Kinetic properties of hexose-monophosphate dehydrogenases. II. Isolation and partial purification of 6-phosphogluconate dehydrogenase from rat liver and kidney cortex.
6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely isolated (more than 95%) from glucose-6-phosphate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 60-75% ammonium-sulphate fractionation, by which both hexose-monophosphate dehydrogenases activities were separated, and finally the protein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of the isolated partially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-Menten kinetic, with no evidence of co-operativity. The optimum pH for both liver and kidney-cortex 6PGDH was 8.0. The Km values of liver 6PGDH for 6-phosphogluconate (6PG) and for NADP were 157 microM and 258 microM respectively, while the specific activity measured at optimum conditions (pH 8.0 and 37 degrees C) was 424.2 mU/mg of protein. NADPH caused a competitive inhibition against NADP with an inhibition constant (Ki) of 21 microM. The Km values for 6PG and NADP from kidney-cortex 6PGDH were 49 microM and 56 microM respectively. The specific activity at pH 8.0 and 37 degrees C was 120.7 mU/mg of protein. NADPH also competitively inhibited 6PGDH activity, with a Ki of 41 microM. This paper describes a quick, easy and reliable method for the separation of the two dehydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurements of their activities.
Corpas
F J
FJ
Unit of Vegetable Biochemistry, Zaidín Experimental Station [Consejo Superior de Investigaciones Científicas (CSIC)], Granada, Spain.
García-Salguero
L
L
Barroso
J B
JB
Aranda
F
F
Lupiáñez
J A
JA
D000818:Animals; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D005942:Gluconates / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009249:NADP / Q000378:metabolism; D010427:Pentose Phosphate Pathway; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D005942:Gluconates; D009249:NADP; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; C008884:6-phosphogluconic acid