Effects of N-ethylmaleimide on the ATPase activities of cardiac myosin from thyrotoxic rabbits.

The ATPase activities of cardiac myosin from thyrotoxic and euthyroid rabbits have been compared. The Ca²⁺-ATPase activity of myosin from thyrotoxic animals was elevated by 200%, while the K⁺(EDTA)-ATPase activity was the same as in euthyroid animals. Modification by N-ethyl-maleimide of the most rapidly reacting class of sulfhydryls (SH₁) in myosin from euthyroid animals increased Ca²⁺-ATPase activity about 177% over the unreacted value. Modification of the SH₁ groups in myosin from thyrotoxic animals had no effect on CA²⁺-ATPase activity. We conclude that thyroxin may increase cardiac myosin ATPase activity by a conformational change in the same region as the SH₁ thiols.     

Dinitrophenylation of rabbit skeletal actomyosin.

Dinitrophenylated reconstituted or natural actomyosin effected changes in the Ca2+ sensitivity which were dependent upon the ionic strength of the reaction medium. Dinitrophenylation of reconstituted actomyosin in 0.6 M KCl led to the incorporation of 2-6 mol of the reagent per 5-10(5) g of protein and it possessed considerable Ca2+ sensitivity. Dinitrophenylated natural actomyosin under the same conditions lost most of its Ca2+ sensitivity when 1.3-5.4 mol of the dinitrophenyl group were bound. The myosin from these modified actomyosins did not lose Ca2+ sensitivity and the myosin was labeled only with 0.4-1.7 mol of the dinitrophenyl group. Dinitrophenylation of both kinds of actomyosin in 0.06 M KCl abolished the Ca2+ sensitivity; the myosin from the modified actomyosins also lost Ca2+ sensitivity. Myosin alone was more susceptible to a loss of Ca2+ sensitivity than myosin in actomyosin. Actin protected the ability of myosin to sense Ca2+ regulated actin in modified actomyosin at 0.6 M KCl but not at 0.06 M KCl. Actomyosin dinitrophenylated in the presence of ATP lost Ca2+ sensitivity. However, the myosin from this actomyosin possessed Ca2+ sensitivity. Thiolysis of the dinitrophenylated actomyosin by 2-mercaptoethanol at low ionic strength did not restore the Ca2+ sensitivity of this actomyosin or its myosin although there was a significant loss of the dinitrophenyl group.     

Activation by actin of ATPase activity of chemically modified gizzard myosin without phosphorylation.

The ATPase activity of myosin from chicken gizzard measured in the presence of either Mg²⁺ or Ca²⁺ is increased in the absence of dithiothreitol or upon reaction with Cu²⁺, o-iodosobenzoate, or N-ethylmaleimide. Iodosobenzoate or Cu²⁺ produce no change in K⁺(EDTA)-ATPase while N-ethylmaleimide produces a decrease. These treatments also make the actin-activated ATPase insensitive to Ca²⁺ when assayed in the presence of tropomyosin and a partially purified myosin light chain kinase. Phosphorylation of N-ethylmaleimide modified myosin remains dependent on Ca²⁺ and therefore appears not to be required for activation by actin of the ATPase activity of modified myosin.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Actin and Myosin in pea tendrils

We demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils. The molecular weight of tendril actin is 43,000, the same as rabbit skeletal muscle actin. The native molecular weight of tendril myosin is about 440,000. Tendril myosin is composed of two heavy chains of molecular weight approximately 165,000 and four (two pairs) light chains of 17,000 and 15,000. At high ionic strength, the ATPase activity of pea tendril myosin is activated by K⁺-EDTA and Ca²⁺ and is inhibited by Mg²⁺. At low ionic strength, the Mg²⁺-ATPase activity of pea tendril myosin is activated by rabbit skeletal muscle F-actin. Superprecipitation occurred after incubation at room temperature when ATP was added to the crude actomyosin extract. It is suggested that the interaction of actin and myosin may play a role in the coiling movement of pea tendril.     

The mechanism of vanadium action on selective K+-permeability in human erythrocytes

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO²⁺, +4 oxidation state) to vanadate (VO₃^?, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K⁺ permeability, which is very similar to the Gardos Ca²⁺-induced K⁺ permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K⁺. In ultrapure media with very low Ca²⁺, however, vanadyl has no effect on K⁺ permeability. These experiments suggest that Ca²⁺ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K⁺ permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca²⁺ from EDTA. Vanadate, an inhibitor of Ca²⁺-ATPase, causes a selective increase in K⁺ permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca²⁺. Earlier observations of effects of vanadyl and vanadate on erythrocyte K⁺ permeability can thus be explained on the basis of inhibition of the Ca²⁺ pump by vanadium, leading to an increase in intracellular Ca²⁺ concentration.     

Gel electrophoretic and density gradient analysis of the (K + Ca)-ATPase and the (Na + K)-ATPase activities of cardiac membrane vesicles

The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K⁺ + Ca²⁺)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na⁺ + K⁺)-ATPase, an enzymic activity localized to sarcolemma. The (K⁺ + Ca²⁺)-ATPase activity was stimulated 4- to 5-fold by 100 mM K⁺ in the presence of 50 μM Ca²⁺. Phosphorylation of the (K⁺ + Ca²⁺)-ATPase of intact vesicles with [γ-³²P]ATP was Ca²⁺ dependent, and monovalent cations including K⁺ increased the level of [³²P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K⁺ + Ca²⁺)-ATPase was inactivated, as was Ca²⁺-dependent ³²P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na⁺ + K⁺)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na⁺ + K⁺)-ATPase activity of intact membrane vesicles. ³²P incorporation by the (Na⁺ + K⁺)-ATPase of SDS-extracted vesicles was Na⁺ dependent, and Na⁺-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the ³²P-intermediates of the (K⁺ + Ca²⁺)-ATPase and the (Na⁺ + K⁺)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na⁺ + K⁺)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K⁺ + Ca²⁺)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na⁺ + K⁺)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K⁺ + Ca²⁺)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.     

The effect of phosphorylation of gizzard myosin on actin activation

Gizzard myosin is phosphorylated by a kinase found in chicken gizzards. The 20,000 dalton light chains are the only subunits to show an appreciable extent of ³²P incorporation. Phosphorylation requires trace amounts of Ca²⁺. The Mg²⁺-ATPase activity of gizzard myosin in the phosphorylated form is activated to an appreciable extent by skeletal actin, whereas the activation of the non-phosphorylated myosin is verylow. These results suggest that the Ca²⁺-sensitive regulatory mechanism of gizzard actomyosin is mediated via a kinase. In the presence of Ca²⁺ the onset of contraction and the resultant increase of the Mg²⁺-ATPase activity we suggest is due, at least partly, to the phosphorylation of the 20,000 dalton light chains. Whether or not Ca²⁺ binding by myosin is also essential remains to be established.     

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     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Regulation of Cardiac Muscle Contractility

The heart's physiological performance, unlike that of skeletal muscle, is regulated primarily by variations in the contractile force developed by the individual myocardial fibers. In an attempt to identify the basis for the characteristic properties of myocardial contraction, the individual cardiac contractile proteins and their behavior in contractile models in vitro have been examined. The low shortening velocity of heart muscle appears to reflect the weak ATPase activity of cardiac myosin, but this enzymatic activity probably does not determine active state intensity. Quantification of the effects of Ca⁺⁺ upon cardiac actomyosin supports the view that myocardial contractility can be modified by changes in the amount of calcium released during excitation-contraction coupling. Exchange of intracellular K⁺ with Na⁺ derived from the extracellular space also could enhance myocardial contractility directly, as highly purified cardiac actomyosin is stimulated when K⁺ is replaced by an equimolar amount of Na⁺. On the other hand, cardiac glycosides and catecholamines, agents which greatly increase the contractility of the intact heart, were found to be without significant actions upon highly purified reconstituted cardiac actomyosin.     

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2+-ATPase, the gatekeeper and the pivot

In this review we summarize mutagenesis work on the structure–function relationship of transmembrane segment M1 in the Na⁺,K⁺-ATPase and the sarco(endo)plasmic reticulum Ca²⁺-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca²⁺ interaction/occlusion in Ca²⁺-ATPase and K⁺ interaction/occlusion in Na⁺,K⁺-ATPase. Leu⁶⁵ of the Ca²⁺-ATPase and Leu⁹⁹ of the Na⁺,K⁺-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu³⁰⁹/Glu³²⁹. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca²⁺-ATPase and the Na⁺,K⁺-ATPase. All numbering of Na⁺,K⁺-ATPase amino acid residues in this article refers to the sequence of the rat α₁-isoform.     

Changes in lipids and biochemical properties of actomyosin from pre- and post-spawned hake (Merluccius hubbsi Marini)

The total lipid contents of muscle, the liver somatic index and the total lipid contents of actomyosin in hakes changed with the gonadal condition of the fish. Non-polar lipids in actomyosin from pre-spawned hake were 145% higher than in actomyosin from post-spawned hake; polar lipids were 30% higher. The relative percentage of phospholipids changed from 32% in pre-spawned hake to 48% in post-spawned hake. The Mg²⁺-ATPase activity in actomyosin increased from the pre-spawned to the post-spawned conditions, and this could be related to a higher phospholipids to neutral lipids ratio in post-spawned hake. Gradual decreases in both Ca²⁺-ATPase activity and myosin: actin ratio during the gonadal development of fish were found, suggesting a partial loss of myosin functionality of the actomyosin complex.     

Insight into the role of DL-α-lipoic acid against cyclophosphamide induced alterations in calcium sensitivity of cardiac myofilaments

Cyclophosphamide (CP), a potent antitumor drug, is known to cause severe cardiotoxicity. The present study is aimed at evaluating the role of DL-α-Lipoic acid (LA) on the calcium responsiveness of cardiac myofilaments isolated from CP treated rats. Adult male Wistar rats were divided into four treatment groups. Two groups received single intraperitoneal injection of CP (200 mg/kg b.wt.) to induce cardiotoxicity, one of these groups received LA treatment (25 mg/kg b.wt. for 10 days). A vehicle treated control group and a LA drug control were also included. Cardiotoxicity was evident from increased levels of cardiac Troponin I in serum of CP treated rats. The pCa-actomyosin ATPase relationship of myofilaments demonstrated a rightward shift indicating diminished responsiveness in CP treated rats. The hill coefficient was reduced and the myofibrillar myosin Ca²⁺-ATPase and K⁺-(EDTA) activities were also significantly (P < 0.05) reduced. Ultrastuctural observations were also in agreement with the above abnormal changes, wherein loss of myofilaments occurred. LA effectively normalized these abnormalities and restored the cardiac function in CP administered rats.     

The mechanochemical basis of amoeboid movement: II. Cytoplasmic filament stability at low divalent cation concentrations

The role played by Ca²⁺ in the stability of cytoplasmic actin and myosin filaments was investigated ultrastructurally with negatively stained isolated cytoplasm from Chaos carolinensis. Cytoplasm was incubated in solutions containing 5, 10, 15 and 25 mM EGTA for periods of time varying from 2 to 20 min. As either the EGTA concentration or duration of incubation was increased, the extent of myosin and actin filament depolymerization increased. The actin filaments depolymerized except where they were stabilized by interaction with myosin. With longer incubation times or higher EGTA concentrations complete depolymerization of the actin filaments could be accomplished. Myosin aggregates also disassembled and became shorter, while monomeric myosin labelled adjacent thin filaments to form arrowhead complexes resembling myosin enriched actomyosin [1]. These actomyosin complexes were relatively stable at low Ca²⁺ concentrations. In addition, the complexes showed a characteristic 35 nm periodicity and were dissociable in the presence of Mg²⁺-ATP. The actin containing filaments were more labile at low Ca²⁺ concentrations than the myosin aggregates. These results suggest that in cells capable of regulating their Ca²⁺ concentrations efficiently, filament polymerization-depolymerization could play a role in the control of cytoplasmic streaming.     

Sensitivity and reversibility of Ca-dependent inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na⁺ + K⁺)-ATPase to inhibition by Ca was increased 30-fold by a partially purified extract of human red cell hemolysate. The hemolysate fraction reduced the concentration of free Ca required for 50% inhibition from 30 μM to approx. 1 μM. Ca-dependent inhibition of the (Na⁺ + K⁺)-ATPase in the presence and absence of the hemolysate fraction was completely reversible. The hemolysate fraction also stimulated the Ca²⁺-ATPase and increased its affinity for Ca. In the presence of the hemolysate fraction, the concentration of free Ca that inhibited the (Na⁺ + K⁺)-ATPase by 50% was similar to that which half-maximally stimulated the Ca²⁺-ATPase. Boiling the fraction destroyed its effect on the (Na⁺ + K⁺)-ATPase, but did not impair its stimulation of the Ca²⁺-ATPase.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.