動物細胞抽出液

Concerning the estimation of DNA synthesizing enzyme (DNA nucleotidyltransferase, DNA polymerase) in extracts of animal cells, some problems in preparation of cell extracts and estimation of their enzymatic activities were investigated. (1) HeLa cells were disrupted by osmotic shock and centrifuged with or without addition of KCl at 0.05 M. Although the concentration of salts in the final extracts was adjusted to be the same, higher activiy of DNA nucleotidyltransferase and DNase in both specific and total activities was estimated in the extract with KCl, suggesting that KCl protects the activity. (2) Activities of DNA nucleotidyltransferase and DNase were compared in extracts of HeLa cells prepared after disruption of cells by osmotic shock or 20 KC sonic treatment. By osmotic disruption, the extract of higher specific activity of DNA nucleotidyltransferase and lower one of DNase were obtained. However, the extract after sonication contained higher total activities of both enzymes. (3) Similar effect of KCl and sonic treatment in preparing cell extract was also established from Landschutz ascites tumor cells. (4) The cell extract was obtained by centrifuging the disrupted cell suspension of small amount of HeLa cell at 105,000 × g for 15 or 30 minutes, contrary to the common condition of centrifugation at 105,000 × g for 60 to 90 minutes. (5) From HeLa cells, the extracts of cell nuclei, small particles and cell sap were prepared after osmotic shock followed by differential centrifugation at isotonic sucrose concentration and sonic treatment. It was found that most of both enzymes were contained in cell sap and the lowest activities were estimated in cell nuclei. (6) The extracts of HeLa cells or ascies tumor cells were precipitated at pH 4.8 with 0.2 N acetic acid and enzyme solution of higher DNA nucleotidyltransferase activity was usually prepared. However, DNase activity was decreased by acid precipitation. (7) DNA nucleotidyltransferase of HeLa cell, especially in cell nuclei, was activated after infection with herpes simplex virus. Similar slight activation after virus infection was observed in DNase too. In addition, activating ratios varied depending on time after infection. (8) Enzyme solution was adsorbed on small pieces of filter paper and the estimation of DNA nucleotidyltransferase with the enzyme-paper was discussed. Linear incorporation of TMP was observed for 3 hours, although the activity was decreased by adsorbing the enzyme on paper and by drying it. However, the activity of DNA nucleotidyltransferase was more or less inhibited by crushed cyanogum gel, but not by non-crushed one.