Cytosolic calcium dependent neutral proteinase of human erythrocytes: the role of calcium ions on the molecular and catalytic properties of the enzyme

The soluble neutral proteinase of human erythrocytes dissociates into constituent subunits of 80k and 30k in the presence of mM concentrations of Ca²⁺. Similarly the soluble natural inhibitor of this proteinase, of approximate molecular weight 240k, is dissociated into 60k subunits by mM concentrations of Ca²⁺. Removal of Ca²⁺ restores the native oligomeric structure of the proteinase and of the natural inhibitor. The formation of the native active enzyme or of the inactive enzyme-inhibitor complex depends on reversible association-dissociation processes mediated by Ca²⁺ concentration.     

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I

DNase I and proteinase K are two enzymes commonly used in the purification of highly polymerized RNA. In the presence of EDTA DNase I is rapidly inactivated by proteinase K while in 10 mm Ca²⁺ DNase is totally immune to proteinase K inactivation even at protease concentrations of up to 1 mg/ml. RNase A, a common contaminant of “RNase-free” DNase was inactivated by proteinase K in the presence or absence of Ca²⁺. Treatment of DNase I with proteinase K in the presence of Ca²⁺ selectively removed RNase A activity as judged by rRNA and poly(A⁺ RNA ribosomal RNA degradation monitored by sucrose gradient centrifugation. These results suggest that (i) DNase A and proteinase K can be used together in the presence of Ca²⁺ to obtain better digestion of nucleoprotein complexes, and (ii) proteinase K treatment of Ca²⁺ DNase can be used to selectively remove contaminating RNase.     

The effect of calciums on molecular motions of proteinase K

The native serine protease proteinase K binds two calcium cations. It has been reported that Ca²⁺ removal decreased the enzyme’s thermal stability and to some extent the substrate affinity, but has discrepant effects on catalytic activity of the enzyme. Molecular dynamics simulations were performed on the Ca²⁺-bound and Ca²⁺-free proteases to investigate the mechanism by which the calciums affect the structural stability, molecular motions, and catalytic activity of proteinase K. Very similar structural properties were observed between these two forms of proteinase K during simulations; and several long-lived hydrogen bonds and salt bridges common to both forms of proteinase K were found to be crucial in maintaining the local conformations around these two Ca²⁺ sites. Although Ca²⁺ removal enhanced the overall flexibility of proteinase K, the flexibility in a limited number of segments surrounding the substrate-binding pockets decreased. The largest differences in the equilibrium structures of the two simulations indicate that, upon the removal of Ca²⁺, the large concerted motion originating from the Ca1 site can transmit to the substrate-binding regions but not to the catalytic triad residues. In conjunction with the large overlap of the essential subspaces between the two simulations, these results not only provide insight into the dynamics of the underlying molecular mechanism responsible for the unchanged enzymatic activity as well as the decreased thermal stability and substrate affinity of proteinase K upon Ca²⁺ removal, but also complement the experimentally determined structural and biochemical data.     

Trypsin and calcium ions elicit changes of the membrane potential in pig blood platelets.

The addition of trypsin or thrombin or of Ca²⁺ ions to pig blood platelets was followed by a K⁺- dependent change of the membrane potential similar to that produced by the ionophore valinomycin. The effect of trypsin and of Ca²⁺, but not of valinomycin, was prevented by La³⁺ and by EGTA. It is proposed that upon the modification of the platelet surface by trypsin (and by thrombin under physiological conditions) membrane Ca²⁺ move from the external to the internal side of the platelet surface membrane and open the gates of K⁺ - specific channels.     

Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca²⁺-activated phospholipid-dependent protein kinase. Autoradiography showed that Ca²⁺-activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca²⁺-activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca²⁺-dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.     

Staphylococcal serine proteinase increases intracellular free calcium concentration in human polymorphonuclear leukocytes

Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 µg/ml. The highest Ca²⁺ rise (104 ± 47 nM) was observed for 10 µg/ml SSP. It was mainly dependent (81 ± 11%) on extracellular calcium influx, however, SSP mobilized 68 ± 7% of Ca²⁺ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca²⁺ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca² concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways.     

Docking of calcium ions in proteins with flexible side chains and deformable backbones

A method of docking Ca²⁺ ions in proteins with flexible side chains and deformable backbones is proposed. The energy was calculated with the AMBER force field, implicit solvent, and solvent exposure-dependent and distance-dependent dielectric function. Starting structures were generated with Ca²⁺ coordinates and side-chain torsions sampled in 1000 Å³ cubes centered at the experimental Ca²⁺ positions. The energy was Monte Carlo-minimized. The method was tested on fourteen Ca²⁺-binding sites. For twelve Ca²⁺-binding sites the root mean square (RMS) deviation of the apparent global minimum from the experimental structure was below 1.3 and 1.7 Å for Ca²⁺ ions and side-chain heavy atoms, respectively. Energies of multiple local minima correlate with the RMS deviations from the X-ray structures. Two Ca²⁺-binding sites at the surface of proteinase K were not predicted, because of underestimation of Ca²⁺ hydration energy by the implicit-solvent method.     

Efficient degradationin vitro of all intermediate filament subunit proteins by the Ca2+-activated neutral thiol proteinase from Ehrlich ascites tumor cells and porcine kidney

Vimentin, desmin, glial fibrillary acidic protein, neurofilament triplet proteins, and a mixture of cytokeratins were digested with Ca²⁺-activated neutral thiol proteinase isolated from Ehrlich ascites tumor (EAT) cells and porcine kidney. All intermediate filament proteins were degraded by the proteinase, although with different rates and Ca²⁺ optima. These results are in part at variance with our previous statement that the Ca²⁺-activated proteinase from EAT cells is specific for vimentin and desmin.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Calcium and thiol reactivity of human plasma clotting factor XIII

1. The reaction of iodoacetate, 2-chloromercuri-4-nitrophenol and 5,5′-dithiobis-(2-nitrobenzoate) with thrombin-cleaved Factor XIII (i.e. Factor XIII_a) was accompanied by enzyme inhibition. 2. The reaction with iodoacetate and 5,5′-dithiobis-(2-nitrobenzoate) was absolutely dependent on Ca²⁺, and the rate of reaction increased with the Ca²⁺ concentration up to very high, non-physiological concentrations. 3. 2-Chloromercuri-4-nitrophenol reacted with Factor XIII_a in the absence of Ca²⁺, but at a much slower rate. 4. Stopped-flow methods were used to quantify the reaction with 5,5′-dithiobis-(2-nitro-benzoate) because of the Ca²⁺-dependent dissociation of Factor XIII_a (a′₂b₂) and subsequent aggregation of the a′ chains into turbid precipitates. 5. The 3-carboxy-4-nitrothio-phenolate released was consistent with the reaction of 2 thiol groups/molecule of Factor XIII_a. The isolated b chains of Factor XIII did not react with either of the chromophoric reagents. This indicated that the a′ chains of Factor XIII_a were responsible for the thiol reactivity of the enzyme. 6. The Ca²⁺ dependence of the enzyme inhibition by these thiol reagents was very dependent on protein concentration. This is discussed in relation to the Ca²⁺-induced dissociation of Factor XIII_a. 7. The acceptor substrate, casein, decreased the Ca²⁺ concentration required for enzyme inhibition by both the mercurial and the aromatic disulphide compounds. Dansylcadaverine did not affect Ca²⁺ dependence of inhibition.     

Role of yessotoxin in calcium and cAMP‐crosstalks in primary and K‐562 human lymphocytes: The effect is mediated by Anchor kinase a mitochondrial proteins

Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3′,5′‐cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca²⁺), whereas the contrary effect has been observed in a Ca²⁺‐free medium. In the present article, the effect of YTX in K‐562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K‐562 cell line are completely opposite than in fresh human lymphocytes, since in K‐562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K‐562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K‐562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca²⁺ increase in fresh human lymphocytes but no effect was observed on Ca²⁺ pools depletion in these cells. However, our results show that, in K‐562 cells, YTX has no effect on cytosolic Ca²⁺ levels in a medium with Ca²⁺ and induces an increase on Ca²⁺ pools depletion followed by a Ca²⁺ influx. As far as Ca²⁺ modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca²⁺ reservoirs affected by YTX are different than thapsigargin‐sensible pools. Furthermore, YTX‐dependent Ca²⁺ pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase‐4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p‐(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca²⁺, YTX and cAMP pathways. Also, results obtain demonstrate that YTX‐dependent Ca²⁺ influx was only abolished by FCCP pre‐treatment, which indicates a link between YTX and mitochondria in K‐562 cell line. Cytosolic expression of A‐kinase anchor proteins (AKAPs), the proteins which integrates phosphodiesterases (PDEs) and PKA to the mitochondria, was determined in both cell models. On the one hand, in human fresh lymphocytes, YTX increases AKAP149 cytosolic expression. This fact is accompanied with a decrease in cAMP levels, and therefore PDEs activation, which finally leads to cell survival. On the other hand, in tumoural lymphocytes, YTX has an opposite effect since decreases AKAP149 cytosolic expression and increase cAMP levels which leads to cell death. This is the first time that YTX and mitochondrial AKAPs proteins relationship is characterised. J. Cell. Biochem. 113: 3752–3761, 2012. © 2012 Wiley Periodicals, Inc.     

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation

Human platelets use a rise in cytosolic Ca²⁺ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca²⁺ signals to trigger each of these independently is unknown. Other cells create local Ca²⁺ signals to activate Ca²⁺-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca²⁺ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca²⁺ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca²⁺ to isolate a cytosolic Ca²⁺ nanodomain created by Ca²⁺ release from the DTS. In the absence of any detectable rise in global cytosolic Ca²⁺ concentration, thrombin stimulation triggered Na⁺/Ca²⁺ exchanger (NCX)-dependent Ca²⁺ removal into the extracellular space, as well as Ca²⁺-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca²⁺ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca²⁺ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca²⁺ nanodomain contained within the MC that can control shape change independently of platelet aggregation.     

Involvement of STIM1 in the proteinase‐activated receptor 1‐mediated Ca2+ influx in vascular endothelial cells

Thrombin increases the cytosolic Ca²⁺ concentrations and induces NO production by activating proteinase‐activated receptor 1 (PAR₁) in vascular endothelial cells. The store‐operated Ca²⁺ influx is a major Ca²⁺ influx pathway in non‐excitable cells including endothelial cells and it has been reported to play a role in the thrombin‐induced Ca²⁺ signaling in endothelial cells. Recent studies have identified stromal interaction molecule 1 (STIM1) to function as a sensor of the store site Ca²⁺ content, thereby regulating the store‐operated Ca²⁺ influx. However, the functional role of STIM1 in the thrombin‐induced Ca²⁺ influx and NO production in endothelial cells still remains to be elucidated. Fura‐2 and diaminorhodamine‐4M fluorometry was utilized to evaluate the thrombin‐induced changes in cytosolic Ca²⁺ concentrations and NO production, respectively, in porcine aortic endothelial cells transfected with small interfering RNA (siRNA) targeted to STIM1. STIM1‐targeted siRNA suppressed the STIM1 expression and the thapsigargin‐induced Ca²⁺ influx. The degree of suppression of the STIM1 expression correlated well to the degree of suppression of the Ca²⁺ influx. The knockdown of STIM1 was associated with a substantial inhibition of the Ca²⁺ influx and a partial reduction of the NO production induced by thrombin. The thrombin‐induced Ca²⁺ influx exhibited the similar sensitivity toward the Ca²⁺ influx inhibitors to that seen with the thapsigargin‐induced Ca²⁺ influx. The present study provides the first evidence that STIM1 plays a critical role in the PAR₁‐mediated Ca²⁺ influx and Ca²⁺‐dependent component of the NO production in endothelial cells. J. Cell. Biochem. 108: 499–507, 2009. © 2009 Wiley‐Liss, Inc.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Palmitic and Stearic Acids Bind Ca2+ with High Affinity and Form Nonspecific Channels in Black-Lipid Membranes. Possible Relation to Ca2+-Activated Mitochondrial Pores

A mitochondrial hydrophobic component that forms Ca²⁺-induced nonspecific ion channels in black-lipid membranes (Mironova et al., 1997) has been purified and its nature elucidated. It consists of long-chain saturated fatty acids—mainly palmitic and stearic. These fatty acids, similar to the mitochondrial hydrophobic component, bind Ca²⁺ with high affinity in comparison with unsaturated fatty acids, saturated fatty acids with shorter aliphatic chains, phospholipids, and other lipids. Ca²⁺-binding is inhibited by Mg²⁺ but not by K⁺. For palmitic acid, the K _d for Ca²⁺ was 5 M at pH 8.5 and 15 M at pH 7.5, with the B _max of 0.48 ± 0.08 mmol/g. This corresponds to one Ca²⁺ ion for eight palmitic acid molecules. The data of IR spectroscopy confirm that Ca²⁺ does not form ionic bonds with palmitic and stearic acids under hydrophobic conditions. It has been found that in the presence of Ca²⁺, palmitic and stearic acids, but not unsaturated FFA induce a nonspecific permeability in black-lipid membranes. Addition of Ca²⁺ in order to induce the permeability transition, increases the extractable amount of palmitic and stearic acids, the effect being prevented by a phospholipase A₂ inhibitor. The possible involvement of palmitic and stearic acids in the mitochondrial nonspecific permeability is discussed.     

Ca2+-dependent effect of acetylphosphate on spin-labeled sarcoplasmic reticulum.

Acetylphosphate produces a definite change in the spectrum of an iodoacetamide spin probe covalently bound to sarcoplasmic reticulum ATPase. The observed change, which is Ca²⁺ dependent and reversible, is attributed to a protein conformational change occurring during the Ca²⁺ transport cycle.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle