Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (Octopus vulgaris Cuvier, 1797)

Abstract

The NAD + dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K m for both NAD + and D-G3P were 66 lM and 320 lM, respectively. The maximal velocity V max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.

Influence of heavy metals on glyceraldehyde‐3‐phosphate dehydrogenase interactions in Chironomus riparius larvae

“…Kinetic analysis of the enzyme activity showed that Vmax of the purified GAPDH ranged between the values of the other known species cytosolic GAPDHs from 4 U/mg to 50 U/mg and Km for G3P was similar to those from the other organisms . The results suggest that catalytic properties of GAPDH of C. riparius were similar to those of other organisms.…”

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

“…Purification and characterization of sardine and octopus GAPDHs GAPDH was purified to electrophoretic homogeneity from a soluble protein fraction of S. pilchardus skeletal muscle and O. vulgaris arm muscle, using a simple procedure involving a fractioned precipitation in the 60-88% (W/V) saturation range of ammonium sulfate and only one column chromatography step, namely dye-affinity chromatography on Cibacron Blue-Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ, USA) [22,23].…”

Cloning, expression and characterization of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase of adult Haemonchus contortus†

“…This NAD + -dependent glycolytic enzyme is widely distributed and conserved in organisms from bacteria to humans (Fothergill-Gilmore & Michels, 1993) and is approximately 35–37 kDa in size (Fothergill-Gilmore & Michels, 1993). In some species, four such proteins may combine to form a tetramer structure with a native molecular weight in the range of 140–150 kDa (Oukhattar et al , 2008).…”