ISSN:
1432-0983
Keywords:
Ammonium assimilation
;
Glutamate metabolism
;
Enzyme kinetics
;
Glutamate dehydrogenase
;
S. pombe
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract The initial velocity, pH and temperature optima, and Km values of Schizosaccharomyces pombe NADP-glutamate dehydrogenase (NADP-GDH: EC 1.4.1.4) have been determined. NADP-GDH was found to be specific for the substrates used in the reaction mixtures. NADP-GDH activity showed a sigmoidal response to changes in α-ketoglutarate concentrations, following Hill kinetics with a coefficient nH=2. A two-fold and a three-fold increase in activity was found in extracts of cells grown on a medium containing cytosine or histidine as a sole nitrogen source, respectively, relative to the activity found in cells grown on other sole nitrogen sources including ammonium, adenine, arginine, aspartate, asparagine, glutamate, glutamine, leucine, lysine, proline, uridine and urea. Five NADP-GDH-defective mutants were isolated on the basis of no growth on ammonium plus allantoin as sole nitrogen sources. The mutants also failed to grow on allantion alone but, in contrast, they were phenotypically indistinguishable from the wild-type growing on solid minimal medium with ammonium. Additionally, the mutants were found to grow as wild-type on minimal medium with alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline in the absence or presence of allantoin. In liquid minimal medium with ammonium as sole nitrogen source they had a slower growth than the wild-type. Normal growth was observed in cells grown on alanine, arginine, asparagine, aspartate, glutamate, glutamine, leucine, ornithine and proline. The mutants had undetectable levels of NADP-GDH activity, but retained wild-type levels of NAD-GDH, glutame synthase (GOGAT) and glutamine synthetase (GS). Mutants were found to map in two unlinked genetic loci by recombinational analysis. One locus was designated as gdh1 and was represented by four mutant alleles (gdh1-1, gdh1-2, gdh1-3 and gdh1-4), whilst the second locus, gdh2, had one mutant allele (gdh2-1). No differences were observed in growth tests or enzymic studies between these loci.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00310495
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