Evidence that a phosphorylated intermediate in a brain transport adenosine triphosphatase is an acyl phosphate.

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Abstract
A phosphorylated intermediate can be demonstrated in a brain "transport ATPase" preparation incubated with ATP- [gamma] -P32. This Intermediate shows many of the properties previously shown in kidney and electric organ "transport ATPase" preparations; i.e., Na+-dependent formation, K+-dependent dephosphorylation, and inhibition of Na+ and K+ effects by ouabain. Pretreatment with DFP [diisopropyl fluorophosphate], which was previously shown to inhibit irreversibly the "transport ATPase," markedly reduces the formation of the p32-labeled phosphorylated intermediate. The phosphorylated intermediate is more labile to alkali than to acid. Digestion with pepsin of the protein releases 2 major radioactive "peptides" which migrate slowly toward the cathode. Treatment of the peptic digests with hydroxylamine acetate or with acyl phosphatase liberates most of the radioactivity from these peptides as inorganic phosphate. One phosphorylated intermediate in the "transport ATPase" is probably an acyl phosphate. Hydroxylamine or alkali treatment of the labeled protein releases about 70% of the transport related phosphorylation as orthophoshate. The other 30% of the transport-related phosphorylation may be due to other phosphorylated intermediates in the ATPase preparation.