Ca binding of intestinal smooth muscle myosin B

By means of both centrifugation and filtration techniques, the Ca binding activity of intestinal myosin B was studied. The binding capacity of myosin B was Ca dependent and was approximately linear when the concentration of Ca in the medium ranged from 10(-4) to 10(-7) M. The Ca sensitivity of ATPase activity in the same range of Ca concentration exhibited a sigmoid curve. The Scatchard plot of Ca binding showed that intestinal myosin B had at least two types of binding sites. One of these was defined as a high affinity site with an apparent affinity constant of 2.5 x 10(6) M-1. The other was supposed to be a low affinity site of Ca binding. Mild trypsin treatment reduced the Ca binding capacity of intestinal myosin B by 1.45-2.44 nmol/mg protein. These values are approximately the concentration of the high affinity Ca binding sites in the intestinal myosin B. A major concern regarding the effect of trypsin is that the reduction of Ca binding surely accompanied the elimination of Ca sensitivity of myosin B ATPase activity. From these results, it seems likely that the high affinity sites of Ca binding identified in this study are based on the troponin-like component included in intestinal myosin B.