UNCOUPLING OF OCCLUSION FROM ATP HYDROLYSIS ACTIVITY IN SARCOPLASMIC-RETICULUM (CA2-ATPASE(+MG2+))

The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasm ic reticulum (Ca2++Mg2+)-ATPase by trypsin digestion was re-investigat ed by comparing ATPase activity with the ability of the enzyme to occl ude Eu3+ (a transport parameter) after various tryptic digests. With t his method, I e-examination of uncoupling by tryptic digest of the ATP ase revealed that TD2 cleavage (Arg-198) had no effect on either occlu sion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca 2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presenc e of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activi ty, but completely lost the ability to occlude Eu3+. Digest past TD2 i n the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP a nalog) at 25 degrees C resulted in no loss of occlusion,, thus reveali ng the absolute requirement of ATP during the digest to eliminate occl usion. From these findings we conclude that uncoupling of Ca2+ transpo rt from ATPase activity is possible by tryptic digestion of the (Ca2+Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.