Oxidation of reactive sulfhydryl groups of sarcoplasmic reticulum ATPase

Abstract

The role of reactive sulfhydryl groups of sarcoplasmic reticulum ATPase has been investigated. Incubation of ATPase with 17 mol o-iodosobenzoic acid per mol ATPase results in a 15% inhibition of Ca 2+ uptake with only a 5% loss of ATPase activity. When ATPase is treated with 15 mol KMnO 4 per mol ATPase, Ca 2+ uptake is completely inhibited. From the measurement of remaining SH groups using 5,5′-dithiobis(2-nitrobenzoic acid), it is found that the oxidation of approximately four SH groups per ATPase molecule with KMnO 4 leads to a complete loss of Ca 2+ uptake, while the oxidation of five SH groups per ATPase with o-iodosobenzoic acid results in only 15% inhibition of Ca 2+ uptake. The results of amino acid analysis indicate that KMnO 4 oxidizes the reactive SH groups to sulfonic acid groups. Among the five o-iodosobenzoic acid-reactive SH groups, at least one shows a distinct Ca 2+ dependence. Addition of o-iodosobenzoic acid to the reaction medium containing KMnO 4 does not increase the number of oxidized SH groups, indicating that both o-iodosobenzoic acid and KMnO 4 oxidize the same SH groups of the enzyme. The different effects of two oxidizing agents on sarcoplasmic reticulum ATPase eliminate the possibility of direct involvement of SH group(s) in the ATPase reaction.