| Popis: |
The specific activity of plant NADH-dependent glutamate synthase (NADH- GOGAT) in root nodules of Phaseolus vulgaris L., is over three-fold higher than the specific activity of ferredoxin-dependent GOGAT. NADH-GOGAT is composed of two distinct isoenzymes which can be separated from crude nodule extracts by ion-exchange chromatography. Both NADH-GOGAT isoenzymes have been purified to apparent homogeneity and an anti-NADH-GOGAT antibody has been raised against purified NADH-GOGAT eluted from a denaturing polyacrylamide gel. They both have been shown to be monomeric proteins with similar MfS of about 200,000 and are both specific for NADH as reductant. An investigation of their kinetic characteristics has shown slight differences in their Kms for L-glutamine, 2-oxoglutarate and NADH, and they have different pH optima, with NADH-GOGAT I exhibiting a broad pH optimum of 8.0 whereas NADH-GOGAT II has a much narrower pH optimum of 8.5. Subcellular localization studies have shown that both isoenzymes are located in plastids but in different cell types: NADH-GOGAT I is mainly prerent in the plastids of outercortex cells whilst NADH-GOGAT II is found to be located in the plastids of inner central cells of root nodules of P.vulgaris. NADH-GOGAT II has been found to be only present in root nodules and not other organs of P.vulgaris. During nodulation both isoenzymes increase in activity but the major increase is due to NADH-GOGAT II which increases over a time course similar to the increase in nitrogenase activity. The results suggest that the two isoenzymes of NADH-GOGAT play different physiological roles in nodule cells: NADH-GOGAT II is mainly responsible for assimilating ammonium produced during dinitrogen fixation whilst NADH-GOGAT I could assimilate ammonium from other metabolic pathways. In nodules grown under Ar : 02and nodules formed with Rhizobium Fix- mutan* CE108, both NADH-GOGAT isoenzymes, especially NADH-GOGAT II, have been observed to be expressed at early stages of nodulation but the specific activities of the two isoenzymes are low and remain constant at later stages of nodulation. However, exogenous NH4+ supplied to nodulated root systems has no effect on increasing the activities of the two NADH-GOGATsbut can induce the senescence of the nodules. It could be proposed that the initial expression of NADH-GOGAT is independent of nitrogen fixation but the level of the two NADH-GOGAT isoenzymes, especially NADH-GOGAT II is affected by nitrogen fixation. |
