عزل أنزيمي كلوتامين سنثيتيز و كلوتاميت ديهيدروجينيز من نسيج كلى إنسان سليم و آخر مصاب بسرطان خلايا الكلى

الملخص EN

The research included isolation of glutamine synthetase and glutamate dehydrogenase from normal & tumor human kidney’s tissue using different biological techniques, These included ammonium sulfate precipitation, dialysis, gel filtration chromatography on sephadex G-75 and sephadex G-200.

It was shown that, the comparative molecular weight of the second proteinous peak (produced from sephadex G-200 colum, also it contain glutamine synthetase and glutamate dehydrogenase activity) for normal & tumor kidney’s tissue was found to be (304,000 ± 3,000 Da) and (297,000 ± 3,000 Da) respectively, And the purification of glutamine synthetase and glutamate dehydrogenase in this peak was (42.48 fold) and (42.29 fold) respectively for normal kidney’s tissue, (92.23 fold) and (86.13 fold) respectively for tumor kidney’s tissue.

Further more, the comparative molecular weight of the subunit of glutamine synthetase and glutamate dehydrogenase were determined by sodium dodecyl sulfate-polyacrylamide electrophoresis techniques for normal and tumor kidney’s tissue and found to be (42,600 Da) and (53,900 Da) respectively .

The results also showed that the optimum conditions of glutamine synthetase were obtained using imidazole-HCl (100 mM) as a buffer at pH (7.2), (45 C°) and (15 mM) of glutamate as a substrate.

Using Linweaver-Burk plot, it was found that Vmax and Km have the values of (0.0818 U / ml), (11.97 mM) respectively.

The effect of ammonium chloride and adenosine-5́-triphosphate concentration on the enzyme activity were also preformed.

The optimum concentration of ammonium chloride is (1 mM) with Vmax (0.065 U / ml) and Km (0.419 mM).

On the other hand, the optimum concentration of adenosine-5́-triphosphate is (4 mM) with Vmax (0.0609 U / ml) and Km (1.64 mM).

Finally, The optimum conditions of glutamate dehydrogenase were obtained using Tris-HCl (100 mM) as a buffer at pH (8.8), (40 C°) and (50 mM) of glutamate as a substrate.

Using Linweaver-Burk plot, it was found that Vmax and Km have the values of (0.6916 U / ml), (29.5 mM) respectively.

The effect of nicotinamide adenine dinucleotide concentration on the enzyme activity was also preformed.

The optimum concentration of nicotinamide adenine dinucleotide is (4 mM) with Vmax (0.424 U / ml) and Km (0.91 mM).