Calmodulin dissociation regulates brush border myosin I (110-kD-calmodulin) mechanochemical activity in vitro.
110-kD-calmodulin, when immobilized on nitrocellulose-coated coverslips, translocates actin filaments at a maximal rate of 0.07-0.1 micron/s at 37 degrees C. Actin activates MgATPase activity greater than 40-fold, with a Km of 40 microM and Vmax of 0.86 s-1 (323 nmol/min/mg). The rate of motility mediated by 110-kD-calmodulin is dependent on temperature and concentration of ATP, but independent of time, actin filament length, amount of enzyme, or ionic strength. Tropomyosin inhibits actin binding by 110-kD-calmodulin in MgATP and inhibits motility. Micromolar calcium slightly increases the rate of motility and increases the actin-activated MgATP hydrolysis of the intact complex. In 0.1 mM or higher calcium, motility ceases and actin-dependent MgATPase activity remains at a low rate not activated by increasing actin concentration. Correlated with these inhibitions of activity, a subset of calmodulin is dissociated from the complex. To determine if calmodulin loss is the cause of calcium inhibition, we assayed the ability of calmodulin to rescue the calcium-inactivated enzyme. Readdition of calmodulin to the nitrocellulose-bound, calcium-inactivated enzyme completely restores motility. Addition of calmodulin also restores actin activation to MgATPase activity in high calcium, but does not affect the activity of the enzyme in EGTA. These results demonstrate that in vitro 110-kD-calmodulin functions as a calcium-sensitive mechanoenzyme, a vertebrate myosin I. The properties of this enzyme suggest that despite unique structure and regulation, myosins I and II share a molecular mechanism of motility.
Collins
K
K
Department of Biology, Massachusetts Institute of Technology, Cambridge.
Sellers
J R
JR
Matsudaira
P
P
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology*; D002645:Chickens; D004533:Egtazic Acid / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*
D000199:Actins; D002147:Calmodulin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D002118:Calcium