TITLE:
Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain Idiomarina loihiensis
AUTHORS:
Ilham Mardad, Tarik Baibai, Emna Ammar, Abdelaziz Soukri
KEYWORDS:
Glyceraldehyde-3-Phosphate Dehydrogenase; Idiomarina loihiensis; Purification; NAD+; Kinetics; Saline Strain
JOURNAL NAME:
Advances in Biological Chemistry,
Vol.3 No.2,
April
30,
2013
ABSTRACT: Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until
now, the characterization of the GAPDH phosphorylante was never studied. Here,
we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate
dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the
basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification
method consisted of two steps, ammonium sulfate fractionation followed by one
chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal
antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The
optimum pH of the purified enzyme was 8.5. Studies on the effect of
temperatures revealed an enzyme increasing activity of about 45?C. The
molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels
yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated
was 19 μM
and 3.1 μM, respectively. The maximal velocity of the purified enzyme was
estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity
and a final yield of approximately 32.5%.
The physicochemical properties of this GAPDH, being characterized, could
be used in further studies.