Molecular Pharmacology, Vol 6, 557-566, Copyright © 1970 by the American Society for Pharmacology and Experimental Therapeutics

Inhibitory Sites on Sodium- and Potassium-Activated Adenosine Triphosphatase for Chlorpromazine Free Radical and Ouabain

¹ Department of Pharmacology, Michigan State University, East Lansing, Michigan 48823

The effects of chlorpromazine free radical and/or ouabain on (Na⁺ + K⁺)-ATPase activity [Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, EC 3.6.1.3], on free sulfhydryl concentration, and on the ability to bind ³H-ouabain were studied in deoxycholate- and sodium iodide-treated rat brain microsomal fractions to determine the inhibitory sites of these compounds on (Na⁺ + K⁺)-ATPase. The treatment of the enzyme preparation with chlorpromazine free radical resulted in inhibition of (Na⁺ + K⁺)-ATPase activity and a proportionate decrease in free sulfhydryl concentration. Chlorpromazine, per se, affected neither free sulfhydryl concentration nor enzyme activity. Incubation of the enzyme with ouabain, NaCl, MgCl₂, and Tris-ATP at 37° for 20 min resulted in irreversible inhibition of the enzyme activity. Free sulfhydryl concentration was unaffected. Prior treatment with ouabain did not prevent chlorpromazine free radical from reacting with sulfhydryl groups of the enzyme preparation. Prior treatment with chlorpromazine free radical also did not prevent ouabain from reacting with the enzyme but did prevent p-hydroxymercuribenzoate from reacting with the inhibited enzyme, indicating that chlorpromazine free radical and p-hydroxymercuribenzoate inhibit the enzyme by interacting with free sulfhydryl groups whereas ouabain reacts at a different site. The ability of chlorpromazine free radical to inhibit ³H-ouabain bindng on (Na⁺ + K⁺)-ATPase was considerably less than its ability to reduce (Na⁺ + K⁺)-ATPase activity. We conclude that chlorpromazine free radical inhibits enzyme activity by interacting with sulfhydryl groups on the (Na⁺ + K⁺)-ATPase whereas ouabain is bound to a different site on the enzyme.

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ACKNOWLEDGMENT We thank Mrs. Lillian C. Edney for expert technical assistance.