Regulation of Pyrimidine Ribonucleoside Incorporation in Isolated Bone Cells: STIMULATION BY INSULIN AND BY 2,3-DIHYDROXY-1,4-DITHIOBUTANE (DITHIOTHREITOL)

Abstract

Abstract We have extended our previous observation that insulin enhances the incorporation of pyrimidine ribonucleosides into nucleotide pools and into RNA in bone cells (Peck, W. A., and Messinger, K., J. Biol. Chem., 245, 2722 (1970)), and have examined the role of cellular sulfhydryl groups in this effect. Stimulation was not due to impurities in the insulin preparations used. Porcine single component and low zinc amorphous insulins were as effective as zinc-containing crystalline insulin when compared on a weight basis. Isolated A chains were ineffective, and the stimulatory ability of insulin was markedly diminished by previous exposure to anti-insulin antiserum. Dithiothreitol, in concentrations ranging from 0.6 to 2.0 mm, also stimulated uridine incorporation. Like insulin, dithiothreitol exerted its major effect on pyrimidine rather than purine ribonucleoside incorporation, appeared to stimulate uridine phosphorylation rather than uptake, was effective within 15 min of treatment, acted even in the absence of metabolizable extracellular substrates and at high as well as low starting concentrations of uridine in the incubation medium. The effects of insulin and dithiothreitol were additive in concentrations which caused less than maximal stimulation, but were nonadditive in maximal stimulatory concentrations. Their effects differed significantly in that dithiothreitol stimulation, but not that of insulin, was still present 45 min after cessation of treatment. Dithioerythritol, the erythro isomer of dithiothreitol, was equally effective, but other thiols (reduced glutathione, thioglycolate, cysteine, 2-mercaptoethanol, and 2,3-dimercaptopropanol), as well as disulfide-containing hormones (arginine and lysine vasopressin), were nonstimulatory. However, the effect of dithiothreitol was related to its sulfhydryl groups, since it was abolished by their prior (a) oxidation or (b) blockage with N-ethylmaleimide. These results suggest that cellular sulfhydryl groups participate in pyrimidine ribonucleoside phosphorylation and in the insulin stimulation of this pathway. The specific molecular configuration of dithiothreitol (dithioerythritol) may allow its sulfhydryl groups to interact with an insulin-sensitive site.