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 sites.
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.
Kawamura
T
T
Higuchi
W
W
Emoto
Y
Y
Tawada
K
K
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D017301:Ca(2+) Mg(2+)-ATPase; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D015879:Myosin Subfragments / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013849:Thimerosal / Q000494:pharmacology; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D000199:Actins; D015879:Myosin Subfragments; D014639:Vanadium; D013849:Thimerosal; D014638:Vanadates; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase