Arginine as a Precursor of Pyrimidines in Strain L-929 Fibroblasts Infected with Pleuropneumonia-like Organisms

Abstract

All metazoan cells in culture require either arginine or citrulline for growth (1, 2).The importance of arginine is emphasized by the fact that it is rapidly depleted from the medium in many primary and established cell strains (3).The L-929 fibroblasts maintained in this laboratory and shown to harbor a pleuropneumonia-like infection also demonstrate a.n elevated requirement for this amino acid.This information, plus the facts that ornithine failed to replace t,he arginine requirement and that arginine was converted to ornithine without equivalent urea formation, prompted us to evaluate the role of the urea cycle in the metabolism of these cells.The purpose of this investigation was to determine the metabolic fates of arginine, urea, and ornithine, and to assess the enzymatic activities of arginase, arginine deiminase, carbamyl phosphate synthetase, and ornithine transcarbamylase.Experiments reported here indicate that the guanidino carbon of arginine provides a C-2 carbon source for pyrimidines. EXPERIMENTAL PROCEDUREJlaf&aZs-Cell strains' were obtained from Microbiological Associates2 and maintained in Eagle's medium (4) as described in a previous report (5).Uniformly labeled L-arginine-14C, urea-14C, uniformly labeled nL-ornithine-14C, uniformly labeled L-glutamine14C, NaH14C03, uniformly labeled nL-norleucine-14C, uniformly labeled L-proline-'4C, and labeled nL-arginine-guanidino-'4C were obtained from New England Nuclear Corporation and assayed for purity.The activity of the guanidino carbon of the uniformly labeled L-arginine was determined by degradation according to Strassman and Weinhouse (6).All experiments were corrected for isotopic impurities, which were found to range from 0.3 to 3%.The Dowex 50 microbead column (17 to 18 /JCL) of Spackman, Stein, and Moore (8), modified by the met,hod of Piez and Morris (7), was used to resolve the amino acids according to the pro-