On auxotrophy for pyrimidines of respiration‐deficient chick embryo cells

Abstract

Chick embryo cells treated with chloramphenicol are inherently resistant to the growth-inhibitory effect of the drug when cultured in the presence of tryptose phosphate broth. The cells were auxotrophic for pyrimidines and the presence in the broth of compounds of pyrimidine origin is demonstrated by chromatographic procedures and mass spectral analyses. They are in the form of ribonucleosides, ribonucleotides and pyrimidine-containing oligoribonucletoides. To understand the mechanism responsible for pyrimidine auxotrophy, the activity of enzymes involved in the pyrimidine biosynthetic pathway was determined. Measurement of the conversion of dihydroorotic acid to orotic acid in cell-free extracts revealed that chloramphenicol-treated chick embryo cells are deficient in dehydroorotate dehyrogenase activity. The data in vitro are supported by studies on the nutritional requirements of the respiration-deficient cells and by the incorporation in vivo of the labeled dihydroorotic acid into the acid-insoluble fraction of the cells. Although the activity of the dehydrogenase in vitro is described by 95%, the enzyme is present in chloramphenicol-treated cells and its activity is unmasked by the artificial electron acceptor meandione. A study of the activity of other enzymes of the pyrimidine biosynthetic pathways demonstrated that their activity is comparable to that in control cells. Evidently, auxotrophy for pyrimidines results from the inhibition of the flow of electrons along the mitochondrial electron transport chain.