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.
Nilsson
A
A
Department of Marine Microbiology, University of Göteborg, Sweden.
Adler
L
L
D002412:Cations; D002845:Chromatography; D004361:Drug Tolerance; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D005990:Glycerol / Q000494:pharmacology; D005993:Glycerolphosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009994:Osmolar Concentration; D011188:Potassium / Q000494:pharmacology; D004718:Saccharomycetales / Q000201:enzymology*; D012492:Salts / Q000494:pharmacology*; D013379:Substrate Specificity
D002412:Cations; D005971:Glutamates; D012492:Salts; D009243:NAD; D018698:Glutamic Acid; D005993:Glycerolphosphate Dehydrogenase; D008274:Magnesium; D005990:Glycerol; D011188:Potassium