MUHAMMET GÜLER, VEDAT TÜRKOĞLU, MEHMET RIZA KIVANÇ

Properties and some in vitro studies of 6-phosphogluconate dehydrogenase purified from the liver of Chalcalburnus tarichi, the only fish living in Lake Van's highly alkaline water (pH 9.8)

In this study, some enzymology parameters and some antibiotics affecting the enzyme activity of 6-phosphogluconate dehydrogenase (6PGD) extracted from t the livers of fish from Lake Van (\textit{Chalcalburnus tarichi}) were investigated because it is an important enzyme-producing NADPH, a reductive power, protecting the cell against the oxidative agents by producing reduced glutathione. The crude enzyme solution was obtained by using the affinity chromatography method. The native molecular weight of the enzyme is $\sim $90,000 $\pm $ 3000 Da and it is composed of two subunits with identical molecular weights of $\sim $46,000 Da as exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH and temperature for optimal conditions of 6PGD were about 8.5 and 40 $^{\circ}$C, respectively, and the enzyme showed optimal activity in 40 mM ammonium sulfate solution. In addition, in vitro effects of clindamycin phosphate, streptomycin sulfate, and lincomycin antibiotics on the enzyme activity were investigated.

Anahtar Kelimeler:

Enzyme purification, affinity chromatography, antibiotic, enzyme inhibition

Properties and some in vitro studies of 6-phosphogluconate dehydrogenase purified from the liver of Chalcalburnus tarichi, the only fish living in Lake Van's highly alkaline water (pH 9.8)

Keywords:

Enzyme purification, affinity chromatography, antibiotic, enzyme inhibition,

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where ∆ OD is the increase in the absorbance at 340?nm per minute, Vc (mL) is total reaction volume, Ve (mL) is the volume of enzyme solution (diluted sample) added, f is the dilution coeﬃcient, and 6.22 is the millimolar absorption coeﬃcient of NADPH molecules.28
So as to find out the Kiconstants of the antibiotics, Lineweaver–Burk graphs were drawn. The IC50 (half maximal inhibitory concentration) values of the antibiotics were obtained from activity (%) vs. drug concentration plots. These antibiotics can bind to diﬀerent sites of the enzyme because the drugs used here are not similar to the substrate of the enzyme at the molecular level. 4. Conclusion
In this study, 6PGD has been purified 446-fold with a specific activity of 23,173 EU/mg protein. This work has demonstrated that 6PGD has two subunits with identical molecular weights.17 −19