletal activities associated with cell proliferation. To achieve high-efficiency expression, an adenovirus encoding the CMV–calponin construct (Adv-CaP) was generated by homologous recombination in 293 cells. With greater than 90% of BT and 3T3 cells infected with Adv-CaP, calponin expression (32 and 11 μg/mg total protein, respectively) was similar to that in smooth muscle tissues (51 μg/mg). Cells were infected with Adv-CaP for 48 h, replated at low density and proliferation rates were assessed by cell density and [ 3H]thymidine incorporation. Cell growth and DNA synthesis by Adv-CaP-infected cells were inhibited to one-third control values for both BT and 3T3 cells. Expressed calponin was localized primarily on stress fibers in both cell types. Calponin may act at the cytoskeletal level to retard signaling pathways that normally lead to tight coupling between cell shape and DNA synthesis.

Troponin from smooth adductor muscle of Ezo-giant scallop.

Troponin which can confer Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity has been prepared from the smooth adductor muscle of Ezo-giant scallop (Patinopecten yessoensis). The troponin comprises 40-, 20-, and 19-kDa components. In order to characterize the components, they were separated from each other by CM-Toyopearl column chromatography in the presence of 6 M urea. Consequently, the 20-kDa component was identified as troponin C, based on the Ca2+-binding ability. The amount of Ca2+ bound to the troponin C was estimated to be 0.75 mol/mol at 10(-4) M Ca2+ by the equilibrium dialysis method. The 19-kDa component was identified as troponin I on the basis of not only its inhibitory effect on rabbit actomyosin Mg-ATPase activity along with the smooth adductor tropomyosin, but also the releasing effect of the smooth adductor troponin C on the inhibition. On the other hand, the 40-kDa component was regarded as troponin T on the basis that it bound to F-actin-tropomyosin filament and was indispensable for conferring Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity, along with troponin C and troponin I. The above assignments were confirmed by both amino acid analysis and immunoblotting using rabbit antisera raised against counterparts of scallop striated muscle troponin.

Accumulation of Unphosphorylated Calponin in the Submembranous Cytoskeletons of Arachidonic Acid-stimulated Human Platelets

Calponin, a basic smooth-muscle protein capable of binding to F-actin, tropomyosin and calmodulin in vitro, was tested for its expression and subcellular localization in resting and stimulated human platelets. Using immunoblotting techniques calponin was revealed as a single protein band with a molecular weight of 34 kDa. Although calponin has been shown to be proteolytically degraded by calpain, in the presence of the calpain inhibitor E-64 and EGTA a significant hydrolysis of calponin could not be detected. Upon stimulation with 10 microM arachidonic acid calponin became increasingly incorporated into Triton X-100 insoluble cytoskeletal fractions reaching a plateau after 15 s. The accumulation of calponin in the cytoskeletons of stimulated platelets paralleled the polymerization of actin into newly formed microfilaments. Immunofluorescence microscopy revealed a submembranous co-localization of calponin and actin in aggregated platelets. Since isolated calponin is phosphorylated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II thereby losing its inhibitory effect on the actomyosin MgATPase activity, we examined whether changes in cell shape due to platelet stimulation are accompanied by a phosphorylation of calponin. By performing immunoblotting analysis on either resting or stimulated platelets phosphorylation of calponin on tyrosine, serine or threonine residues could not be demonstrated. In line, [32P]radiolabeling experiments were unable to detect phosphate incorporation into calponin. These observations support the hypothesis that calponin plays a physiological role in regulating contraction and secretion of human platelets even in the absence of its phosphorylation.

Amino Acid Sequence of C-Terminal 17 kDa CNBr-Fragment of Akazara Scallop Troponin-I1

The M(r) 52,000 subunit of Akazara scallop striated muscle troponin, which was tentatively identified as troponin I, was cleaved into two major fragments with CNBr: C-terminal 17 kDa fragment (CN17K) and N-terminal 35 kDa fragment (CN35K) [J. Biochem. 108, 519-521 (1990)]. CN17K inhibits rabbit reconstituted actomyosin Mg-ATPase activity, weakly in the absence of troponin T but strongly in its presence, together with Akazara tropomyosin. CN35K, however, hardly shows such inhibition. Thus, the amino acid sequence of the CN17K was determined by the Edman method. CN17K comprises 135 amino acid residues and its calculated molecular mass is 15,732 Da. A computer search of the SWISS-PROT data base revealed the TnIs of crayfish tail muscle, rabbit skeletal muscle, and bovine cardiac muscle to be homologous proteins with total sequence homologies of 39, 30, and 30%, respectively, to CN17K. Significantly high homology was observed among these TnIs in the regions around residues 75-95, 99-114, and 135-151 of the rabbit TnI. From these facts, we conclude that the 52K subunit is a TnI.

Thermal Denaturation of &amp;beta;-Connectin Isolated from Carp Muscle

β-Connectin, a 2, 100 kDa fragment of connectin, was extracted with 0.1M phosphate buffer at pH 6.6 and purified on a Bio-Gel A 50m gel filtration from carp skeletal myofibrils. The isolated connectin was in the native state judging from the fact that it enhanced aggregation of both myosin and actin filaments and appreciably elevated actomyosin Mg-ATPase activity. The thermal denaturation of connectin was measured in 0.1M and 0.6M KCl solutions of neutral pH by means of turbidity. Increase in turbidity was observed at 40°C and occurredmarkedly above 50°C. A physiological concentration of Ca2+ and Mg2+ did not affect turbidity. Theloss of interaction of connectin with myosin was caused by the denaturation of myosin at lower temperatures rather than that of connectin.