Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol
An improved purification procedure yielded a homogeneous preparation of sn-glycerol-3-phosphate dehydrogenase (GPD) from commercially available baker's yeast. The enzyme had an apparent molecular weight of 42,000 by SDS-polyacrylamide gel electrophoresis. This differs from the 31,000 reported earlier on the basis of its elution from a calibrated Sepharose 6B column. When denatured by guanidine (6M) and chromatographed on a Sephadex G-100 column with 6M guanidine in 0.1M phosphate buffer, pH 6.5, containing 0.1M ..beta..-mercaptoethanol, GPD eluted with the approximately 42,000 mw proteins. S. cerevisiae GPD is an NAD-dependent oxidoreductase. With NADH as the variable substrate the GPD-catalyzed reduction of dihydroxacetone phosphate (DHAP) had a K/sub M/ of 0.018 mM and was competitively inhibited by ethanol. With DHAP as the variable substrate and NADH constant GPD catalyzed the reduction with a K/sub M/ of 0.37 mM and was noncompetitively inhibited by ethanol. The calculated K/sub i/ for the non-competitive inhibition was 3.4M. K/sub i/ for the competitive inhibition of NADH by ethanol varied with increasing concentrations of ethanol indicating a more complex mechanism than a truly competitive one.
- Research Organization:
- Lehigh Univ., Bethlehem, PA
- OSTI ID:
- 5089955
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ETHANOL
BIOLOGICAL EFFECTS
OXIDOREDUCTASES
ENZYME ACTIVITY
PURIFICATION
CHROMATOGRAPHY
ELECTROPHORESIS
GUANIDINES
MOLECULAR WEIGHT
NADH2
SACCHAROMYCES CEREVISIAE
ALCOHOLS
CARBONIC ACID DERIVATIVES
COENZYMES
ENZYMES
FUNGI
HYDROXY COMPOUNDS
MICROORGANISMS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PLANTS
SACCHAROMYCES
SEPARATION PROCESSES
YEASTS
560302* - Chemicals Metabolism & Toxicology- Microorganisms- (-1987)