Steady-state kinetics of smooth muscle myosin

Abstract

The kinetic properties of purified chicken gizzard smooth muscle myosin, free of actin, were examined. Analysis of the steady-state kinetic data revealed an intermediary plateau region on the substrate saturation curves. These data, when analyzed by Hill and Lineweaver and Burk plots, indicate positive and negative cooperativity, suggesting at least 4 substrate binding sites. The plateau region was abolished when the kinetic measurements were made at pH 5.5 and 9.0. Positive and negative cooperative effects were absent at pH 9.0, and hyperbolic kinetics was observed. At pH 5.5, although the plateau region was abolished, the enzyme exhibited positive cooperativity of substrate binding. When either heated or urea treated enzyme was used for kinetic measurements the plateau region shifted towards a higher substrate concentration range; the cooperativity of binding sites was lost at low substrate concentrations, but was instead seen at higher concentrations; and the Vmax was doubled.