The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1 mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28~

Myosin was isolated from the main pulmonary artery of swine and was phosphorylated or dephosphorylated by utilizing the endogenous kinase or phosphatase, respectively. The myosins, phosphorylated to various degrees, were purified free of kinase and phosphatase activities by gel filtration on Sepharose CL-4B agarose columns. The level of actin-activated ATPase activity was dependent upon the degree of myosin light chain phosphorylation. Fully phosphorylated myosin reconstituted with actin and tropomyosin (actin/tropomyosin = 6:1) had the highest ATPase activity (0.1 umol of PJ/mg-min). The actin-activated ATPase activity showed maximal (60-65%) Ca2+ sensitivity at 2 mol of Ca2+ bound per mol of myosin. The actin-activated ATPase activity, Ca2+ binding, and Ca + sensitivity ofarterial myosin were also dependent upon Mg2+ concentration. The ATPase activity was maximal at 2-3 mM Mg2+ and, at low (0.5 mM) Mg2+ concentration, the activity was only one-third of the maximal activity. Increasing the Mg2+ above 3 mM was not associated with a further increase in ATPase activity, but the Ca2+ binding and Ca2+ sensitivity decreased with increasing Mg2+ concentration. The maximal Ca2+ sensitivity was observed at 2-3 mM Mg2+, a concentration at which the myosin bound 2 mol of Ca2+/mol. Both the ATPase activity and the Ca2+ sensitivity were more remarkable when actin that contained tropomyosin was used to activate the ATPase activity. The data indicate that calcium regulates the actinactivated ATP hydrolysis not only by its effects on the phosphorylation system but also by direct binding to the myosin.Phosphorylation of myosin is correlated with actomyosin ATPase in gizzard (1, 2), vas deferens (3), and in porcine and bovine stomach (4, 5). Using purified myosins in phosphorylated and unphosphorylated states, Chacko et aL (6) showed that the myosin ATPase measured at high ionic strength (0.5 M KCl) is the same for phosphorylated and unphosphorylated myosin.However, the actin-activated ATPase activity measured under physiological conditions is severalfold higher for phosphorylated myosin. This agrees with experiments in which platelet myosin was used (7). The degree of actin-activated ATP hydrolysis by myosin isolated from mammalian smooth muscle is linearly correlated with the amount ofphosphate covalently bound to the 20,000-dalton light chain (5). Proponents of the phosphorylation mechanism agree that Ca2+ activates actomyosin by its effect on Ca2+-dependent kinase. Kerrick and co-workers (8), using skinned fiber preparations from avian gizzard and rabbit ileum to determine the relationship between phosphorylation and muscle tension, showed that the light chain became phosphorylated when tension was developed by the muscle strips. A relationship between phosphorylation and development of tension has also been reported when intact smooth muscle strips were used (9-11). The phosphorylation and tension development were correlated linearly by using hog carotid (11).In contrast to findings that the phosphorylation is corr...

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RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polycytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing conditions. These results demonstrate that specific polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.

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