Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules

Abstract

Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N2) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in specific activity to 13 micromole per minute per milligram of protein and an activity yield of 37%. Further purification to near homogeneity was achieved by fast protein liquid chromatography, but with substantial loss of activity. Enzymic activity was highly labile, losing 3% per hour even when substrates, stabilizers, and reducing agents were included in buffers. However, activity could be partially stabilized for up to 1 month by storing GOGAT at -800C in 50% glycerol. The subunit molecular weight of GOGAT was estimated at 200 ± 7 kilodaltons with a native molecular weight of 235 ± 16 kilodaltons, which suggested that GOGAT is a monomer of unusually high molecular weight. The pi was estimated to be 6.6. The Km values for glutamine, a-ketoglutarate, and NADH were 466, 33, and 4.2 micromolar, respectively. Antibodies were produced to NADH-GOGAT. Specificity of the antibodies was shown by immunotitration of GOGAT activity. Alfalfa nodule NADH-GO-GAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Westem blots probed with anti-GOGAT showed that the high GOGAT activity of nodules as compared to roots was associated with increased levels of GOGAT polypeptides. Nodule NADH-GOGAT appeared to be highly expressed in effective nodules and little if any in other organs.

Molecular Characterization of NADH-Dependent Glutamate Synthase from Alfalfa Nodules

“…Thus, these enzymes, which are part of a common metabolic pathway, appear to be regulated differently. The fact that expression of the NADH-GOGAT gene shows the strictest requirement for effective nodulation, as well as the fact that NADH-GOGAT is present in effective nodules in much lower amounts than the other three enzymes (Groat and Schrader, 1982;Vance and Stade, 1984;Anderson et al, 1989;Griffith and Vance, 1989), leads us to consider the possibility that it may be the rate-limiting or regulated step of nitrogen assimilation. We are currently in the process of characterizing genomic clones for NADH-GOGAT, AAT2, and noduleenhanced PEPC to elucidate the regulatory mechanisms involved in the expression of these enzymes, each of which plays an important role in the nitrogen assimilatory pathway in root nodules.…”

“…GOGAT protein levels were determined by reaction of these filters with anti-GOGAT antiserum as described previously . The amino acid sequence of the amino terminus of the GOGAT protein, purified as described by Anderson et al (1989), was determined by the University of Minnesota lnstitute of Human Genetics protein sequencing facility, Minneapolis.…”

“…Bacterial NADPH-GOGATs (Trotta et al, 1974;Adachi and Suzuki, 1977;Hemmila and Mantsala, 1978;Oliver et al, 1987) and yeast (Sacc/raromyces cerew's/ae) NADH-GOGAT (Masters and Meister, 1982) are comprised of two nonidentical subunits. In contrast, all higher plants studied thus far (Suzuki and Gadal, 1984;Anderson et al, 1989;Chen and Cullimore, 1989), as well as Chlamydomonas reinhardtii (Cullimore and Sims, 1981) and Neurospora crassa (Hummelt and Mora, 1980), contain NAD(P)H-GOGATs consisting of a single polypeptide. A comparison of the deduced alfalfa and £ co// GOGATs (Figures 1 and 2) indicates that the alfalfa protein is comprised of sequences related to both prokaryotic subunits.…”

“…This is in part because root and nodule forms of NAD(P)H-GOGAT are highly unstable and occur in Iow abundance. Root nodule NADH-GOGAT from lupine (Benny and Boland, 1977), common bean Cullimore, 1988, 1989), and alfalfa has been isolated and characterized, and its activity has been found to increase markedly during development of Np-fixing (effective) nodules (Benny and Boland, 1977;Chen and Cullimore, 1988;Anderson et al, 1989). Plant-controlled, non-N2-fixing (ineffective) nodules and nodules with reduced N2 fixation, however, have very low to nondetectable NADH-GOGAT activities (Groat and Vance, 1982;Vance and Johnson, 1983;Egli et al, 1989).…”

Nitrogen assimilation in Citrus based on CitEST data mining

“…NADH-GOGAT is located primarily in plastids of non-photosynthetic tissues such as roots (Suzuki and Gadal, 1984). In root nodules of legumes, NADH-GOGAT is involved in the assimilation of nitrogen fixed by Rhizobium (Anderson et al, 1989). It has been hypothesized that NADH-GOGAT catalyzes the rate-limiting step of ammonia assimilation in these root nodules (Gregerson et al, 1993).…”