The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells.

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Abstract
Ehrlich ascites tumor cell [mouse] extracts form a gel when warmed to 25.degree. C at pH 7.0 in sucrose solution and the gel rapidly becomes a sol when cooled to 0.degree. C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl and a reducing agent. Gelation was inhibited reversibly by 0.5 .mu.M free Ca2+, and 25-50 ng/ml of either cytochalasin B or D, but was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of 6 major proteins with MW > 300,000, 270,000, 89,000, 51,000, 48,000 and 42,000 daltons. The last component was identified as cell actin because it had the same MW as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter and it restored gelation after it inhibition by DNase I. Gelation apparently depends on actin. The extracts showed temperature-dependent, cytochalasin-sensitive and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.