Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase.
Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1,384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2', 5'-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K(m) values for G-6-P and NADP(+) and V(m) were determined to be 0.041+/-0.0043 mM, 0.0147+/-0.001 mM and 28.23+/-0.86 microMol min(-1) mg protein(-1), respectively. The pH optimum was 7.4 and the optimum temperature was 45 degrees C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys 'Ordered Bi Bi' mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a 'ping pong' mechanism.
Ulusu
Nuray N
NN
Hacettepe University, Faculty of Medicine, Department of Biochemistry, 06100 Ankara, Turkey. nnulusu@hacattepe.edu.tr
Tandogan
Berivan
B
D000375:Aging; D000818:Animals; D002846:Chromatography, Affinity; D005954:Glucosephosphate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D012756:Sheep; D013696:Temperature; D014461:Ultracentrifugation
D005954:Glucosephosphate Dehydrogenase