Assay, purification and properties of mammalian D-2-hydroxy acid dehydrogenase

Biochem J. 1969 Oct;115(1):55-64. doi: 10.1042/bj1150055.

Abstract

1. A new method is described for the measurement of d-2-hydroxy acid dehydrogenase in samples of animal tissues. 2. The distribution of the enzyme in a number of animals was determined. Of the animal tissues tested, the most active source of the enzyme was found to be rabbit kidney cortex. 3. The enzyme was purified from rabbit kidney to a stage at which it appears to be homogeneous in the analytical ultracentrifuge and on polyacrylamide-gel electrophoresis. 4. The molecular weight was estimated by gel filtration to be approx. 102000; combination of gelfiltration data and the sedimentation coefficient gave a value of 95000. 5. The purified enzyme has a spectrum typical of a flavoprotein. The change induced in the spectrum on addition of d-malate or d-lactate suggests the formation of a flavin semiquinone. 6. Flavin can be removed by treatment with acid ammonium sulphate, and activity can be restored to the inactive apoenzyme by addition of FAD, but not of FMN or riboflavin. 7. Studies of acceptor specificity showed that the enzyme has a relatively weak d-2-hydroxy acid oxidase activity.

MeSH terms

  • Alcohol Oxidoreductases / analysis*
  • Alcohol Oxidoreductases / isolation & purification
  • Animals
  • Brain / enzymology
  • Cattle
  • Chromatography, Gel
  • Electrophoresis
  • Flavin Mononucleotide / pharmacology
  • Flavin-Adenine Dinucleotide / pharmacology
  • Flavins
  • Guinea Pigs
  • Kidney / enzymology*
  • Lactates / pharmacology
  • Liver / enzymology
  • Malates / pharmacology
  • Methods
  • Molecular Weight
  • Muscles / enzymology
  • Myocardium / enzymology
  • Quaternary Ammonium Compounds / pharmacology
  • Rabbits
  • Rats
  • Riboflavin / pharmacology
  • Sheep
  • Spectrum Analysis
  • Spleen / enzymology
  • Swine
  • Ultracentrifugation

Substances

  • Flavins
  • Lactates
  • Malates
  • Quaternary Ammonium Compounds
  • Flavin-Adenine Dinucleotide
  • Flavin Mononucleotide
  • Alcohol Oxidoreductases
  • Riboflavin