It is known that after a short digestion with trypsin myosin A yields two major fragments. One of these fragments, called HMM, retained the actin-combining ability of the parent molecule (1, 2) and in media containing very low concentrations of salts (<0.02 M) actin increased the Mg2+ activated ATPase activity of the HMM enzyme (3). In spite of the well documented complex formation between actin and HMM, previous authors were unable to show the superprecipitation reaction (“test tube contraction”) with acto-HMM in mixtures which were found to be suitable for superprecipitation with the actomyosin complex (2, 3). It has to be emphasized, however, that the superprecipitation of actomyosin is observed only in mixtures containing low salt concentrations (<0.3 M) and under such conditions myosin A is insoluble. The HMM, unlike myosin, is also soluble in mixtures containing low concentration of salts (<0.3M) and therefore one important condition for superprecipitation was not fulfilled in the experiments of previous authors. By reducing the solubility of HMM we have studied the effect of ATP on acto-HMM suspensions. The results of these experiments are described below. Materials and Methods. The ATP was purchased from the Sigma Company, polyethylene glycol having a mol wt of 4000 was obtained from the J. T. Baker Chemical Company, all other reagents used were of reagent grade. Actin and myosin A was prepared from rabbit skeletal muscle as described by Mommaerts (4). The HMM was prepared according to the method of Szent-Gyorgyi (5). Superprecipitation was measured using the method of Ebashi (6) as described in an earlier paper (7). HMM and actin [3:1, (w/w), HMM:actin] were mixed in 0.067 M phosphate buffer at pH 6.1 and incubated at 0° at least for 15 min prior to the experiments.
Read full abstract
May 1, 2008
S W H Cowley + 3
Open Access
