Biomedical subjects
Publications and source records attributed to P Apontoweil.
A mutation affecting the activity of the first enzyme in glutathione biosynthesis in E. coli K 12 has been mapped. The mutant allele called gshA is located by transduction in the pheA-nalB segment. The linkages with pheA and tyrA provide a convenient method for transfer of gshA to other strains and so introduce a very low level of non-protein sulfhydryl groups into the E. coli cells.
The synthesis of glutathione in Escherichia coli K 12 was studied in crude, cell-free extracts. The pH optima and the apparent Km values for the substrates have been determined for both synthesizing enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase. gamma-Glutamylcysteine synthetase was found to be approximately twice as active as glutathione synthetase. In a growing culture, the cellular level of GSH showed a considerable increase up to 6.6 mumol per ml cell pellet in the stationary growth phase. GSSG was not detectable. The levels of the enzymes remained constant, indicating that glutathione biosynthesis depends at least in the beginning on the availability of the component amino acids. The pathway is controlled by feedback inhibition and not by repression.
The thiol-oxidizing agent "diamide" (CH3)2NCON equal to NCON(CH3)2 was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic process. In one mutant, glutathione synthetase was entirely inactive. Three mutants were deficient in gamma-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide rang of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.
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