Purification and Properties of an NADP-specific 6-phosphogluconate Dehydrogenase From Streptococcus Faecalis

J Biol Chem. 1975 Aug 10;250(15):6093-100.

Abstract

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.

MeSH terms

  • Amino Acids / analysis
  • Animals
  • Binding Sites
  • Chromatography, DEAE-Cellulose
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Enterococcus faecalis / enzymology*
  • Immunoelectrophoresis
  • Kinetics
  • Mathematics
  • Molecular Weight
  • NADP
  • Organ Specificity
  • Phosphogluconate Dehydrogenase / immunology
  • Phosphogluconate Dehydrogenase / isolation & purification
  • Phosphogluconate Dehydrogenase / metabolism*
  • Protein Binding
  • Rabbits / immunology
  • Species Specificity
  • Sulfhydryl Compounds / analysis
  • Time Factors
  • Ultracentrifugation

Substances

  • Amino Acids
  • Sulfhydryl Compounds
  • NADP
  • Phosphogluconate Dehydrogenase