Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. I. Phosphoenzyme with bound Ca2+ which is exposed to the external medium.
Sarcoplasmic reticulum vesicles were phosphorylated with [gamma-32P]ATP in the presence of external Ca2+ without added Mg2+. The phosphoenzyme (EP) formed had tightly bound Ca2+ and was dephosphorylated by ADP. When the external Ca2+ was chelated after phosphorylation, Ca2+ dissociated from the EP and ADP addition no longer induced dephosphorylation. Subsequent addition of CaCl2 caused rapid recombination of Ca2+ and restoration of the ADP sensitivity. These findings show that the dissociation and recombination of Ca2+ took place on the outer surface of the membranes, indicating the existence of EP with bound Ca2+ which was exposed to the external medium (Caout.EP). The Ca2+ affinity of the Ca2+ binding site in Caout.EP was comparable to that of the high affinity Ca2+ binding site in the dephosphoenzyme (E). This shows that phosphorylation is not accompanied by an appreciable reduction in the Ca2+ affinity of the Ca2+ binding site, provided this site is exposed to the external medium. The transition from ADP-sensitive EP to ADP-insensitive induced by Ca2+ chelation was unaffected by Mg2+ in the medium. Mg2+ did not activate hydrolysis of the ADP-sensitive EP with bound Ca2+, whereas it markedly accelerated hydrolysis of the ADP-insensitive EP without bound Ca2+.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium