Purification and properties of the flavine-stimulated anaerobic L- -glycerophosphate dehydrogenase of Escherichia coli.
The anaerobic l-alpha-glycerophosphate (l-alpha-GP) dehydrogenase of Escherichia coli was purified approximately 40-fold. The activity of the dehydrogenase, although not affected by the addition of pyridine nucleotides, was stimulated three- to fourfold by flavine adenine dinucleotide (K(m) about 10(-7)m) and up to 10-fold by flavine mononucleotide (K(m) about 10(-4)m). Maximal activity of the enzyme was found only in the combined presence of saturating concentrations of both flavines (stimulation by a factor of 10 to 15). The dependence of the rate of the reaction on the concentration of l-alpha-GP was complex in the presence of both flavines, but in the presence of flavine adenine dinucleotide alone the kinetics were of the Michaelis-Menten type with the K(m) for l-alpha-GP being about 10(-4)m. The product of the reaction was identified as dihydroxyacetone phosphate, and the molecular weight of the dehydrogenase was estimated to be 80,000 +/- 10,000. Phenazine methosulfate, menadione and ferricyanide served as artificial acceptors for the dehydrogenase. The enzyme was sensitive to iodoacetate, p-chloromercuribenzoate, and N-ethymaleimide.
D000645:Ammonium Sulfate; D000693:Anaerobiosis; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000494:pharmacology*; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology*; D005993:Glycerolphosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005994:Glycerophosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D007535:Isomerases / Q000378:metabolism; D008970:Molecular Weight; D011742:Pyrimidine Nucleotides / Q000494:pharmacology; D013237:Stereoisomerism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014306:Trioses
D005994:Glycerophosphates; D007461:Iodoacetates; D011742:Pyrimidine Nucleotides; D013439:Sulfhydryl Reagents; D014306:Trioses; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D005993:Glycerolphosphate Dehydrogenase; D007535:Isomerases; D000645:Ammonium Sulfate