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. 1987 Jan;83(1):75–84. doi: 10.1104/pp.83.1.75

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

Newell F Bascomb 1,2, Robert R Schmidt 1
PMCID: PMC1056302  PMID: 16665219

Abstract

Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either α- or β-subunits with molecular weights of 55,500 or 53,000, respectively. The α-isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the β-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 α- and β-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 β-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 Km values. The ammonium Km of the β-isoenzyme remained constant at approximately 75 millimolar, whereas this Km of the α-isoenzyme ranged from 0.02 to 3.5 millimolar, depending upon nicotinamide adenine dinucleotide phosphate concentration.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahmad I., Hellebust J. A. Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica. Plant Physiol. 1984 Nov;76(3):658–663. doi: 10.1104/pp.76.3.658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bascomb N. F., Prunkard D. E., Schmidt R. R. Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells. Plant Physiol. 1987 Jan;83(1):85–91. doi: 10.1104/pp.83.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bascomb N. F., Turner K. J., Schmidt R. R. Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA. Plant Physiol. 1986 Jun;81(2):527–532. doi: 10.1104/pp.81.2.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bascomb N. F., Yeung A. T., Turner K. J., Schmidt R. R. Turnover of ammonium-inducible glutamate dehydrogenase during induction and its rapid inactivation after removal of inducer from Chlorella sorokiniana cells. J Bacteriol. 1981 Mar;145(3):1266–1272. doi: 10.1128/jb.145.3.1266-1272.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beudeker R. F., Tabita F. R. Characterization of glutamine synthetase isoforms from chlorella. Plant Physiol. 1985 Apr;77(4):791–794. doi: 10.1104/pp.77.4.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang J. Y., Knecht R., Ball R., Alkan S. S., Braun D. G. A sensitive peptide mapping method. Identification of three amino acid substitutions within two anti-azobenzenearsonate monoclonal antibody light chains. Eur J Biochem. 1982 Oct;127(3):625–629. [PubMed] [Google Scholar]
  7. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  8. Gronostajski R. M., Yeung A. T., Schmidt R. R. Purification and properties of the inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana. J Bacteriol. 1978 May;134(2):621–628. doi: 10.1128/jb.134.2.621-628.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Israel D. W., Gronostajski R. M., Yeung A. T., Schmidt R. R. Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella. J Bacteriol. 1977 May;130(2):793–804. doi: 10.1128/jb.130.2.793-804.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Meredith M. J., Gronostajski R. M., Schmidt R. R. Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana. Plant Physiol. 1978 Jun;61(6):967–974. doi: 10.1104/pp.61.6.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  13. Perlman D., Raney P., Halvorson H. O. Cytoplasmic and secreted Saccharomyces cerevisiae invertase mRNAs encoded by one gene can be differentially or coordinately regulated. Mol Cell Biol. 1984 Sep;4(9):1682–1688. doi: 10.1128/mcb.4.9.1682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Prunkard D. E., Bascomb N. F., Molin W. T., Schmidt R. R. Effect of Different Carbon Sources on the Ammonium Induction of Different Forms of NADP-Specific Glutamate Dehydrogenase in Chlorella sorokiniana Cells Cultured in the Light and Dark. Plant Physiol. 1986 Jun;81(2):413–422. doi: 10.1104/pp.81.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Prunkard D. E., Bascomb N. F., Robinson R. W., Schmidt R. R. Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana. Plant Physiol. 1986 Jun;81(2):349–355. doi: 10.1104/pp.81.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sumar N., Casselton P. J., McNally S. F., Stewart G. R. Occurrence of Isóenzymes of Glutamine Synthetase in the Alga Chlorella kessleri. Plant Physiol. 1984 Feb;74(2):204–207. doi: 10.1104/pp.74.2.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Talley D. J., White L. H., Schmidt R. R. Evidence for NADH- and NADPH-specific isozymes of glutamate dehydrogenase and the continuous inducibility of the NADPH-specific isozyme throughout the cell cycle of the eucaryote Chlorella. J Biol Chem. 1972 Dec 25;247(24):7927–7935. [PubMed] [Google Scholar]
  18. Turner K. J., Bascomb N. F., Lynch J. J., Molin W. T., Thurston C. F., Schmidt R. R. Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells. J Bacteriol. 1981 May;146(2):578–589. doi: 10.1128/jb.146.2.578-589.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Turner K. J., Gronostajski R. M., Schmidt R. R. Regulation of initial rate of induction of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase during the cell cycle of synchronous Chlorella. J Bacteriol. 1978 Jun;134(3):1013–1019. doi: 10.1128/jb.134.3.1013-1019.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wilchek M., Lamed R. Immobilized nucleotides for affinity chromatography. Methods Enzymol. 1974;34:475–479. doi: 10.1016/s0076-6879(74)34058-x. [DOI] [PubMed] [Google Scholar]
  21. Yeung A. T., Turner K. J., Bascomb N. F., Schmidt R. R. Purification of an ammonium-inducible glutamate dehydrogenase and the use of its antigen affinity column-purified antibody in specific immunoprecipitation and immunoadsorption procedures. Anal Biochem. 1981 Jan 1;110(1):216–228. doi: 10.1016/0003-2697(81)90138-x. [DOI] [PubMed] [Google Scholar]

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