Phosphorylation and functional modifications of sarcoplasmic reticulum and myofibrils in isolated rabbit hearts stimulated with isoprenaline.

Isoprenaline stimulation of perfused rabbit hearts was associated with simultaneous phosphorylation of proteins in the myofilaments and phospholamban in the sarcoplasmic reticulum (SR). Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi, freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline, and myofibrils and SR were prepared from the same hearts. Stimulation of 32P incorporation in troponin I (TnI) and C-protein by isoprenaline was associated with a decrease in Ca2+-sensitivity of the myofibrillar Mg2+-dependent ATPase activity. Stimulation of 32P incorporation in SR by isoprenaline was associated with an increase in the initial rates of oxalate-facilitated Ca2+ transport, assayed with SR vesicles in either microsomal fractions or homogenates from the perfused hearts. These findings provide evidence that phosphorylation of TnI, C-protein and phospholamban in the intact cell is associated with functional alterations of the myofibrils and SR which may be responsible in part for the effects of catecholamines on the mammalian myocardium.     

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation.

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically 'frozen' during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca2+ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromatography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18--20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3--0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 microM epinephrine.     

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically ‘frozen’ during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca²⁺ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromotography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18?20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3–0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 μM epinephrine.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle.

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca2+-dependent ATPase and are also able to accumulate up to 10 mumol Ca2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a3 and an increase in the content of [32P]phosphoenzyme. The basic components in "calcium-oxalate preparation" from hearts are proteins with molecular weights of about 100000 (Ca2+-dependent ATPase) and 55000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12-16 mumol Pi/min per mg protein. Enzyme activity of purified Ca2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca2+-dependent ATPases isolated from heart and skeletal muscles.     

Mg-ATPase and CA2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts – effects of calcium sensitizing agents MCI-154, DPI 201–106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca²⁺ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca²⁺ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10^–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca²⁺ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca²⁺ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca²⁺ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.     

Phosphorylated cardiac myofibrils and their effect on ATPase activity.

Control guinea pig cardiac myofibrils were isolated in the presence of Triton X-100. Experimental myofibrils, prepared in the presence of Triton X-100, NaF, cyclic AMP and ATP, possessed a reduced myofibrillar ATPase activity. When myofibrils isolated under control conditions were incubated for two hours at 25°C with NaF, ATP and cyclic AMP, the ATPase activity was also decreased; however, the ATPase activity was not reduced as much as that of myofibrils isolated under experimental conditions. Incubation of myofibrils with $\text{E. coli}$ aklaline phosphatase and guinea pig heart phosphoprotein phosphatase resulted in an increase in ATPase activity and a decrease in phosphoprotein phosphate. Thus there appeared to be an inverse relationship between myofibrillar ATPase activity and phosphoprotein phosphate content. The results indicated that a protein kinase is associated with the Triton X-100 purified myofibrils and supports the notion that intact myofibrils can exist in at least two catalytic forms.     

Changes in myocardial contractile protein ATPases in chronically adrenalectomized rats with and without glucocorticoid replacement

Studies were conducted to examine the effects of chronic adrenalectomy (Adx) and adrenalectomy plus glucocorticoid replacement therapy on rat cardiac contractile protein ATPase activities. The Ca2+-dependent Mg-ATPase activity of myofibrils isolated from rat ventricles 3 weeks postadrenalectomy (Adx) was significantly decreased at all pCa2+ concentrations (P less than 0.01), compared to sham-operated (SO) rats. Similarly, Ca2+-, K+-EDTA, and actin-activated myosin ATPase activities of Adx rat hearts were markedly decreased below that of SO rats (P less than 0.01). Dexamethasone administration to Adx rats prevented the decrease of Ca2+- and K+-ATPase activities of myosin, but not of myofibrillar Ca2+-dependent Mg-ATPase or actin-activated myosin Mg-ATPase activities. These studies suggest that glucocorticoid insufficiency induced by adrenalectomy results in altered myocardial contractile protein ATPase activity which may underlie impaired cardiac performance.     

Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts.

Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca²⁺-dependent ATPase and are also able to accumulate up to 10 μmol Ca²⁺ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a₃ and an increase in the content of [³²P]phosphoenzyme. The basic components in “calcium-oxalate preparation” from hearts are proteins with molecular weights of about 100 000 (Ca²⁺-dependent ATPase) and 55 000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca²⁺-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12–16 μmol P_i/min per mg protein. Enzyme activity of purified Ca²⁺-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca²⁺-dependent ATPases isolated from heart and skeletal muscles.     

Time dependent response of cardiac myofibrillar ATPase activity to exercise

1. The purpose of the present study was to investigate the time course of run training effects on the Ca2+ kinetics of the cardiac myofibrillar ATPase activity in female Sprague-Dawley rats. 2. The cardiac myofibrillar ATPase activity was measured at varying Ca2+ levels, and the Hill-n and pCa50 were measured in the hearts of rats after 3, 6 and 9 weeks of running training with a training program that began with an initially high intensity (HINT) and a training program with a more progressive increase in intensity (PROG). 3. After 3 and 6 weeks of training cardiac myofibrillar ATPase activity in the hearts of the trained rats in both training programs was elevated by 28-40% over the control group (P less than 0.05) at a pCa5 but was not different from the control groups after 9 weeks of training (P greater than or equal to 0.05). 4. Also the Ca2+ co-operativity as measured by the Hill-n was elevated in the hearts of the trained rats after 6 and 9 weeks of training when compared to control groups suggesting changes in the regulatory proteins of the myofibrils of hearts from trained rats. 5. The elevations in cardiac myofibrillar ATPase activity suggest that the myocardium responded to the training stimulus in a phasic manner. 6. The regression of cardiac myofibrillar ATPase in the late weeks of training might be related to a reduction or a loss of a specific training stimulus for the myocardium.     

Coordination of cardiac sarcoplasmic reticulum and myofibrillar function by protein phosphorylation

Adrenergic stimulation alters functional dynamics of the heart by mechanisms most likely involving cyclic AMP (cAMP)-dependent protein phosphorylation. In vitro studies indicate that the myofibrils and sarcoplasmic reticulum (SR) may act as effectors of the adrenergic stimulation. cAMP-dependent phosphorylation of troponin I (TnI), one of the regulatory proteins of cardiac myofibrils, results in a decreased steady-state affinity of troponin C (TnC) for calcium, an increase in the off-rate for Ca2+ exchange with TnC, and a rightward shift of the relation between free Ca2+ and myofibrillar force or ATPase. Phosphorylation of phospholamban, a regulatory protein of cardiac SR, results in an increased velocity of Ca2+ transport by SR vesicles, an increased affinity of the transport protein for Ca2+, and an increased turnover of elementary steps of the ATPase reaction. These in vitro findings support the hypothesis that the inotropic response of the heart to catecholamine stimulation involves phosphorylation of TnI and phospholamban. Our in vivo studies with perfused rabbit hearts show that during the peak of the inotropic response to isoproterenol there is a simultaneous phosphorylation of TnI and an 11,000-dalton protein in the SR, most likely the monomeric form of phospholamban.     

Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms

Phospholamban, a putative regulator of cardiac sarcoplasmic reticulum Ca2+ transport, has been shown to be phosphorylated in vitro by cAMP-dependent protein kinase and an intrinsic Ca2+-calmodulin-dependent protein kinase activity. This study was conducted to determine if Ca2+-calmodulin-dependent phosphorylation of phospholamban occurs in response to physiologic increases in intracellular Ca2+ in intact myocardium. Isolated guinea pig and rat ventricles were perfused with 32Pi after which membrane vesicles were isolated from individual hearts by differential centrifugation. Administration of isoproterenol (10 nM) to perfused hearts stimulated 32P incorporation into phospholamban, Ca2+-ATPase activity, and Ca2+ uptake of sarcoplasmic reticulum isolated from these hearts. These biochemical changes were associated with increases in contractility and shortening of the t 1/2 of relaxation. Elevated extracellular Ca2+ produced comparable increases in contractility but failed to stimulate phospholamban phosphorylation or Ca2+ transport and did not alter the t 1/2 of relaxation. Inhibition of trans-sarcolemmal Ca2+ influx by perfusing the ventricles with reduced extracellular Ca2+ (50 microM) attenuated the increases in 32P incorporation produced by 10 nM isoproterenol. Trifluoperazine (10 microM) also attenuated isoproterenol-induced increases in 32P incorporation into phospholamban. In both cases, Ca2+ transport was reduced to a degree comparable to the reduction in phospholamban phosphorylation. These results suggest that direct physiologic increases in intracellular Ca2+ concentration do not stimulate phospholamban phosphorylation in intact functioning myocardium. Ca2+-calmodulin-dependent phosphorylation of phospholamban may occur in response to agents which stimulate cAMP-dependent mechanisms in intact myocardium.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Role of regulatory proteins (troponin-tropomyosin) in pathologic states

In vertebrate striated muscle, troponon-tropomyosin is responsible, in part, not only for transducing the effect of calcium on contractile protein activation, but also for inhibiting actin and myosin interaction when calcium is absent. The regulatory troponin (Tn) complex displays several molecular and calcium binding variations in cardiac muscles of different species and undergoes genetic changes with development and in various pathologic states.Extensive reviews on the role of tropomyosin (Tm) and Tn in the regulation of striated muscle contraction have been published describing the molecular mechanisms involved in contractile protein regulation. In our studies, we have found an increase in Mg²⁺ ATPase activity in cardiac myofibrils from dystrophic hamsters and in rats with chronic coronary artery narrowing. The abnormalities in myofibrillar ATPase activity from cardiomyopathic hamsters were largely corrected by recombining the preparations with a TnTm, complex isolated from normal hamsters indicating that the TnTm, may play a major role in altered myocardial function. We have also observed down regulation of Ca²⁺ Mg²⁺ ATPase of myofibrils from hypertrophic guinea pig hearts, myocardial infarcted rats and diabetic-hypertensive rat hearts. In myosin from diabetic rats, this abnormality was substantially corrected by adding troponin-tropomyosin complex from control hearts. All of these disease models are associated with decreased ATPase activities of pure myosin and in the case of rat and hamster models, shifts of myosin, heavy chain from alpha to beta predominate.In summary, there are three main troponin subunit components which might alter myofibrillar function however, very few direct links of molecular alterations in the regulatory proteins to physiologic and pathologic function have been demonstrated so far.     

Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria.

1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3.The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump.     

Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column.

The (Ca2+-Mg2+)-ATPase from human erythrocyte membranes has been solubilized in Triton X-100 and purified on a calmodulin affinity chromatography column in the presence of phosphatidylserine, to limit the inactivation of the enzyme. The enzyme was purified at least 150 times when compared with the original ghosts and showed a specific activity of 3.8 mumol.mg-1.min-1. In sodium dodecyl sulfate-polyacrylamide gels, a single major band was visible at a position corresponding to a molecular weight of about 125,000; a minor band (11% of the total protein) was present at a position corresponding to Mr = 205,000. Upon incubation of the purified preparation with [32P]ATP, both bands were phosphorylated in proportion to their mass, suggesting that both were active forms of purified ATPase.     

A vacuolar-type ATPase, partially purified from potassium transporting plasma membranes of tobacco hornworm midgut

An azide- and vanadate-insensitive, N-ethylmaleimide-sensitive ATPase has been partially purified from a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. The properties of the membrane-bound ATPase activity were identical to those of the ATPase activity of highly purified goblet cell apical membranes (Wieczorek, H., Wolfersberger, M. G., Cioffi, M., and Harvey, W. R. (1986) Biochim. Biophys. Acta 857, 271-281). 90% of the azide- and vanadate-insensitive ATPase activity was solubilized by C12E10, leaving 90% of the contaminating azide-sensitive mitochondrial ATPase activity in the pellet after centrifugation at 100,000 x g for 1 h. After discontinuous sucrose gradient centrifugation of the supernatant at 220,000 x g for 1 h nearly all of the azide- and vanadate-insensitive ATPase activity was found in the 30% sucrose fraction without contaminating azide- or vanadate-sensitive ATPase activity. Two prominent bands with relative molecular masses (Mr) of about 600,000 and 900,000, both displaying azide-insensitive and N-ethylmaleimide-sensitive ATPase activity, were found in native microgradient polyacrylamide gel electrophoresis of the 30% sucrose fraction. The two bands could not be separated by anion exchange chromatography. Denaturation of both bands resulted in the same polypeptide pattern (five major bands with Mr 70,000, 57,000, 46,000, 29,000 and 17,000) in sodium dodecylsulfate-polyacrylamide gel electrophoresis, indicating that they represented oligomers of the same protein unit. Substrate and inhibitor specificities of the partially purified ATPase were similar to those of the membrane-bound ATPase activity, whereas salt selectivity differed partly. Altogether, structural and functional properties of the ATPase strongly resemble those of vacuolar-type ATPases.     

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.     

Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.