Contributed by Philip W Miller, Waltraud I Dunn, and Robert R Schmidt,
University of Florida, Dept. Microbiology and Cell Science, Gainesville,
FL 32611 USA
Florida Agricultural Experiment Station Serial Number T-00256
One of the research goals of this laboratory is to elucidate the pre- and posttranslational mechanisms that regulate both carbon and nitrogen metabolism in plants. Chlorella sorokiniana, a unicellular green alga, has been used extensively as a model system to study enzymes involved in higher plant metabolism. Research in this laboratory has shown that Chlorella sorokiniana possesses seven ammonium inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) isoenzymes that are regulated by both nitrogen and carbon metabolites (Prunkard et al. 1986). Biochemical, immunochemical, and physical characterization of purified GDH homohexamers revealed that the subunits (α, 55.5 kD; , 53 kD) composing these holoenzymes are nearly identical; however, kinetic characterization of the enzymes showed them to have strikingly different Km values, allosteric properties, and turnover rates (Bascomb and Schmidt 1987). Recent molecular genetic analysis indicates that minor modifications in the primary protein sequences may account for these observed properties. In order to further elucidate the regulation of these isoenzymes, it was necessary to purify to homogeneity a large quantity of active enzyme for polyclonal/monoclonal antibody production and additional protein analysis.
We report here the use of the Model 491 prep cell for the purification
of native NADP-GDH protein by nondenaturing PAGE with <2% loss