This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Web of Science (24) Citing Articles via Google Scholar Google Scholar Articles by Bascomb, N. F. Articles by Schmidt, R. R. Search for Related Content PubMed PubMed Citation Articles by Bascomb, N. F. Articles by Schmidt, R. R. Agricola Articles by Bascomb, N. F. Articles by Schmidt, R. R.

Metabolism and Enzymology

Purification and Partial Kinetic and Physical Characterization of Two Chloroplast-Localized NADP-Specific Glutamate Dehydrogenase Isoenzymes and Their Preferential Accumulation in Chlorella sorokiniana Cells Cultured at Low or High Ammonium Levels ¹

Department of Microbiology and Cell Science, 1059 McCarty Hall, University of Florida, Gainesville, Florida 32611

Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either - or -subunits with molecular weights of 55,500 or 53,000, respectively. The -isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the -isoenzyme accumulated after cells were fully induced (120 minutes) at high ammonium concentrations (29 millimolar). Purification of isoenzymes was achieved by (NH₄)₂SO₄ fractionation, gel-filtration, anion-exchange fast protein liquid chromatography, and affinity chromatography. The - and -isoenzymes were separated by their differential binding to Type 4 nicotinamide adenine dinucleotide phosphate-Sepharose. Both isoenzymes bound to an antibody affinity column to which purified antibody (prepared against -isoenzyme) was covalently attached. Peptide mapping of the subunits showed them to have a high degree of sequence homology. Both subunits were synthesized in vitro from precursor protein(s) with a molecular weight of 58,500. Although the subunits have similar chemical, physical, and antigenic properties, their holoenzymes have strikingly different ammonium K_m values. The ammonium K_m of the -isoenzyme remained constant at approximately 75 millimolar, whereas this K_m of the -isoenzyme ranged from 0.02 to 3.5 millimolar, depending upon nicotinamide adenine dinucleotide phosphate concentration.

¹ Supported by United States Public Health Service Grant GM 29733 from the National Institutes of Health. Florida Agriculture Experiment Station Journal Series, No. 7259.