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| Studies on Purification and Properties of Rabbit Muscle Glycerol 3-Phosphate Dehydrogenase |
| WANG Zhen-wei, LI Gang-rui, LI Lin-li, WANG Shuai-kun, MENG Yan-fa |
| College of Life Sciences, Key Laboratory of Bio-Resources and Eco-Environment Ministry of Education, Sichuan University, Chengdu 610064, China |
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Abstract Objective: To explore the purification and properties of glycerol 3-phosphate dehydrogenase(GPDH) from rabbit muscle and provide theoretical support for its applications on enzyme linked diagnosis reagents in vitro. Methods: Glycerol 3-phosphate dehydrogenase from rabbit muscle was purified by ammonium sulfate fractional precipitation, DEAE-sepharose, Blue-sepharose and Hydroxyapatite. Sephacryl S-200HR chromatography and gradient PAGE were used to determine its molecular weight, kinetic properties such as effect of pH, temperature, substrate concentration and chemicals were also determined. Results: The purified enzyme showed a prominent single band on PAGE, and its molecular weight was estimated to be 115～122 kDa. The enzyme was stable below 45℃ and pH 6.0～9.0 with maximal activity at 45℃, pH 9.0. Km value of the enzyme was calculated to be 7.4×10-3mol/L for substrate glycerol 3-phosphate, and to be 1.47×10-4mol/L for substrate NAD+. Ba2+, Mn2+, Fe2+, Al3+, Cu2+, Ni2+, Ag+, Hg2+, NaN3 and EDTA inhibited the activity of GPDH at various degree, whereas Mg2+, Ca2+, Co2+, Zn2+ could stimulate the enzyme activity, NaF had no obvious effect on glycerol 3-phosphate dehydrogenase.
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Received: 24 August 2012
Published: 25 February 2013
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