Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate Tetrahymena thermophila

In the present study, we purified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase （GAPDH） which is involved in cellular energy production and has important housekeeping functions, from the ciliate Tetrahymena thermophila using a three-step procedure. The enzyme was purified -68 folds by ammonium sulfate precipitation, followed by two steps of column chromatography （DEAE-cellulose and Mono-S）. The puri- fied enzyme is a homotetramer with a molecular weight of -120kDa. Isoelectric focusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8. Western blot analysis showed a single 32-kDa band corresponding to the enzyme subunit using a monospecific polyclonal antibody against the T. thermophila GAPDH. The maximum of enzyme activity occurred at pH 8.0 and at 30-35℃. The apparent Km values for both NAD＋ and D-glyceraldehyde-3-phosphate were 0.102± 0.012 and 0.360 ± 0.018 mM, respectively. The maximal velocity （Vmax） was 39.40 ± 2.95 U/mg. The T. thermophila GAPDH is inhibited by oxidative and nitrosative stress reagents.