Independent conformational changes caused by calmodulin and calcium in the cardiac microsomal (Ca2+, Mg2+)-ATPase during its ATP hydrolysis-ATP synthesis cycle.
The cardiac sarcoplasmic reticulum preparation employed in this study presented two fractions of associated calmodulin, one easily removable and the other tightly bound. The latter one was resistant to treatment with EGTA and high ionic strength. The forward or hydrolytic component of the (Ca2+, Mg2+)-ATPase cycle of calmodulin-free vesicles was stimulated when preincubated with trypsin, as opposed to the inhibition of this activity when the intact or calmodulin containing vesicles were employed in parallel assays. Interestingly, the reverse reaction of the ATPase cycle or ATP in equilibrium Pi exchange reaction found in both types of vesicles, was more sensitive to exposure to trypsin. Although a drastic inhibition of this exchange reaction was observed independent of the presence of the modulator, this reaction was transiently stimulated when the calmodulin-free vesicles were preincubated with a low trypsin to protein ratio. The differential effect of trypsin upon each reaction indicates that the equilibrium between the E1 and the E2 states of the enzyme is displaced. [125I]calmodulin was found to bind equally to the Ca X E1 approximately P and the E2 - P intermediates of the isolated enzyme formed with the ATP and Pi respectively. It is suggested the formation of an independent overall conformational state for the (Ca2+, Mg2+)-ATPase in the presence of calmodulin.
D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D000242:Cyclic AMP / Q000032:analysis; D006868:Hydrolysis; D008861:Microsomes / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014357:Trypsin / Q000494:pharmacology
D002147:Calmodulin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium