Purification and characterization of glycerol-3-phosphate dehydrogenase (NAD+) in the salt-tolerant yeast Debaryomyces hansenii.

The NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of the salt-tolerant yeast Debaryomyces hansenii was purified by poly(ethylene glycol) precipitation and a combination of chromatographic procedures. The enzyme existed in two forms with different ionic characters and specific activity. On SDS-polyacrylamide gel electrophoresis, both forms yielded one predominant band with an apparent molecular weight of 42,000. The specific activity of the enzyme was dependent on the concentration of the enzyme and on the ionic strength of the dissolving medium. All ions tested stimulated the enzyme activity in the ionic strength range 0-100 mM, with glutamate yielding the highest activity. Above these concentrations, the dehydrogenase showed high tolerance for glutamate in concentrations up to 0.9 M, whereas malate, sulfate and chloride were inhibitory. Enzyme activity showed little sensitivity to the type of cation present and was only slightly affected by 5 M glycerol. The true Km values for the substrates were 6.6 microM for NADH, 130 microM for dihydroxyacetone phosphate, 0.3 mM for NAD and 1.2 mM for glycerol-3-phosphate, and the enzyme showed specificity for these four substrates only. It is proposed that the enzyme functions in cellular osmoregulation by providing glycerol 3-phosphate for the biosynthesis of glycerol, the main compatible solute in D. hansenii, and that the enzyme is well adapted to function in yeast cells exposed to osmotic stress.

Further investigations on M. leprae. Growth promotion and inhibition by organic substances and observations on antagonistic and syntergistic effects.

The multiplication of 2 strains of M. leprae on a medium containing a sonic extract (SE), prepared from M. smegmatis, was promoted by cysteine, tryptophane and dimethylasulfoxide (DMSO), while glutamic acid, glutamine and histidine exerted variable effects. The final effects of glutamic acid and glutamine were determined by the total concentration of both compounds together. The presence of cysteine and glutamic acid alone or together with DMSO abolished all inhibitory effects. Desferal did not enable the multiplication of M. leprae on media devoid of SE prepared from M. smegmatis. However with SE and 0.005 per cent and 0.002 per cent concentrations of Desferal its initial growth was accelerated. Its final counts, noted after an 8-month incubation, did not exceed those observed without Desferal. Puring and pyrimidine compounds promoted markekly the multiplication of M. leprae (counts greater than 3 times 10-7/ml). The highest counts were observed with pyrimidines (thymine, thymidine, cytosine) applied single or combined.