PURIFICATION AND PARTIAL CHARACTERIZATION OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE FROM COW ADRENAL CORTEX

Bovine adrenal glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) was purified by isoelectric precipitation, calcium phosphate gel adsorption, ammonium sulfate precipitation, and column chromatography on diethylaminoethyl Sephadex, carboxymethyl Sephadex, and Sephadex G-200. The enzyme was purified 2500-fold with a 20% yield. The average specific activity at pH 8.0 and 37° of the crystalline enzyme was 340 µmoles of oxidized nicotinamide-adenine dinucleotide phosphate (NADP+) reduced/min per mg of protein. The preparation was demonstrated to be homogeneous by diethylaminoethylcellulose column chromatography, electrophoresis on cellulose acetate and Polyacrylamide gel, velocity ultracentrifugation, and three consecutive crystallizations to a constant specific activity. The molecular weight was calculated to be 235,000, derived from a Ve/V0 ratio of 1.58 on a calibrated Sephadex G-200 column and to be 238,700 by a sedimentation constant (S2o,w) of 9.8 S on the ultracentrifuge. The optimum pH in the absence of Mg2+ was 8.5; with Mg2+it was the same but with a 20% greater activity. The Km for NADP+ at pH 8.0 and 25° in the presence of Mg2+ was 5.6 × 10−6 m as compared to a Km of 4.7 × 10−2 m for NAD+. The Km for glucose 6-phosphate with NADP+ at pH 8.0 and 25° in the presence of Mg2+ was 4.2 × 10−5 m compared to 1.9 × 10−4 m with NAD+. © 1968, American Chemical Society. All rights reserved.