Purification and characterization of cytosolic glycerol-3-phosphate dehydrogenase from skeletal muscle of jerboa (Jaculus orientalis).
Cytosolic glycerol-3-phosphate dehydrogenase was purified from jerboa (Jaculus orientalis) skeletal muscle and its physical and kinetic properties investigated. The purification method consisted of a multi-step procedure and this procedure is presented. The specific activity of the purified enzyme is 53.6 U/mg of protein, representing a 77-fold increase in specific activity. The apparent Michaelis constant (Km) for dihydroxyacetone is 137.39 (+/- 25.56) microM whereas the Km for glycerol-3-phosphate is 468.66 (+/- 27.59) microM. The kinetic mechanism of purified enzyme is 'ordered Bi-Bi' and this result is confirmed by the product inhibition pattern. Under the conditions of assay, the pH optimum occurs at pH 7.7 for the reduction of dihydroxyacetone phosphate and at pH 9.0 for glycerol-3-phosphate oxidation. In the direction of dihydroxyacetone phosphate, the optimal temperature is 35 degrees C. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 33,000 (+/- 1000), whereas non-denaturing polyacrylamide gel yields a molecular weight of 72,000 (+/- 2000), suggesting that the enzyme may exist as a dimer. A polyclonal antiserum raised against the purified enzyme was used to localize the enzyme in different jerboa tissues by Western blot method. The purified enzyme is sensitive to N-ethylmaleimide, and incubation of the enzyme with 20 mM N-ethylmaleimide resulted in a complete loss of catalytic activity. The purified enzyme is inhibited by several metal ions including Zn2+ and by 2,4-dichlorophenoxyacetic acid.
Berrada
W
W
Département de Biologie, Faculté des Sciences, Université Hassan II-Ain Chock, Maârif, Casablanca, Morocco.
Naya
A
A
Iddar
A
A
Bourhim
N
N
D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis / Q000187:drug effects; D003600:Cytosol / Q000201:enzymology*; D004795:Enzyme Stability; D005033:Ethylmaleimide / Q000494:pharmacology; D020869:Gene Expression Profiling; D005993:Glycerolphosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D009928:Organ Specificity; D012377:Rodentia; D013696:Temperature
D015084:2,4-Dichlorophenoxyacetic Acid; D005993:Glycerolphosphate Dehydrogenase; D005033:Ethylmaleimide