Uncoupling of occlusion from ATP hydrolysis activity in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.
The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ 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 presence 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 activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport 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.
Lockwich
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T
Clinical Investigations and Patient Care Branch NIDR, National Institutes of Health, Bethesda, MD 20892.
Dunigan
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CD
Shamoo
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AE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D005063:Europium / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D005063:Europium; D000255:Adenosine Triphosphate; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium