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.
Cite this paper
Mardad, I. , Baibai, T. , Ammar, E. and Soukri, A. (2013) Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain Idiomarina loihiensis. Advances in Biological Chemistry, 3, 170-176. doi: 10.4236/abc.2013.32022.
 Forthergill-Gilmore, L.A. and Michels, P.A.M. (1993) Evolution of glycolisis. Progress in Biophysics and Molecular Biology, 59, 105-135.
 Sirover, M.A. (2005) New nuclear functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells. Cell Biochemistry, 95, 45-52.
 Cerff, R. (1995) The chimeric nature of nuclear genomes and the antiquity of introns as demonstrated by GAPDH gene system. In: Go, M. and Schimmel, P., Eds., Tracing Biological Evolution in Protein and Gene Structures, Elsevier, Amsterdam,
 Marangos, P.J. and Constantinides, S.M. (1974) Multiple forms of flounder muscle glyceraldehyde-3-phosphate dehydrogenase. Subunit composition, properties, and tissue distribution of the forms. Biological Chemistry, 249, 951-958.
 Wolny, M. (1977) Effect of borate on the catalytic activities of muscle glyceraldehyde 3-phosphate dehydrogenase. European Journal of Biochemistry, 80, 551-556.
 Allison, W.S. and Kaplan, N.O. (1964) The comparative enzymology of triosephosphate dehydrogenase. Biological Chemistry, 239, 2140-2152.
 Harris, I. (1964) Structure and catalytic activity of alcohol dehydrogenases. Nature, 203, 30-34.
 Perham, R.N. (1969) The comparative structure of mammalian glyceraldehyde-3-phosphate dehydrogenases. Biochemical Journal, 111, 17-21.
 Iddar, A., Valverde, F., Serrano, A. and Soukri, A. Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum.
 Soukri, A., Valverde, F., Hafid, N., Elkebbaj, M.S. and Serrano, A. (1995) Characterization of muscle glyceraldehyde-3-phosphate dehydrogenase isoforms from euthermic and induced hibernating Jaculus orientalis. Biochimica et Biophysica Acta, 1243, 161-168.
 Soukri, A., Hafid, N., Valverde, F., Elkebbaj, M.S. and Serrano, A. (1996) Evidence for a posttranslational covalent modification of liver glyceraldehyde-3-phosphate dehydrogenase in hibernating jerboa (Jaculus orientalis). Biochimica et Biophysica Acta, 1292, 177-187.
 Heinz, F. and Freimuller, B. (1982) Glyceraldehyde-3phosphate dehydrogenase from human tissues. Methods in Enzymology, 89, 301-305.
 Serrano, A., Mateos, M.I. and Losada, M. (1991) Differential regulation by trophic conditions of phosphorylating and non phosphorylating NADP(+)-dependent glyceroldehyde-3-phosphate dehydrogenases in Chlorella fusca. Biochemical and Biophysical Research Communications, 181, 1077-1083. doi:10.1016/0006-291X(91)92047-N
 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0
 Hedrick, J.L. and Smith, A.J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Archives of Biochemistry and Biophysics, 126, 155-164.
 Vaitukaitis, J.L. (1981) Production of antisera with small doses of immunogen: Multiple intradermal injections. Methods in Enzymology, 73, 46-52.
 Cleland, W.W. (1963) The kinetics of enzyme catalysed reaction with two or more substrates or products, nomenclature and rate equations. Biochimica et Biophysica Acta, 67, 104-137. doi:10.1016/0926-6569(63)90211-6
 Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254.
 Mountassif, D., Baibai, T., Fourrat, L., Moutaouakkil, A., Iddar, A., Soukri, A. and El Kebbaj, M.S. (2009) Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes. ABBS, 41, 399-406.
 Hafid, N., Valverde, F., Villalobo, E., Elkebbaj, M., Torres, A., Soukri, A. and Serrano, A. (1998) Glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena pyriformis: Enzyme purification and characterization of a gap C gene with primitive eukaryotic features. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 119, 493-503.
 Mounaji, K., Erraiss, N., Iddar, A., Wegnez, M., Serrano, A. and Soukri, A. (2002) Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 131, 411-421.
 Thompson, S.T., Cass, K.H. and Stellwagen, E. (1975) Blue dextran-sepharose: An affinity column for the dinucleotide fold in proteins. Proceedings of the National Academy of Sciences, 72, 669-672.
 Nickells, R.W., Browder, L.W. and Wang, T.I. (1989) Factors influencing the heat shock response of Xenopus laevis embryos. Biochemistry and Cell Biology, 67, 687695. doi:10.1139/o89-103
 Iddar, A., Serrano, A. and Soukri, A. (2002) Phosphate-stimulated NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus. FEMS Microbiology Letters, 211, 29-35.
 Fourrat, L., Iddar, A. and Soukri, A. (2007) Purification and characterization of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the dromedary camel. Acta Biochimica et Biophysica Sinica, 39, 148-154.
 Serrano, A., Mateos, M.I. and Losada, M. (1993) ATPdriven transhydrogenation and ionization of water in a reconstituted glyceraldehyde-3-phosphate dehydrogenase (phosphorylating and non-phosphorylating) model system. Biochemical and Biophysical Research Communications, 197, 1348-1356. doi:10.1006/bbrc.1993.2625