Cold inactivation of vacuolar proton-ATPases.
Incubation of the reconstituted H+-ATPase from chromaffin granules on ice resulted in inactivation of the proton-pumping and ATPase activities of the enzyme. Inactivation was dependent on the presence of Mg2+, Cl-, and ATP during the incubation at low temperature. Approximately 1 mM ATP, 1 mM Mg2+, and 200 mM Cl- were required for maximum inactivation. Incubation for about 10 min on ice was required to achieve 50% inactivation. A much smaller decline in activity was observed when the enzyme was incubated at room temperature with the same chemicals. Inactivation in the cold resulted in the release of five polypeptides from the membrane with apparent molecular masses of 72, 57, 41, 34, and 33 kDa on sodium dodecyl sulfate gels. Three of the polypeptides of 72, 57, and 34 kDa were identified as subunits of vacuolar H+-ATPases by antibody cross-reactivity. Similar results were obtained with several other vacuolar H+-ATPases including those from plant sources. It was concluded that the catalytic sector of the enzyme is released from the H+-ATPase complex by cold treatment, resulting in inactivation of the enzyme.
Moriyama
Y
Y
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Nelson
N
N
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000838:Anions; D002412:Cations; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D002837:Chromaffin Granules / Q000201:enzymology; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007668:Kidney / Q000201:enzymology / Q000648:ultrastructure; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009566:Nitrates / Q000494:pharmacology; D010944:Plants / Q000201:enzymology / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014617:Vacuoles / Q000201:enzymology*
D000838:Anions; D002412:Cations; D002712:Chlorides; D009566:Nitrates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium