In vitro actomyosin motility in deuterium oxide.
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 microm/sec, n=11) at pD 8.5 in D2O was about 60% of that (7.1 +/- 1.1 microm/sec, n=11) at pH 8.5 in H2O. The Km values of 95 and 80 microM and Vmax values of 3.2 and 5.1 microm/sec 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 Km value of actin-activated Mg-ATPase activity of myosin subfragment 1(S1) was decreased from 50 microM[actin] in H2O to 33 microM[actin] in D2O without any significant changes in Vmax (9.4 s(-1) in D2O and 9.3 s(-1) in H2O). The rate constants of ADP release from the acto-S1-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 degrees C. 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.
Chaen
Shigeru
S
Department of Applied Physics, College of Humanities and Sciences, Nihon University, Tokyo 156-8550, Japan.
Yamamoto
Naoto
N
Shirakawa
Ibuki
I
Sugi
Haruo
H
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D017666:Deuterium Oxide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D009068:Movement; D018482:Muscle, Skeletal / Q000378:metabolism; D009218:Myosins / Q000737:chemistry; D011817:Rabbits; D014867:Water / Q000737:chemistry
D000199:Actins; D014867:Water; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D009218:Myosins; D017666:Deuterium Oxide