A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens.
This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.
Ulusu
N N
NN
Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Kus
M S
MS
Acan
N L
NL
Tezcan
E F
EF
D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D008970:Molecular Weight; D013816:Thermodynamics
D005954:Glucosephosphate Dehydrogenase