Effect of deuterium oxide on actomyosin motility in vitro

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D2O) and water (H2O) to examine the effect of D2O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D2O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H2O assay solution, but the maximum velocity (4.1 +/- 0.5 mum/s, n = 11) at pD 8.5 in D2O was about 60% of that (7.1 +/- 1.1 mum/s, n = 11) at pH 8.5 in H2O. The K-m values of 95 and 80 muM and V-max values of 3.2 and 5.1 mum/s for the D2O and H2O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The K-m value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 muM [actin] in H2O to 33 muM [actin] in D2O without any significant changes in V-max (9.4 s(-1) in D2O and 9.3 s(-1) in H2O). The rate constants of ADP release from the acto-SI-ADP complex measured by the stopped flow method were 361 +/- 26 s(-1) (n = 27) in D2O and 512 +/- 39 s(-1) (n = 27) in H2O at 6 degreesC. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D2O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D2O. (C) 2001 Elsevier Science B.V. All rights reserved.