KINETIC-PROPERTIES OF HEXOSE-MONOPHOSPHATE DEHYDROGENASES .2. ISOLATION AND PARTIAL-PURIFICATION OF 6-PHOSPHOGLUCONATE DEHYDROGENASE FROM RAT-LIVER AND KIDNEY CORTEX

6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cor tex 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 centrifugati on, 60-75% ammonium-sulphate fractionation, by which both hexose-monop hosphate dehydrogenases activities were separated, and finally the pro tein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate a ny contaminating metabolites. The kinetic properties of the isolated p artially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-M enten 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 6PG DH for 6-phosphogluconate (6PG) and for NADP were 157 mu M and 258 mu M respectively, while the specific activity measured at optimum condit ions (pH 8.0 and 37 degrees C) was 424.2 mU/mg of protein. NADPH cause d a competitive inhibition against NADP with an inhibition constant (K -i) of 21 mu M. The Km values for 6PG and NADP from kidney-cortex 6PGD H were 49 mu M and 56 mu M respectively. The specific activity at pH 8 .0 and 37 degrees C was 120.7 mU/mg of protein. NADPH also competitive ly inhibited 6PGDH activity, with a K-i of 41 mu M. This paper describ es a quick, easy and reliable method for the separation of the two deh ydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurement s of their activities.