Difference of the Inhibitor of DNA Synthesis in Liver Extract from

The presence of a protein-like inhibitor of DNA synthesis in liver extract has been reported earlier. This inhibi tor is different from liver arginase, which is known as a potent growth inhibitor of tissue culture cells. Some evidence is also presented that the inhibition is not due to the destruction of essential nutrients in the medium but to the inhibitor's direct effect on the cells. Introduction. An extract of liver was previously found to inhibit DNA synthesisof AH-414 asciteshepatomacellsafter a short incubation period of 1—2hours (1 1). Other investigators had demonstrated, earlier, that similar liver extracts inhibited the growth of tissue culture cells; subsequent studies revealed that this inhibition of growth could be ascribed to the pres ence of arginase which hydrolyzes arginine, an essential amino acid for cell growth (1, 3, 7—9). This finding led to the imme diate question as to whether the inhibitor of DNA synthesis, mentioned above, could also be due to arginase in the liver extract; indeed, Holley (6) obtained inhibition ofDNA synthe sis by incubating cultured cells with purified liver arginase for relatively prolonged periods of time. The purpose of this communication is to show that the in hibition of DNA synthesis in malignant cells produced by short-term incubation with liver extracts is not due to the presence of arginase. Materials and Methods. Beef liver was homogenized in two volumes of 0.05 M Tris-HC1 buffer (pH 7.6) in a Waring blend er and then centrifuged at 70,000 X g for two hours in a Spinco L ultracentrifuge. The supernatant was dialyzed against io@ Mmanganeseulfateandusedastheliverextract. Arginase was extracted and purified from acetone powder of beefliver according to the method of Bach and Killips (2) with a slight modification in the final step. After the isopropanol treatment (step G of Bach and Killips), diethylaminoethyl cel lulose column chromatography was used for further purifica tion. Arginase was eluted from the column with 0.01 M Tris HCI buffer (pH 7.6) without any retention by diethylamino ethyl cellulose ; the solution eluted can be kept at —20°Cfor several weeks without significant loss of arginase activity. It was also dialyzed against iO@ M manganese sulfate. Arginase was assayed by the method of Nadai (10). The