MODULATION OF THE SUBSTRATE BINDING-SITES OF PLATELET MYOSIN BY ACTIN

Studies were undertaken to compare steady-state kinetics of ATPase of purified [bovine] platelet actomyosin and myosin free of actin. Actomyosin exhibited highly sigmoid kinetics with at least 2 interacting ATP or UTP binding sites. These studies were done at 0.6 M KCl where actin and myosin are supposedly dissociated in the presence of these nucleotides. When the dissociation of platelet actomyosin was actually investigated by a sucrose density gradient technique under conditions similar to those of steady-state kinetic experiments, only partial dissociation of actin from myosin was observed. This was especially true at low nucleotide concentrations where differences in the sigmoidicity of saturation curves of actomyosin and actin-free myosin were observed. In platelet actomyosin, actin apparently enhances cooperativity of nucleotide binding sites of myosin by reducing the Km for ATP or UTP. Saturation curves of platelet myosin using ATP or UTP as substrates were less sigmoidal and possessed an intermediary plateau region, when analyzed by Hill and reciprocal plots, these data indicated positive and negative cooperativity suggesting more than 2 substrate binding sites. Platelet myosin also hydrolyzed other nucleotides (the order of rates being ITP > UTP > UTP > ATP > CTP > GTP). ITP kinetics diffeFed from those of ATP or UTP in that no plateau region was observed on the saturation cuHve. No ITP binding site cooperativity was seen at low substrate concentrations (up to 0.2 mM) but was instead observed at high ITP concentratin. Conformational myosin changes induced by ITP may not be necessarily be identical to those induced by ATL or UTP.