Chicken gizzard heavy meromyosin that retains the two light-chain components, including a phosphorylatable one

Abstract

A method was developed to obtain a preparation of chicken gizzard heavy meromyosin (HMM) that retains the two light-chain components of parent myosin: the 20,000-dalton and 17,000-dalton light-chains. The HMM preparation was also shown to retain two characteristics of the ATPase activity of the parent myosin: the characteristic effect of phosphorylation of the 20,000-dalton light-chain component on the ATPase activity, and the characteristic dependence of the ATPase activity on the KCl concentration. 1. Two distinct stages were observed in the Mg-ATPase reaction catalyzed by gizzard HMM and rabbit skeletal actin in the presence of gizzard "native" tropomyosin (NTM) and Ca2+ ions: an early lag phase, in which the reaction rate gradually increased, and a subsequent steady state, in which the reaction proceeded at a high, constant rate. Urea-gel electrophoresis revealed that the 20,000-dalton light-chain component was gradually phosphorylated in the lag phase, and was fully phosphorylated in the steady state. It was also observed that addition of EGTA (to remove Ca2+ ions) at various times in the lag phase caused neither a further increase nor a decrease in the reaction rate, and that addition of EGTA in the steady state caused no change in the reaction rate. These observations imply that the ATPase activity increased as the amount of phosphorylated 20,000-dalton light-chain component increased, and also that Mg-ATPase of acto-phosphorylated HMM was no longer calcium-sensitive. 2. The Mg-ATPase activity of HMM in the presence of gizzard NTM and Ca2+ ions or EGTA was studied as a function of the concentration of rabbit skeletal actin. The maximal activity (Vmax) and the apparent affinity constant of acto-HMM (KA) were thus estimated from the double-reciprocal plot of Eisenberg-Moos: the Vmax and KA values for phosphorylated HMM (in the presence of Ca2+ ions) were 5 S(-1) and 5.5 mg/ml actin, respectively, and the Vmax value for unphosphorylated HMM (in the presence of EGTA) was 0.3 S(-1), assuming that the KA value with unphosphorylated HMM is equal to that with phosphorylated HMM.