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Metabolism and Enzymology

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells ¹

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

The kinetics of accumulation (per milliliter of culture) of the - and - subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The -subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the -subunit holoenzyme(s). After 120 minutes, the -subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using ³⁵SO₄ and immunochemical procedures, the rate of synthesis of the -subunit was shown to be greater than the -subunit during the first 80 minutes of induction. The - and -subunits had different rates of degradation during the induction period (t = 50 versus 150 minutes, respectively) and during the deinduction period (t = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

³ Present address: ZymoGenetics, Inc., 2121 North 35th St., Seattle, WA 98103.

¹ Supported by United States Public Health Service Grant GM 29733 from the National Institutes of Health. Florida Agriculture Experiment Station Journal Series, No. 7301.