Human granulocyte 6 phosphogluconate dehydrogenase. Purification by elective elution with NADP+, immunological and kinetic properties.
Human granulocyte 6 phosphogluconate dehydrogenase has been totally purified from a single patient with chronic granulocytic leukaemia. 48 mg of protein, of specific activity 20 IU per mg of protein, have been obtained in the course of three different steps only. The overall yield was 30 p. cent and the purification was 100 folds. Purified 6 phosphogluconate dehydrogenase was homogeneous when tested in acrylamide and acrylamide SDS gel electrophoresis or in immunodiffusion. The enzyme was immunologically identical in red blood cells, blood platelets and normal leukocytes. The fixation of both substrates, NADP-+ and 6 phosphogluconate, seemed to proceed through a non ordered mechanism. NADPH was an inhibitor strictly competitive with respect to NADP-+ and non competitive with respect to 6 phosphogluconate. 2-3 Diphosphoglycerate seemed to be able to bind on both the fixation sites of NADP-+ and 6 phosphogluconate. The inhibition by ATP was competitive with 6 phosphogluconate and non competitive with NADP-+. 6 phosphogluconate dehydrogenase was inactivated by SH reagents and was partially protected against this inactivation by both substrates. Both substrates protected the enzyme against thermal inactivation. The influence of ionic strength, pH and ions have been studied, and the results have been compared to those reported by other authors for erythrocyte enzyme.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D003864:Depression, Chemical; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D004912:Erythrocytes / Q000201:enzymology; D006098:Granulocytes / Q000201:enzymology*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D005779:Immunodiffusion; D007700:Kinetics; D007951:Leukemia, Myeloid / Q000201:enzymology*; D007962:Leukocytes / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D009994:Osmolar Concentration; D010734:Phosphogluconate Dehydrogenase / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D011189:Potassium Chloride / Q000494:pharmacology
D004791:Enzyme Inhibitors; D006902:Hydroxymercuribenzoates; D009249:NADP; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D010734:Phosphogluconate Dehydrogenase