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Title: Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol

Abstract

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.

Authors:
; ; ;
Publication Date:
Research Org.:
Lehigh Univ., Bethlehem, PA
OSTI Identifier:
5089955
Report Number(s):
CONF-8606151-
Journal ID: CODEN: FEPRA
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 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
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 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)

Citation Formats

Merkel, J R, Chen, S M, Osinchak, J, and Trumbore, M. Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol. United States: N. p., 1986. Web.
Merkel, J R, Chen, S M, Osinchak, J, & Trumbore, M. Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol. United States.
Merkel, J R, Chen, S M, Osinchak, J, and Trumbore, M. 1986. "Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol". United States.
@article{osti_5089955,
title = {Improved purification of sn-glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae and its inhibition by ethanol},
author = {Merkel, J R and Chen, S M and Osinchak, J and Trumbore, M},
abstractNote = {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.},
doi = {},
url = {https://www.osti.gov/biblio/5089955}, journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:6,
place = {United States},
year = {Thu May 01 00:00:00 EDT 1986},
month = {Thu May 01 00:00:00 EDT 1986}
}

Conference:
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