Purification of nicotinamide adenine dinucleotide phosphate- specific glutamate dehydrogenase from Chlorella sorokiniana and partial characterization of its physical, kinetic, and immunological properties

• Main Author: Gronostajski, Richard Mark
• Other Authors: Biochemistry and Nutrition
• Format: Others
• Published: Virginia Tech 2014
• Subjects: LD5655.V855 1977.G747; Chlorella sorokiniana; Dehydrogenases; Isoenzymes
• Online Access: http://hdl.handle.net/10919/43944; http://scholar.lib.vt.edu/theses/available/etd-07282010-020210/

<p>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.</p> <p> 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.</p> <p> 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.</p> === Master of Science