Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes  

作　　者：Driss Mountassif Tarik Baibai Latifa Fourrat Adnane Moutaouakkil Abdelghani Iddar M'Hammed Said El Kebbaj Abdelaziz S oukri 

机构地区：[1]Laboratoire de Biochimie et Biologie Moléculaire, Université Hassan II-Ain Chock, Facult6 des Sciences A~n Chock, km 8 route d'EI Jadida BP. 5366, Maarif, Casablanca, Morocco [2]Laboratoire de Physiologie et Génétique Moléculaire, Université Hassan II - Ain Chock, Faculté des Sciences Ain Chock, km 8 routed'E1 Jadida BP. 5366, Maarif, Casablanca, Morocco [3]Unité de Radio-Immuno-Analyse, Département des Applications aux Sciences du Vivant, Centre National de l'Energie, des Sciences etdes Techniques Nucléaires, BP 1382 RP, 10001 Rabat, Morocco

出　　处：《Acta Biochimica et Biophysica Sinica》2009年第5期399-406,共8页生物化学与生物物理学报（英文版）

摘　　要：A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase （GAPDH, EC 1.2.1.12） from human erythrocytes. The comparison between this rapid method （one step） and the tra- ditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of -150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide （NAD＋）. Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43℃ and 8.5, respectively. The kinetic parameters were also calculated： the Vmax was 4.3 U/mg and the Km values against G3P and NAD＋ were 20.7 and 17.8 μM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase （GAPDH, EC 1.2.1.12） from human erythrocytes. The comparison between this rapid method （one step） and the tra- ditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of -150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide （NAD＋）. Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43℃ and 8.5, respectively. The kinetic parameters were also calculated： the Vmax was 4.3 U/mg and the Km values against G3P and NAD＋ were 20.7 and 17.8 μM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.

关 键 词：glyceraldehyde-3-phosphate dehydrogenase immunoaffinity purification human erythrocytes CHARACTERIZATION 

分 类 号：Q556[生物学—生物化学] Q959.223.6