Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2006-02, Vol.143 (2), p.249-255
• Main Authors: Ulusu, Nuray N., Tandogan, Berivan
• Format: Article
• Language: English
• Subjects: Aging; Animals; Chromatography, Affinity; Glucose-6-phosphate dehydrogenase; Glucosephosphate Dehydrogenase - isolation & purification; Glucosephosphate Dehydrogenase - metabolism; Hydrogen-Ion Concentration; Kidney - enzymology; Kinetic mechanism; Kinetics; Optimum pH; Optimum temperature; Ping Pong Bi Bi system; Purification; Purification and aging; Sheep; Temperature; Ultracentrifugation
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2005.11.018

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 1384-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 μMol min − 1 mg protein − 1 , respectively. The pH optimum was 7.4 and the optimum temperature was 45 °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.