Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes.
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 traditional 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 degrees C 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 muM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.
Laboratoire de Biochimie et Biologie Moleculaire, Universite Hassan II-Ain Chock, Faculte des Sciences Ain Chock, km 8 route d'El Jadida BP. 5366, Maarif, Casablanca, Morocco. drissmountassif@yahoo.fr
Driss
D
Mountassif
Tarik
T
Baibai
Latifa
L
Fourrat
Adnane
A
Moutaouakkil
Abdelghani
A
Iddar
M'hammed Saïd
MS
El Kebbaj
Abdelaziz
A
Soukri
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004912:Erythrocytes / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D015203:Reproducibility of Results; D013696:Temperature
D000911:Antibodies, Monoclonal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases