Relationship between the ATPase Activity and the ATP-Induced Fluorescence Enhancement of SH-Modified Heavy Meromyosin during Its Fractional Inactivation by Vanadate plus ADP: Evidence for Heterogeneity in the Active Sites1

Abstract

We have examined whether heavy meromyosin (HMM) consists of a single kind of active site by analyzing the changes in the relative MgATPase activity and the relative amplitude of the ATP-induced fluorescence enhancement of the protein when the fraction of HMM “affinity”-labeled by vanadate plus ADP was varied. The analysis is based on a prediction that these two changes should be proportional to each other if myosin consists of a single kind of active site and generates the rate-limiting myosin^**product complex emitting enhanced fluorescence. Although the difference between these two changes was very small with native HMM, it was large with HMM in which 5 fast-reactive sulfhydryl-groups per head were pre-modified with thimerosal. The difference indicated the existence of heterogeneous active sites in the SH-modified HMM. The results were best explained in terms of the hypothesis that fifty percent of the active site splits MgATP by a mechanism giving a fluorescence enhancement whereas the other fifty percent splits MgATP by another mechanism giving no fluorescence enhancement. Two possible explanations for the existence of heterogeneous active sites in the SH-modified HMM are discussed. One assumes the pre-existence of some sort of 1: 1 heterogeneity in the micro-environment of the active sites and the other, which is considered less likely, assumes the introduction of the heterogeneity as a result of the SH-modification.