Different phosphorylation patterns of cardiac myosin light chains using ATP and ATP gamma S as substrates

Controversial views have been reported regarding the role of myosin light chain phosphorylation in the regulation of cardiac contractility (for review see. In the past, adenosine 5'-(-thio)triphosphate) (ATP gamma S) instead of ATP has frequently been used to study mechanical and biochemical consequences of myosin P-light chain (P-LC, LC-2) phosphorylation since thiophosphorylated sites are not significantly attacked by phosphatases. Unlike thiophosphorylation phosphorylation of myosin by myosin light chain kinase did neither decrease maximal (unloaded) shortening velocity of cardiac skinned fibres nor ATPase activity of cardiac myofibrils. We have accordingly investigated the phosphorylation pattern of purified cardiac myosin light chains using radioactive labeled ATP gamma S and ATP. We found that both the 28 kDa myosin light chain (LC-1) and the 18 kDa myosin light chain (LC-2, P-LC) were phosphorylated when ATP gamma S was present. In the presence of ATP, however, only LC-2 was found to be phosphorylated.     

The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively

While the structures of skeletal and smooth muscle myosins are homologous, they differ functionally from each other in several respects, i.e., motor activities and regulation. To investigate the molecular basis for these differences, we have produced a skeletal/smooth chimeric myosin molecule and analyzed the motor activities and regulation of this myosin. The produced chimeric myosin is composed of the globular motor domain of skeletal muscle myosin (Met¹–Gly⁷⁷³) and the C-terminal long α-helix domain of myosin subfragment 1 as well as myosin subfragment 2 (Gly⁷⁷³–Ser¹¹⁰⁴) and light chains of smooth muscle myosin. Both the actin-activated ATPase activity and the actin-translocating activity of the chimeric myosin were completely regulated by light chain phosphorylation. On the other hand, the maximum actin-activated ATPase activity of the chimeric myosin was the same as skeletal myosin and thus much higher than smooth myosin. These results show that the C-terminal light chain-associated domain of myosin head solely confers regulation by light chain phosphorylation, whereas the motor domain determines the rate of ATP hydrolysis. This is the first report, to our knowledge, that directly determines the function of the two structurally separated domains in myosin head.     

Alterations in myofibrillar function and protein profiles after complete global ischemia in rat hearts.

We studied changes in myofibrillar function and protein profiles after complete global ischemia with anoxia in rat hearts. Hearts were exposed to global ischemia and anoxia (CGI) for 30 or 60 minutes at 37 degrees C, and myofibrils were prepared for measurement of Ca(2+)-dependent Mg(2+)-ATPase activity at pH 7.0 and 6.5. Hearts incubated in cold saline (1 +/- 1 degrees C) and nonincubated hearts served as controls. Maximum ATPase activity was unchanged at pH 7.0 and pH 6.5 in myofibrils from hearts treated with 30 or 60 minutes of CGI. At pH 7.0, the Hill coefficient, which is an index of cooperative interactions among thin-filament proteins, was unchanged after 30 minutes of CGI but was significantly increased after 60 minutes of CGI. A similar trend for increased cooperativity was observed when myofibrillar ATPase activity was measured at pH 6.5 in myofibrils from rat hearts made ischemic for 30 or 60 minutes. Both 30 and 60 minutes of CGI resulted in increased pCa50 values (half-maximally activating free [Ca2+]) at pH 7.0 and pH 6.5. Densitometric analysis of myofibrillar proteins separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that troponin I and troponin T were degraded during 60 minutes of CGI. Two new protein bands appearing in ischemia-treated myofibrils were identified as partially degraded troponin I and troponin T with Western blots. The troponin I fragment could be phosphorylated by cAMP-dependent protein kinase. In addition, we observed phosphorylation of a protein band that corresponded to myosin light chain-2 in myofibrils from CGI-treated hearts. These results suggest that degradation of thin-filament proteins may contribute to the changes in cooperativity of Ca2+ regulation of ATPase activity observed in the myofibrils from rat hearts exposed to CGI.     

Phosphorylation of myosin light chain kinase from vascular smooth muscle by cAMP- and cGMP-dependent protein kinases

The enzyme, myosin light chain kinase, has been purified to homogeneity from bovine aortic vascular smooth muscle. Approximately 10 mg of enzyme could be obtained from 1 kg of fresh aortas with an overall yield of 26% of the original activity. The vascular myosin light chain kinase has a molecular weight of 160 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antiserum raised to the aortic myosin light chain kinase in rabbits strongly inhibited phosphotransferase activity. In addition, the antiserum was used to identify myosin kinase in a crude homogenate of vascular smooth muscle by radioimmunoblotting. A single species of the enzyme (Mr = 160 000) was identified. The bovine aortic myosin kinase could be phosphorylated by both cyclic AMP- and GMP-dependent protein kinases. Approximately 2 mols PO4/mole of enzyme could be incorporated by the cyclic AMP-dependent protein kinase in the absence of calmodulin. If Ca2+ and calmodulin were included in the reaction mixture, phosphate incorporation by the cyclic AMP-dependent protein kinase was reduced to 1 mol and phosphorylation by cyclic GMP-dependent protein kinase was completely inhibited. These results were confirmed by tryptic peptide mapping. Two distinct phosphopeptides were identified: site-1 and site-2. Both could be phosphorylated by the cyclic AMP-dependent protein kinase but only site-1 was phosphorylated by the cyclic GMP-dependent enzyme. In the presence of Ca2+ and calmodulin, phosphorylation by cAMP-dependent protein kinase was restricted to site-1. The effect of phosphorylation on myosin light chain kinase activity was determined. Only phosphorylation by cyclic AMP-dependent protein kinase was found to alter the requirement of myosin kinase for calmodulin. The K0.5 (i.e. the concentration of calmodulin required for half-maximal enzyme activation) for calmodulin was 5 nM for the unphosphorylated myosin kinase. With 2 mol PO4/mol myosin kinase incorporated, the K0.5 for calmodulin was increased to 82 nM. When only 1 mol PO4/mol myosin kinase was incorporated, no effect on calmodulin requirement was observed. Moreover, single site phosphorylation had no effect on other activity parameters, including Km for ATP and for light chains. Our studies suggest that cyclic AMP-dependent protein kinase may play an important role in the regulation of vascular myosin kinase activity. Moreover, our results indicate that cyclic GMP-dependent protein kinase does not affect calmodulin-activation of myosin kinase or several other activity parameters.     

Unchanged myosin kinase activity in hypertrophied rat heart

The decrease in myosin ATPase activity observed in cardiac hypertrophy induced by cardiac overload has been related to an isoenzymic redistribution of myosin. To test the hypothesis of an additional regulation of myosin ATPase through light chain phosphorylation, we measured the myosin kinase activity together in sham-operated and 50% to 100% hypertrophied rat hearts. The myosin kinase were purified approximately 600 fold with 6% yield by ion exchange chromatography and calmodulin-affinity chromatography. The presence of very important levels of proteolytic activity in the rat heart resulted in a partial loss of the myosin kinase calmodulin-dependency. The major component from both myosin kinase purified fractions was a 63 kdaltons protein. The protein content was identical in myosin kinase purified fractions from sham-operated and hypertrophied hearts. The calmodulin-dependent activity of myosin kinase, assayed in the presence of 0.1 mM Ca2+ and 10(-6) M calmodulin (about 6.6 nmol P X min-1 X mg-1), was identical in sham-operated and 50% to 100% hypertrophied hearts. Thus, myosin kinase specific activity, in these conditions, was unchanged in rat heart chronic hypertrophy. This result suggests that no direct functional relationship exists between the enzymatic properties of myosin and myosin kinase during the chronic phase of cardiac hypertrophy.     

Alteration of cross-bridge kinetics by myosin light chain phosphorylation in rabbit skeletal muscle: implications for regulation of actin-myosin interaction.

Myosin light chain phosphorylation in permeable skeletal muscle fibers increases isometric force and the rate of force production at submaximal levels of calcium activation; myosin light chain phosphorylation may underlie the increased rate and extent of force production associated with isometric twitch potentiation in intact fibers. To understand the mechanism by which myosin light chain phosphorylation manifests these effects, we have measured isometric force, isometric stiffness, rate of isometric force redevelopment after isotonic shortening, and isometric ATPase activity in permeabilized rabbit psoas muscle fibers. These measurements were made in the presence and absence of myosin light chain phosphorylation over a range of calcium concentrations that caused various levels of activation. The results were analyzed with a two-state cross-bridge cycle model as suggested by Brenner [Brenner, B. (1988) Proc. Natl. Acad. Sci. USA 85, 3265-3269]. The results indicate that myosin light chain phosphorylation exerts its effect on force generation and the isometric rate of force redevelopment in striated muscle through a single mechanism, namely, by increasing the rate constant describing the transition from non-force-generating cross-bridges to force-generating states (fapp). gapp, the reverse rate constant, is unaffected by phosphorylation as are the number of cycling cross-bridges. Since both calcium and myosin light chain phosphorylation increase fapp, the possibility is considered that modulation of fapp may represent a general mechanism for regulating force in actin-myosin systems.     

Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells.

A fraction has been obtained from baby hamster kidney (BHK-21) cells that will stimulate the actin-moderated ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of both BHK-21 myosin and gizzard smooth muscle myosin. This activation is associated with the specific phosphorylation of the myosin 20,000-dalton light chain. The BHK-21 myosin light chain kinase preparation contains a major protein of approximately 105,000 molecular weight as determined by sodium dodecyl sulfate gel electrophoresis. Both the actin activation and phosphorylation events require the presence of Ca2+ and the so-called modulator or calcium-dependent regulator protein that has been isolated from smooth muscle, brain, and other tissues. On the basis of these results we propose that this kinase system constitutes a Ca2+-dependent regulatory mechanism for myosin-actin interactions in nonmuscle mammalian cells.     

Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac, and smooth muscle.

We studied product inhibition of the actin-activated ATPase of myosin subfragment-1 (S-1) from the three types of muscle tissue: skeletal, cardiac, and smooth. Increasing levels of [MgADP] in the 0-1-mM range caused significant inhibition of the actin-activated MgATPase activity of cardiac and gizzard but not skeletal muscle S-1. When total nucleotide concentration ([ATP] + [ADP]) was kept constant at 1 mM, ATPase activity was inhibited by 50% at an ADP/ATP ratio of 6:1 for cardiac S-1 and 3:1 for gizzard S-1. For skeletal S-1, however, even a 19:1 ratio did not cause 50% inhibition of ATPase activity. The observed effect was not due to changes in pH or inorganic phosphate concentration, nor could it be explained by substrate (ATP) depletion. In the absence of actin, ADP had little or no inhibitory effect on the ATPase activity of S-1, and these observations imply that ADP is competing directly for the ATP binding site of the actin-S1 complexes of cardiac and smooth muscle S-1. ADP has previously been shown to be a weak competitive inhibitor of the ATPase activity in skeletal muscle. The current data imply that ADP is a very effective competitive inhibitor for the actin-activated ATPase activity of cardiac and gizzard S-1 and, therefore, that ADP may be a physiologically important modulator of contractile activity in cardiac and smooth muscle.     

Dissociation of myosin light chains and decreased myosin ATPase activity with acidification of synthetic myosin filaments: Possible clues to the fate of myosin in myocardial ischemia and infarction

The effect of mild acidification of synthetic (reconstituted) myosin filaments was studied in order to gain insight into some of the possible effects of ischemia-induced intracellular acidosis on the structure and function of myosin following myocardial infarction and myocardial ischemia. Degradation products of myosin that are soluble (at physiologic ionic strength and pH) would be of potential diagnostic value for myocardial infarction. Acidification of rabbit skeletal synthetic myosin filaments led to a pH dependent partial dissociation of the heaviest (LC₁) and lightest (LC₃) of the 3 light chains. Dissociation was detected from pH 5.0 to 6.5 and was maximal at pH 6.0, at which 30% of LC₁ was dissociated. Acidification of canine cardiac synthetic myosin filaments led to partial dissociation of both light chains; but more LC₁ than LC₂ was dissociated. Light chains reassociated with heavy chains upon return of the pH to 7. Light chains of myosin have recently been reported to appear in the peripheral blood after myocardial infarction but the small amount of free light chains in the heart is insufficient to account for the amount that appears in the blood. Acid-mediated dissociation of light chains in vitro suggests that circulating light chains after myocardial infarction may arise as a result of the intracellular acidosis of ischemic myocytes. The mechanisms responsible for the acidification-induced decrease in myofibrillar actomyosin adenosine triphosphatase (ATPase) activity are unclear. One possibility is that the decreased myofibrillar ATPase activity is due in part to an acid-induced decrease of the myosin ATPase of the myofibril irrespective of the effect of acid on the troponin-tropomyosin regulatory system. This possible mechanism is supported by the observations that acidification of rabbit skeletal and human and canine cardiac synthetic myosin filaments resulted in a reduction of ATPase activity (measured at pH 7.5) of the redissolved myosin which was progressive with greater acidification. The reduction in ATPase activity occurred whether the return of the myosin to pH 7.5 was accomplished in the presence or absence of dissociated light chains.     

Regulation of actin-activated ATP hydrolysis by arterial myosin.

Myosin was isolated from the main pulmonary artery of swine and was phosphorylated or dephosphorylated by utilizing the endogenous kinase or phosphatase, respectively. The myosins, phosphorylated to various degrees, were purified free of kinase and phosphatase activities by gel filtration on Sepharose CL-4B agarose columns. The level of actin-activated ATPase activity was dependent upon the degree of myosin light chain phosphorylation. Fully phosphorylated myosin reconstituted with actin and tropomyosin (actin/tropomyosin = 61:1) had the highest ATPase activity (0.1 mumol of Pi/mg . min). The actin-activated ATPase activity showed maximal (60--65%) Ca2+ sensitivity at 2 mol of Ca2+ bound per mol of myosin. The actin-activated ATPase activity, Ca2+ binding, and Ca2+ sensitivity of arterial myosin were also dependent upon Mg2+ concentration. The ATPase activity was maximal at 2--3 mM Mg2+ and, at low (0.5 mM) Mg2+ concentration, the activity was only one-third of the maximal activity. Increasing the Mg2+ above 3 mM was not associated with a further increase in ATPase activity, but the Ca2+ binding and Ca2+ sensitivity decreased with increasing Mg2+ concentration. The maximal Ca2+ sensitivity was observed at 2--3 mM Mg2+, a concentration at which the myosin bound 2 mol of Ca2+/mol. Both the ATPase activity and the Ca2+ sensitivity were more remarkable when actin that contained tropomyosin was used to activate the ATPase activity. The data indicate that calcium regulates the actin-activated ATP hydrolysis not only by its effects on the phosphorylation system but also by direct binding to the myosin.     

Structural and functional properties of myosin associated with the compensatory cardiac hypertrophy in the rabbit.

Right ventricular hypertrophy was produced in rabbits by pulmonary artery constriction. Measurement of intraventricular pressure, ventricular wet and dry weights and histologic examination of the liver demonstrated substantial hypertrophy with no signs of congestive failure 3 to 6 weeks following constriction. Right ventricular papillary muscle from these hearts exhibited a depressed force-velocity relationship and an increased time to peak tension but otherwise were normal. Myosin prepared from control and hypertrophied hearts was of comparable purity and yield. Quantitative densitometry of SDS polyacrylamide gels showed the light chains of hypertrophy myosin were unchanged in number, molecular weight and stoichiometry. Total protein sulfhydryl content was not different for control and hypertrophy myosins. Calcium-activated ATPase activity of hypertrophy myosin was significantly decreased shen measured at varying ATP concentration. By Lineweaver-Burk analysis, V_max of hypertrophy myosin was depressed by 50%, while the apparent K_m was 10% lower. When sulfhydryl-dependent ATPase activity was measured as a function of p-chloromercuribenzoate (PCMB) concentration, hypertrophy myosin exhibited a normal activation profile. The ATPase activity of hypertrophy myosin was initially depressed by 50% and was significantly reduced at low PCMB concentrations. However the difference was absent at peak activation, suggesting that the SH₁ sulfhydryls were capable of activation by PCMB to the same level as for control myosin. These data were consistent with a subtle conformational change at or near the active site, rather than with a change in the composition of major subunits of myosin from hypertrophied hearts.     

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.     

Changes in rat cardiac myosin during development and in culture

Developmental changes in the subunit composition and ATPase activity of myosin isolated from rat ventricular myocardium and 5-day-old myocardial tissue cultures were examined. Electrophoretic analysis of cardiac myosin from 12-week-old adults, 9-, and 16-day neonates, and 5-day tissue cultures demonstrated two light chains (Mol. wt 25 500 and 20 000) with a molar ratio of 1: 1. In 21-day fetal myosin, three light chains were observed (Mol. wt: 25 500; 24 500; 20 000), with a molar ratio of 0.82:0.15: 1.0. Analysis of ATPase activity in the presence of three activators (Ca²⁺, K⁺, and F-actin) showed significant differences between these myosin preparations, though the time course of the change was activator specific. The change in K⁺-activated ATPase activity occurred soon after birth and correlated with the disappearance of the third light chain (Mol. wt 24 500) and a partial isozymic shift from V₃ to V₁ myosin. The Ca²⁺- and actin-activated ATPase activities increased more slowly and were accompanied by continuation of the V₃ to V₁ myosin shift. Thus, it appears that V₃ myosin is heterogeneous. Moreover, kinetic analysis of tissue culture myosin is consistent with the predominance of V₃ myosin with low K⁺-activated ATPase activity.     

Involvement of the C-terminal residues of the 20,000-dalton light chain of myosin on the regulation of smooth muscle actomyosin.

The segment of smooth muscle regulatory light chain essential for the phosphorylation dependent activation of actomyosin motor activity and the binding of myosin heavy chain was identified. The C-terminal domain of the 20-kDa light chain, which is less conserved than the rest of the polypeptide among various muscle types, was mutated by either deletion or substitution of amino acid residues and the mutant light chains were then incorporated into myosin by subunit exchange. Deletion of Lys149-Ala166 markedly reduced the affinity of the light chain for the heavy chain, whereas the C-terminal five residues, Lys167-Asp171, did not contribute to the binding affinity. Deletion of Lys149-Phe158 abolished the phosphorylation-dependent activation of actomyosin ATPase activity as well as superprecipitation activity. These results suggest that the C-terminal domain of the regulatory light chain is critical for transmitting the change in the conformation of the regulatory light chain induced by phosphorylation at Ser19 to the heavy chain.     

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.     

Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity.

In an actomyosin fraction isolated from human platelets, phosphorylation of the 20,000-dalton light chain of myosin is stimulated by calcium and the calcium-binding protein calmodulin. The enzyme catalyzing this phosphorylation has been isolated by using calmodulin-affinity chromatography. Platelet myosin light chain kinase activity was monitored throughout the isolation procedures by using the 20,000-dalton smooth muscle myosin light chain purified from turkey gizzards as substrate. The partially purified myosin kinase requires both calcium and calmodulin for activity and has a specific activity of 3.1 mumol of phosphate transferred to the 20,000-dalton light chain per mg of kinase per min under optimal assay conditions. Km values determined for ATP and myosin light chains are 121 microM and 18 microM, respectively. Of several substrates surveyed as phosphate acceptors (alpha-casein, histone II-A, phosphorylase b, protamine, histone V-S, and phosvitin), only the 20,000-dalton myosin light chain is phosphorylated at a significant rate. These results suggest that platelet myosin light chain kinase is a calcium-dependent enzyme and that the requirement for calcium is mediated by the calcium-binding protein calmodulin.     

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.     

Phosphorylation and thiophosphorylation by myosin light chain kinase: different effects on mechanical properties of chemically skinned ventricular fibers from the pig

The influence of myosin light chain phosphorylation (treatment with myosin light chain kinase = MLCK, calmodulin and ATP) and thiophosphorylation (incubation with MLCK, calmodulin and ATP gamma S) on the maximal shortening velocity (Vmax) and Ca2+ sensitivity of chemically-skinned ventricular fibers from the pig has been studied. Vmax was determined by the slack-test method and by extrapolation of the force-velocity relation by the isotonic quick release method. Vmax was 1.53 muscle length/s (L/s) and 1.94 L/s using the force-velocity relation and the slack-test, respectively. Phosphorylation increased the Ca2+ sensitivity for isometric force development of skinned fibers but had no influence on Vmax. Thiophosphorylation decreased Vmax but had no influence on Ca2+ sensitivity. Phosphorylation pattern of the myosin light chains of the skinned fibers was studied using [gamma-32P]ATP or [gamma-P35S]ATP (250 muCi each) and autoradiography. Incubation of skinned fibers with labeled ATP led to a phosphate incorporation into the 18-kDa myosin light chain (MPLC or regulatory light chain) while incubation with labeled ATP gamma S led to an incorporation of thiophosphate into the 28-kDa myosin light chain (alkali light chain) and tropomyosin. We suggest that the difference in mechanical behavior between phosphorylated and thiophosphorylated skinned fibers are due to differences in the phosphorylation profiles of myofibrillar regulatory proteins.     

Chimeras of Dictyostelium myosin II head and neck domains with Acanthamoeba or chicken smooth muscle myosin II tail domain have greatly increased and unregulated actin-dependent MgATPase activity

Phosphorylation of the regulatory light chain of Dictyostelium myosin II increases V(max) of its actin-dependent MgATPase activity about 5-fold under normal assay conditions. Under these assay conditions, unphosphorylated chimeric myosins in which the tail domain of the Dictyostelium myosin II heavy chain is replaced by either the tail domain of chicken gizzard smooth muscle or Acanthamoeba myosin II are 20 times more active because of a 10- to 15-fold increase in V(max) and a 2- to 7-fold decrease in apparent K(ATPase) and are only slightly activated by regulatory light chain phosphorylation. Actin-dependent MgATPase activity of the Dictyostelium/Acanthamoeba chimera is not affected by phosphorylation of serine residues in the tail whose phosphorylation completely inactivates wild-type Acanthamoeba myosin II. These results indicate that the actin-dependent MgATPase activity of these myosins involves specific, tightly coupled, interactions between head and tail domains.     

The influence of pH on the Ca2+-regulated ATPase of cardiac and white skeletal myofibrils.

An intracellular acidosis depresses the twitch tension of cardiac muscle but in general increases that of skeletal muscle. To determine whether these contrasting effects might be due to a difference in the pH-sensitivities of the Ca²⁺-activation of the myofibrils, we assayed the ATPase activity of rabbit and guinea-pig cardiac, and rabbit white skeletal, myofibrils at 37°C in solutions of different pCa (8.0 to 4.3) and pH (7.20, 7.00, 6.80, 6.40). Care was taken to free the cardiac myofibrils of contaminant membrane ATPases. Reducing the pH from 7.20 to 6.40 decreased the maximum Ca²⁺-regulated portion of the cardiac myofibrillar ATPase by 20% but had no significant effect on that of the skeletal myofibrillar ATPase. This reduction of pH also produced a five-fold increase in the [Ca²⁺] required for half-maximal cardiac ATPase activity at each pH; the corresponding [Ca²⁺] for the skeletal myofibrillar ATPase was increased four-fold. These slight differences in the pH-sensitivities of cardiac and skeletal myofibrillar ATPases probably do not account for more than a small part of the contrasting effects of acidosis on the two types of muscle.