Purification and characterization of glucose-6-phosphate dehydrogenase from sheep erythrocytes and eye lens, and investigation of inhibition or activation kinetics of some drugs and chemical materials on theese enzymes

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes and lens. The purification consisted of three steps, preparation of haemolysate or homogenate, ammonium sulphate fractionation and 2', 5'-ADP Sepharose 4B affinity chromatography. Erythrocytes and lens G6PD enzymes were obtained with a yield of 37.1 % and 66.8 % having a specific activity of 4.64, and 7.8 U/mg proteins respectively. Optimal pH, stable pH, Km and Vmax values for NADP+ and glucose-6- phosphate (G6-P) substrates were also determined for the two enzymes. The overall purification was about 1 189.74-fold for erythrocytes G6PD and 10,400-fold for lens G6PD. Molecular weight of sheep erythrocytes and lens G6PD were determined approximately as 119,662 dal and 56,099 dal, respectively by gel-filtration chromatography. Subunit molecular weight of sheep erythrocytes and lens G6PD were determined approximately as 66,880 dal and 54,957 dal, respectively by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Enzymatic activity was spectrofotometrically measured according to Beutler method at 340 nm. In addition, in vitro effects of some medical drugs on sheep red blood cell and lens G6PD enzymes activity were investigated. 2002 135 pages Key words: Glucose 6-phosphate dehydrogenase, drug, sheep, erythrocyte, lens n