Purification and characterization of glucose 6-phosphate dehydrogenase from sheep erythrocytes and inhibitory effects of some antibiotics on enzyme activity.
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 KM and Vmax 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 +4 degrees C 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. I50 values were determined from Activity%-[Drug] graphs and Ki values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.
Beydemir
Sükrü
S
Atatürk University, Arts and Science Faculty, Department of Chemistry, 25240 Erzurum, Turkey.
Ciftçi
Mehmet
M
Küfrevioğlu
O Irfan
OI
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004912:Erythrocytes / Q000201:enzymology*; D005839:Gentamicins / Q000494:pharmacology; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008970:Molecular Weight; D010400:Penicillin G / Q000494:pharmacology; D012756:Sheep
D000900:Anti-Bacterial Agents; D005839:Gentamicins; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase; D010400:Penicillin G