Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation.
Frozen aqueous suspensions of partially purified membrane-bound renal (Na+ + K+)-ATPase have been irradiated at -135 degrees C with high-energy electrons. (Na+ + K+)-ATPase and K+-phosphatase activities are inactivated exponentially with apparent target sizes of 184 +/- 4 kDa and 125 +/- 3 kDa, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na+ + K+)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na+-K+ exchange, 201 +/- 4 kDa; (ATP + Pi)-activated Rb+-Rb+ exchange, 206 +/- 7 kDa and ATP-independent Rb+-Rb+ exchange, 117 +/- 4 kDa. The apparent size of the alpha-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the beta-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na+ + K+)-ATPase is alpha beta. (2) The inactivation target size for (Na+ + K+)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na+ and K+. (3) The target sizes, for K+-phosphatase (125 kDa) and ATP-independent Rb+-Rb+ exchange (117 kDa) are indistinguishable from that of the alpha-chain itself, suggesting that cation binding sites and transport pathways, and the p-nitrophenyl phosphate binding site are located exclusively on the alpha-chain. (4) ATP-dependent activities appear to depend on the integrity of an alpha beta complex.
D000818:Animals; D001693:Biological Transport, Active; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation; D007679:Kidney Medulla / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism* / Q000528:radiation effects; D013329:Structure-Activity Relationship; D013552:Swine
D010749:Phosphoprotein Phosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium