Nucleotide contents of ascites hepatoma cells and their changes induced by D-galactosamine.

SUMMARY AS-30D and Novikoff rat ascites hepatoma cells were incubated in a medium selected according to the following criteria: (a) low leakage of lactate dehydrogenase; (b) low uptake of trypan blue; (c) high adenosine phosphate content; (‘0 high adenosine triphosphate/adenosine diphosphate ratio and adenylate energy charge. A procedure for defined cooling, freezing, and deproteinization of the incubated cells has been developed. This technique allows enzymatic analy ses of nucleotide contents in both cell lines. and of uridine mono-, di (UDP), and triphosphates revealed a strong predominance of the nucleoside triphosphates. The adenylate energy charge amounts to 0.96 and 0.94 in AS-30D and Novikoff cells, respectively; the energy charge of the uridylate system is 0.94 and 0.90, respectively. The relative ratios of adeno sine phosphatesdetermined by direct enzymatic analysisare significantly higher than those obtained by chromato graphic separation of the nucleotides after labeling with adenosine-8-14C. The amount of uridine triphosphate in both cell lines is 2.6-fold higher than in liver. The analyses ofthe UDP-sugar pattern revealed a selective deficiency of U DP-glucose and of UDP-galactose. UDP-glucuronate and UDP-N-acetyl hexosamines are in the same range as in rat liver. The extremely low UDP-glucose levels in both hepatoma cell lines may explain the virtual absence of glycogen. An incubation of AS-30D cells for 2 hr in the presence of 1 mM uridine doubles their uracil nucleotide content. D-Galactosamine induces a concentration-dependent change in the distribution of uracil nucleotides. No signifi cant decrease of the sum of all uracil nucleotides is observed when the uridine phosphate content is strongly depressed. The uridine triphosphate level decreases to 30 and 15% of 1 Dedicated to the memory of Dr. the controls when AS-30D cells are incubated in the presence of 1 and 2 mM galactosamine, respectively.