Downloaded from www.microbiologyresearch.org by IP: 54.80.173.208 On: Mon, 03 Oct 2016 02:49:54 Journal of' General Microbiology (1 989, 131, 1425--1432. Printed in Great Britain 1425 Regulation by Ammonium of Glutamate Dehydrogenase (NADP+) from Saccharomyces cerevisiae By ELENA BOGONEZ,'? JORGINA SATRUSTEGUI1* AND ALBERT0 MACHADO* Departamento de Bioquimica y Biologia Molecular, Centro de Biologia Molecular, CSIC, Universidad A utbnoma de Madrid, Madrid-34, Spain Departamento de Bioquimica, Facultad de Farmacia, Universidad de Sevilla, Spain (Received 2 August 1984; revised 7 January 1985) The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentration increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls. INTRODUCTION The ability to utilize ammonia as sole nitrogen source is widespread among micro-organisms. Ammonia is assimilated by incorporation into glutamine or glutamate. Glutamine synthetase, an ATP-requiring enzyme, catalyses the first of these reactions, and in many micro-organisms serves together with glutamate synthase as the main ammonia-scavenging pathway which is strongly induced under nitrogen limitation (Magasanik et al., 1974; Vichido et al., 1978). Glutamate dehydrogenase catalyses the second reaction [oxoglutarate + NH,+ + NAD(P)H -+ glutamate + NAD(P)+] and is usually regarded as a high capacity/low affinity enzyme, mainly functional under unlimited ammonia supply (Barratt, 1963; Burn et al., 1974). In organisms having both aminating pathways, glutamine synthetase is drastically decreased upon an increase in ammonia supply, and amination proceeds via glutamate dehydrogenase (Stadtman & Ginsburg, 1974; Magasanik et al., 1974). In Saccharomyces cerevisiae, glutamate dehydrogenase (NADP+) (EC I .4.1.4; NADP- GDH) is the main aminating enzyme and the glutamine synthetase/glutamate synthase pathway has only minor importance in ammonia assimilation (Tempest et al., 1973; Roon et al., 1974). However, the regulatory properties of NADP-GDH are poorly understood. Evidence for specific inactivation of this enzyme has recently been reported (Satrhstegui & Machado, 1978; t Present address : Department of Biochemistry, University of California, Berkeley, CA 94720, USA. Ahhreziation : N A DP-GDH , N A DP+-dependent glutamate dehydrogenase. 0001-2126 0 1985 SGM