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+ greater than Mn2+ greater than Sr2+ greater than Br2+ greater than 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.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology / Q000378:metabolism; D051381:Rats; D012508:Sarcolemma / Q000201:enzymology* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D008670:Metals; D014638:Vanadates; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D005033:Ethylmaleimide