Mechanisms of detergent effects on membrane-bound (Na+ + K+)-ATPase.
Because the nonionic detergent octaethylene glycol dodecyl ether has been used extensively for studies on active solubilized preparations of (Na+ + K+)-ATPase, we tried to see if the detergent alters the properties of the membrane-bound enzyme prior to solubilization. Addition of the detergent, at concentrations below its critical micellar concentration, to reaction mixtures containing the highly purified membrane-bound enzyme reduced the K0.5 of ATP for (Na+ + K+)-dependent ATPase activity without affecting the maximal velocity or abolishing the negative cooperativity of the substrate-velocity curve. Under these conditions, however, the enzyme was not solubilized as evidenced by complete sedimentation of the membrane fragments containing the enzyme upon centrifugation at 100,000 X g for 30 min. Other nonsolubilizing effects of the detergent included an increase in K0.5 of K+, inhibition of Na+-dependent ATPase with no effect on K0.5 of ATP for this activity, and reductions in the spontaneous decomposition rates of the K+-sensitive phosphoenzyme obtained from ATP and the phosphoenzyme obtained from Pi. The nonsolubilizing effects of the detergent on the purified enzyme were obtained with no detectable lag, were readily reversible, and could be distinguished from its vesicle-opening effects on crude membrane preparations. Several other nonionic and ionic detergents had similar effects on the enzyme. The findings indicate (a) detergent binding to hydrophobic sites on extramembranous segments of enzyme subunits; (b) that occupation of these sites mimics the effects of ATP at a low-affinity regulatory site with no effect on high-affinity ATP binding to the catalytic site; and (c) that in studies on detergent-solubilized preparations, it is necessary to distinguish between the effects of solubilization per se and detergent effects at the regulatory site.
Huang
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Kakar
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Askari
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A
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D004285:Dogs; D004789:Enzyme Activation; D007425:Intracellular Membranes / Q000201:enzymology*; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D013501:Surface-Active Agents; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000254:Sodium-Potassium-Exchanging ATPase