The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells.
Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees 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 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 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 molecular weight 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 also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, 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.
D000199:Actins / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002286:Carcinoma, Ehrlich Tumor; D055598:Chemical Phenomena; D002621:Chemistry; D003571:Cytochalasin B / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000494:pharmacology; D005782:Gels; D051379:Mice; D009218:Myosins / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000032:analysis; D009711:Nucleotides / Q000494:pharmacology; D013696:Temperature; D014020:Tissue Extracts / Q000032:analysis
D000199:Actins; D005782:Gels; D009363:Neoplasm Proteins; D009711:Nucleotides; D014020:Tissue Extracts; D003571:Cytochalasin B; D003851:Deoxyribonucleases; D009218:Myosins; D002118:Calcium