Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound.
Brush border myosin I from chicken intestinal microvilli is a membrane-associated, single-headed myosin composed of a 119-kDa heavy chain and several calmodulin light chains. We first describe in detail a new procedure for the rapid purification of brush border myosin I in greater than 99% purity with a yield of 40%, significantly higher than for previous methods. The subunit stoichiometry was determined to be 4 calmodulin light chains/myosin I heavy chain by amino acid compositional analysis of the separated subunits. We have studied the effects of Ca2+ and temperature on dissociation of calmodulin from myosin I and on myosin I Mg2(+)-ATPase and contractile activities. At 30 degrees C the actin-activable ATPase activity is stimulated 2-fold at 10-700 microM Ca2+. Dissociation of 1 calmodulin occurs at 25-50 microM Ca2+, but this has no effect on actin activation. The contractile activity of myosin I, expressed as superprecipitation, is greatly enhanced by Ca2+ under conditions in which 1 calmodulin is dissociated. This calmodulin is thus not essential for actin activation or superprecipitation. Myosin I was found to be highly temperature-sensitive, with an increase to 37 degrees C resulting in dissociation of 1 calmodulin at below 10(-7) M Ca2+ and an additional 1.5 calmodulins at 1-10 microM Ca2+. A complete loss of actin activation accompanies the Ca2(+)-induced calmodulin dissociation at 37 degrees C. Our conclusion is that physiological levels of Ca2+ can either stimulate or inhibit the mechanoenzyme activities of brush border myosin I in vitro, with the mode of regulation determined by the number of associated calmodulin light chains.
Swanljung-Collins
H
H
Department of Biochemistry, Eastern Virginia Medical School, Norfolk 23501.
Collins
J H
JH
D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002645:Chickens; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007413:Intestinal Mucosa / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D002147:Calmodulin; D000205:Actomyosin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D002118:Calcium