Purification and characterization of glucose 6-phosphate dehydrogenase from sheep erythrocytes and inhibitory effects of some antibiotics on enzyme activity


BEYDEMİR Ş., Çiftçi M., KÜFREVİOĞLU Ö. İ.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, vol.17, no.4, pp.271-277, 2002 (SCI-Expanded) identifier identifier identifier

Abstract

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP(+) oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2', 5'-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K-M and V-max values for NADP(+) and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4degreesC was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I-50 values were determined from Activity%-[Drug] graphs and K-i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.