Activation of myosin ATPase by actin isolated from cultured BHK cells and the effect of gelsolin.
Activation of skeletal muscle myosin and myosin subfragment-1 (S1) by actin purified from the cytoplasm of cultured BHK cells was studied using the fluorescence of pyrene-labelled BHK F-actin and its quenching by S1 and by an enzyme-linked ATPase assay. At non-saturating concentrations, both muscle and BHK actin activated skeletal muscle myosin to the same degree: at 30 degrees C and an ionic strength of 108 mM, 1 microM actin approximately doubled the ATPase of myosin or of S1. The association between BHK actin and S1 was also followed in a fluorescence stop flow: the rate of ATP binding monitored by the loss of light scattering upon dissociation of actin was again the same for BHK and muscle actin. The similarity of activation of myosin ATPase by BHK and muscle actin at low actin concentrations (i.e. the similarity of Vmax/Km) suggests that both Vmax and Km are similar for the two types of actin. The effect of varying filament length on actin activation of myosin ATPase was examined using pig plasma or BHK gelsolin to regulate the length. For both types of actin, maximum enhancement of the actomyosin ATPase activity was observed at an actin/gelsolin ratio of about 30:1, whereas inhibition was observed at lower ratios. Both activation and inhibition of actomyosin ATPase were apparent in the absence or presence of calcium; differences were observed only in the extent and the time course of the effect.
Koffer
A
A
MRC Cell Biophysics Unit, London, U.K.
Sleep
J
J
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation; D018260:Gelsolin; D066298:In Vitro Techniques; D007700:Kinetics; D008840:Microfilament Proteins / Q000502:physiology*; D008854:Microscopy, Electron; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011721:Pyrenes
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011721:Pyrenes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium