Acid-labile ATP and/or ADP/P-i binding to the tetraprotomeric form of Na/K-ATPase accompanying catalytic phosphorylation-dephosphorylation cycle

The Na/K-ATPase has been shown to bind 1 and 0.5 mol of P-32/mol of alpha-c hain in the presence [gamma-P-32]ATP and [alpha-P-32]ATP, respectively, acc ompanied by a maximum accumulation of 0.5 mol of ADP-sensitive phosphoenzym e (NaE1P) and potassium sensitive phosphoenzyme (E2P), The former accumulat ion was followed by the slow constant liberation of P-i, but the latter was accompanied with a rapid similar to 0.25 mol of acid-labile P-i burst. The rubidium (potassium congener)-occluded enzyme (similar to 1.7 mol of rubid ium/mol of alpha-chain) completely lost rubidium on the addition of sodium + magnesium, Further addition of similar to 100 mu M [gamma-P-32]ATP and [a lpha-P-32]ATP, both induced 0.5 mol of P-32-ATP binding to the enzyme and c aused accumulation of similar to 1 mol of rubidium/mol of a-chain, accompan ied by a rapid similar to 0.5 mol of P-i burst with no detectable phosphoen zyme under steady state conditions. Electron microscopy of rotary-shadowed soluble and membrane-bound Na/K-ATPases and an antibody-Na/K-ATPase complex , indicated the presence of tetraprotomeric structures (cup), These and oth er data suggest that Na/K-ATP hydrolysis occurs via four parallel paths, th e sequential appearance of (NaE1P:E-ATP)(2), (E2P:E.ATP:E2P:E.ADP/P-i), and (KE2:E.ADP/P-i)(2), each of which has been previously referred to as NaE1P , E2P, and KE2, respectively.