Halenaquinol, a natural cardioactive pentacyclic hydroquinone, interacts with sulfhydryls on rat brain Na+,K+-ATPase

Halenaquinol inhibited the partial reactions of ATP hydrolysis by rat brain cortex Na+,K+-ATPase, such as [3H]ATP binding to the enzyme, Na+-dependent front-door phosphorylation from [γ-33P]ATP, and also Na+- and K+-dependent E1↔E2 conformational transitions of the enzyme. Halenaquinol abolished the positive cooperativity between the Na+- and K+-binding sites on the enzyme. ATP and sulfhydryl-containing reagents (cysteine and dithiothreitol) protected the Na+,K+-ATPase against inhibition. Halenaquinol can react with additional vital groups in the enzyme after blockage of certain sulfhydryl groups with 5,5′-dithio-bis-nitrobenzoic acid. Halenaquinol inhibited [3H]ouabain binding to Na+,K+-ATPase under phosphorylating and non-phosphorylating conditions. Binding of fluorescein 5′-isothiocyanate to Na+,K+-ATPase and intensity of fluorescence of enzyme tryptophanyl residues were decreased by halenaquinol. We suggest that interaction of halenaquinol with the essential sulfhydryls in/or near the ATP-binding site of Na+,K+-ATPase resulted in a change of protein conformation and subsequent alteration of overall and partial enzymatic reactions.