Purification and Characterization of Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase from the Dromedary Camel

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12), a key enzyme of carbon metabolism, was purified and characterized to homogeneity from skeletal muscle of Camelus dromedarius. The protein was purified approximately 26.8 folds by conventional ammonium sulphate fractionation followed by Blue Sepharose CL-6B chromatography, and its physical and kinetic properties were investigated. The native protein is a homotetramer with an apparent molecular weight of approximately 146 kDa. Isoelectric focusing analysis showed the presence of only one GAPDH isoform with an isoelectric point of 7.2. The optimum pH of the purified enzyme was 7.8. Studies on the effect of temperature on enzyme activity revealed an optimal value of approximately 28-32 degrees with activation energy of 4.9 kcal/mol. The apparent K(m) values for NAD(+) and DL-glyceraldehyde-3-phophate were estimated to be 0.025+/-0.040 mM and 0.21+/-0.08 mM, respectively. The V(max) of the purified protein was estimated to be 52.7+/-5.9 U/mg. These kinetic parameter values were different from those described previously, reflecting protein differences between species.

Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate <italic>Tetrahymena thermophila</italic>

“…For purified GAPDHs from S. pilchardus and P. waltl, the V max (37.6 and 33.24 U/mg, respectively) was similar to that obtained for T. thermophila GAPDH, and the K m NAD þ of S. pilchardus GAPDH [28] was also similar to that estimated for T. thermophila GAPDH, but the K m NAD þ of GAPDH from P. waltl [26] was different. Indeed, the K m D-G3P was similar to those found for T. pyriformis [25] and C. dromedarius GAPDHs [30], but was different from those reported for S. pilchardius [28] and P. waltl GAPDHs [26] ( Table 2).…”

“…The purified T. thermophila GAPDH has a homotetrameric structure. This GAPDH differ in a number of instances from those described previously from other sources [25,26,[28][29][30]. Indeed, the effect of two types of stress (oxidative and nitrosative) on this enzyme was studied, showing that oxidative stress is most toxic than nitrosative stress.…”

“…The V max of the purified enzyme was 39.40 + 2.95 U/mg ( Table 2). Therefore, the K m NAD þ of T. thermophila GAPDH is clearly lower, suggesting a higher affinity for the nucleotide co-enzyme, as it was observed for T. pyriformis [25] and Camelus dromedarius [30]. For purified GAPDHs from S. pilchardus and P. waltl, the V max (37.6 and 33.24 U/mg, respectively) was similar to that obtained for T. thermophila GAPDH, and the K m NAD þ of S. pilchardus GAPDH [28] was also similar to that estimated for T. thermophila GAPDH, but the K m NAD þ of GAPDH from P. waltl [26] was different.…”

Comparative molecular analysis of evolutionarily distant glyceraldehyde-3-phosphate dehydrogenase from <italic>Sardina pilchardus</italic> and <italic>Octopus vulgaris</italic>

“…Interestingly, a second cluster of teleost fishes GAPDH was also found, probably due to the occurrence in some species of enzyme isoforms ( paralogs), as has been reported for many other vertebrates [33,36,37].…”

“…A single GAPDH isoform has been previously found in other animal tissues and microorganisms, both prokaryotes and eukaryotes [4,33,44,45]. However, it does not seem to be a general rule, as the presence of several GAPDH isoforms have also been reported in phylogenetically different organisms [37,38,45,46].…”