Identification of a third isoform of Na+, K(+)-ATPase activity in rat brain synaptosomes.
3H-ouabain binding and ouabain-inhibitable 86Rb+ (K+) uptake were investigated as a means to identify a third isoform of Na+, K(+)-ATPase in crude synaptosome preparations. The specific binding of low concentrations (10 nM and 1 uM) of 3H-ouabain, in crude synaptosome preparations, was markedly inhibited by K+ (0.5-5 mM). Accordingly, 86Rb+ (K+) uptake, in the presence of 5 mM K+ was not sensitive to inhibition by low concentrations (10(-11)-10(-7) M) of ouabain. Higher concentrations (10(-6)-10(-2.6) M) of ouabain resulted in a biphasic inhibition of K+ uptake, which distinguished the activities of the presumed alpha 2 and alpha 1 isozymes of Na+, K(+)-ATPase. Reduction of K+ (1.25 mM and 0.5 mM) in the incubation, resulted in the observation of a third component of ouabain-sensitive K+ uptake. This Na+, K(+)-ATPase activity, which was defined, pharmacologically, as very sensitive (VS) to ouabain, exhibited IC50S of 3.6 nM and 92 nM at 1.25 mM K+ and 0.5 mM K+, respectively. Inhibition of ouabain binding and VS-dependent K+ uptake, at a high, physiological concentration (5 mM) of K+, suggests that VS may be an inactive isoform of brain Na+, K(+)-ATPase under resting conditions.
Laboratory of Clinical Studies, DICBR, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20852.
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Foley
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Linnoila
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013574:Synaptosomes / Q000201:enzymology*
D007527:Isoenzymes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium