Inactivation In vivo of Glutamine Synthetase and NAD-specific Glutamate Dehydrogenase: Its Role in the Regulation of Glutamine Synthesis in Yeasts

Abstract

The food yeast, Candida utilis, is a highly adaptable organism possessing efficient and flexible pathways for the assimilation and interconversion of nitrogenous compounds. Earlier studies (Sims, Folkes & Bussey, 1968) indicated some of the ways by which appropriate rates of glutamate and glutamine synthesis could be maintained under different nutritional conditions. We now consider some aspects of the regulation of the enzymes glutamine synthetase (GS) and the deaminating NAD-specific glutamate dehydrogenase Candida utilis is similar to Saccharomyces cerevisiae (Kohlhaw, Dragert & Holzer, I 965) in that when it is grown on glutamate or some other amino acids there is extensive derepression of GS. There is similar derepression of GDHNAD, the enzyme that under such conditions probably supplies the ammonia required for amide synthesis (Hierholzer & Holzer, I 963). The cellular concentration of free ammonia is very low, and it is believed that the increased synthesis of the two enzymes occurs directly or indirectly in response to this reduced availability of ammonia. We have found that the addition of either ammonia or glutamine to yeast so adapted can result in the rapid and extensive inactivation of both GS and GDHNA,. Our reasons for believing that inactivation of both enzymes is necessary for the readaptation of these yeasts to increased supplies of ammonia are discussed. (GDHNAD). METHODS Organisms. Candida utilis (originally designated C. utilis BP 60) was obtained from Professor Lillian Hawker, Department of Botany, University of Bristol ; Saccharomyces cerevisiae x 2 I 80 from Dr W. Duntze, Biochemisches Institut, Universitat Freiburg im Breisgau; and Torulopsis candida N C Y C~~~ from Dr J. A. Barnett, ARC Food Research Institute, Norwich. Growth conditions. Batch cultures were grown at 27' in a Gallenkamp orbital shaker. The medium used was of the following composition: glucose, 55 mM;

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

“…Transferase activity was measured by the y-glutamyl transferase reaction (Ferguson & Sims, 1971). One unit of enzyme activity is defined as the formation of 1 pmol yglutamylhydroxamate min-' at 37 "C. Biosynthetic activity was measured either according to Bender et al (1977), except that cetyltrimethylammonium bromide was omitted, or with the coupled assay system described by Kingdon et al (1968), or by the formation of radioactive glutamine, as described by Prusiner & Milner (1970).…”

The involvement of glutamate dehydrogenase and glutamine synthetase/glutamate synthase in ammonia assimilation by the basidiomycete fungus Stropharia semiglobata

“…In all cases, activity was determined by following the oxidation of NADH at 340nm (30 "C). GOGAT (EC 1.4.7.1) activity was assayed both by the transferase and by the synthetase reactions, as described by Ferguson & Sims (1971. Specific activities of the enzymes are expressed as nmol reduced coenzyme oxidized or y-glutamylhydroxamate formed min-l (mg protein)-'.…”

Regulation of Glutamine Synthetase Isoenzymes in Rhizobium leguminosarum biovar viceae

“…Bacteria (50 ml volumes, unless otherwise stated) were harvested in the exponential phase by centrifugation at 4"C, washed with 0.9% NaCl and frozen as a pellet at -20°C. The pellet was Regulation of GS isoenzymes in R. leguminosarum 63 1 resuspended in 1 ml of extraction buffer (Ferguson & Sims, 1971), sonicated twice for 45 s, with a 1 min interval at 0 "C, and centrifuged at 27000g at 4 "C for 20 min to remove cell debris. Heat treatment.…”

“…For the y-glutamyltransferase activity of GSII, we used the method described by Ferguson & Sims (1971) and measured activity in samples of cell extract before and after heat treatment to assay the heat-stable fraction relative to total activity. The y-glutamyltransferase activity of GSI was assayed according to the protocol of Shapiro & Stadtman (1970) at pH 7.43 (the isoactivity point of the adenylylated and non-adenylylated form of GSI; see below) on cell extracts subjected to heat treatment.…”