The ammonium inducible nicotinamide phosphate-specific glutamate
dehydrogenase from Chlorella sorokiniana has been purified 260-fold to
homogeneity. Depending on the technique used, the native enzyme
appeared to have a molecular mass of 290,000 to 400,000 daltons and to
be composed of subunits with an identical molecular weight of 58,000.
Differences in the molecular weight of the native enzyme, as determined
by sedimentation equilibrium, Sephadex G-200 gel filtration and gradient
polyacrylamide gel electrophoresis, indicate that the native enzyme
may be elliptical in shape.
The amino acid composition of the enzyme is high in glycine,
glutamate, and asparate. Moreover, the arginine to lysine ratio is
similar to those measured in other glutamate dehydrogenases. The Nterminal
amino acid is unavailable to dansylation. All six cysteines
in the enzyme are in the free sulfhydryl form.
The enzyme is very specific for the reduced and oxidized forms of
nicotinamide adenine dinucleotide phosphate and has less than 0.5 percent
of maximal activity, using the oxidized and reduced forms of
nicotinamide adine dinucleotide. With low concentrations of the
substrates, no cooperativity was seen; however severe substrate inhibition
was observed with a-ketoglutarate. Antiserum produced to the
subunits of the enzyme yielded a single precipitin band against
purified enzyme in Ouchterlony double diffusion analysis. "Rocket"
immunoelectrophoresis has been used to quantify the amount of antigen
present in samples of the purified enzyme.
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