The purpose of this project will be to characterize at the molecular level the genetic and metabolic mechanisms which regulate the induction, inactivation, and degradation of an ammonium inducible NADP-specific glutamate dehydrogenase (NADP-GDH) in eucaryotic microorganism, Chlorella. During the previous grant period, accumulation of NADP-GDH antigen and activity was shown to be regulated by both transcriptional and post-transcriptional mechanisms. This six-subunit chloroplast enzyme was shown to be inactivated by covalent modification of its subunits in what appears to be a prerequisite step for their degradation. The covalent modification reaction(s) appear to result in the formation of a covalent bond between two identical subunits. Until modified subunits are characterized completely, they are called putative dimers. The inducer appears to inhibit the rate of the inactivation/""""""""dimerization"""""""" process which presumably results in increased subunit stability and accumulation of active holoenzyme. The level of translatable NADP-GDH mRNA was shown to increase during the induction period. However, the basal level of translatable NADP-GDH mRNA was also observed to increase during the synchronous cell cycle of uninduced cells, reaching a level in uniduced mother cells which was 50-70 percent of the mRNA level in fully induced mother cells.
The specific aims of this proposal will be to use immunochemical, biochemical, and cell synchrony methods (a) to complete chemical characterization of the putative dimer, (b) to identify the different biochemical steps involved in putative dimer formation and degradation in vivo. (c) to use an in vitro inactivation/dimerization and degradation system to identify cofactor, and energy requirements for dimerization and degradation, and to begin fractionation/purification of the different enzymes or proteins involved, (d) to use NADP-GDH constitutive mutants to aid in identification of the sequence of biochemical steps involved in putative dimer formation and degradation, (e) to localize the cellular site (i.e., cytosol or chloroplast) for putative dimer formation and degradation, (f) to show whether the NADP-GDH subunit is synthesized as a precursor (g) to establish the kinetic relationships among the parameters of NADP-GDH activity/antigen, translatable and total sequences during induction/deinduction at different times during a cell cycle.
| Cock, J M; Kim, K D; Miller, P W et al. (1991) A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana. Plant Mol Biol 17:1023-44 |
| Cock, J M; Schmidt, R R (1989) A glutamate dehydrogenase gene sequence. Nucleic Acids Res 17:10500 |
