Effect of divalent cations and chelating agents on the ATPase activity of platelet contractile protein, “thrombosthenin”

Abstract

1. (1) the effect of various divalent cations and EDTA and EGTA on the pig platelet contractile protein “thrombosthenin” has been investigated. Under both low and high ionic strength conditions of assay the Ca 2+ ATPase activity greatly exceeds the Mg 2+ ATPase. With increasing ionic strength, Mg 2+ ATPase decreases and the Ca 2+ ATPase is greatly increased due to dissociation of the actomyosin-like component of thrombosthenin. Mg 2+ inhibits the Ca 2+ ATPase of thrombosthenin and inhibition can be detected at 25 μmolar concentrations of Mg 2+ in the presence of 3 m M Ca 2+. 2. (2) At low chelate concentrations (<0.5 m M), EDTA enhances and EGTA inhibits thrombosthenin ATPase, measured in the absence of added divalent cations. At higher chelate concentrations (>0.5 m M) both EDTA and EGTA were inhibitory towards the Ca 2+ ATPase activity measured in the presence of 3 m M Ca 2+. The findings suggest that thrombosthenin preparations isolated by high ionic strength extraction and low ionic strength precipitation, contain both these divalent cations endogenously associated. 3. (3) ATPase activity profiles with various divalent cations (Ca 2+, Mg 2+, Sr 2+, Mn 2+, Be 2+, Ba 2+, Cd 2+, Co 2+ and Zn 2+) revealed that only Ca 2+ and Mn 2+ significantly activated, Mg 2+ and Sr 2+ weakly activated and Cd 2+, Co 2+ and Zn 2+ were all inhibitory (80–100%, at cation concentrations equimolar with substrate ATP-3 m M). 4. (4) Although on the basis of the ATPase activities, thrombosthenin contains some actomyosin-like protein in the complex the findings suggest that it also includes a considerable excess of an unassociated myosin-like component, rich in Ca 2+ ATPase activity.