Phosphoserine aminotransferase in soybean root nodules : demonstration and localization

Phosphoserine aminotransferase activity was detected in the plant and bacteroid fractions from soybean (Glycine max) root nodules. Both total and specific activities increased in the plant fraction during nodule development. Serine-pyruvate aminotransferase activity was not detectable in the plant or bacteroid fractions of these nodules. Sucrose density gradient fractionation indicated a proplastid localization for phosphoserine aminotransferase. The data presented support a role for this enzyme in carbon supply to purine biosynthesis in the pathway of ureide biogenesis in soybean nodules.     

Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi.

1. The cultured, epimastigote-form of Trypanosoma cruzi contains NADP-linked glutamate dehydrogenase (EC 1.4.1.4), with a molecular weight of about 280,000, similar to the enzyme from Plasmodium chabaudi and different from the enzymes from higher animal sources. 2. T. cruzi also contains aspartate aminotransferase (EC 2.6.1.1), with properties similar to those of the enzyme from mammals. 3. The concerted action of the transaminase and glutamate dehydrogenase might be responsible for the production of NH3 which characterizes the protein catabolism in T. cruzi.     

Immunochemical studies on xanthine dehydrogenase of soybean root nodules

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was purified from root nodules of soybean (Glycine max) and used to prepare a polyclonal rabbit antiserum. Monospecificity of this antiserum was ascertained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipate. During root nodule development of soybean, only one form of XDH was detected on an immunological basis. Titration of XDH by immunoelectrophoresis showed that a remarkable increase in the amount of XDH occurred between two and four weeks after inoculation, in parallel with the increase in enzyme activity. Localization of XDH by immunofluorescence indicated that the enzyme was present exclusively in uninfected cells where it appeared to be associated with discrete organellelsAbbreviations IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis - XDH xanthine dehydrogenase     

Interaction between brain enzymes glutamate dehydrogenase and aspartate aminotransferase.

Fluorescence spectroscopic methods were used to study the interaction between aspartate aminotransferase and glutamate dehydrogenase isolated from pig brain. The conversion of the P-pyridoxal form of the aminotransferase to the P-pyridoxamine form of the enzyme is easily monitored by recording emission spectra upon excitation at 330 nm. Evidence for the interaction between the enzymes was obtained from fluroescence measurements conducted on aspartate aminotransferase label with a fluorescence probe (1-5-AEDANS) attached to one sH residue of the protein. The interaction of the aminotransferase (1μM) with glutamate dehydrogenase (2μM) brings about an enhancement as well as a blue shift in the band position of the fluorescence emitted by the dansyl chromophore. Polarization of fluorescence measurements conducted over a wide range of temperatures reveal that the rotational correlation time of aspartate aminotransferase (35 n.seconds) is increased to a value of 100 n.seconds upon addition of glutamate dehydrogenase.     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

Nanosecond emission anisotropy of interacting enzymes aspartate aminotransferase glutamate dehydrogenase.

Nanosecond fluorescence studies were performed on mitochondrial aspartate aminotransferase from beef liver to determine whether the dimeric enzyme displays any modes of flexibility in the nanosecond range. The most informative quantities calculated from nanosecond fluorescence measurements S(t) and D(t) decay in a monoexponential manner with decay times τ_S = 13 and τ_D = 10 nanoseconds respectively. The observed rotational correlation time θ = 43 M-seconds yields a volume for the dimeric enzyme of 1.97 × 10⁵ A^o3. The rotational correlation time of aspartate aminotransferase is influenced by the presence of the enzyme glutamate dehydrogenase.     

Immunocytochemical localization of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in soybean nodules

Polyclonal antibodies raised against NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase [phosphorylating], EC 1.2.1.12) from the plant cytosolic fraction of soybean [ Glycine max (L.) Merr. cv. Williams] nodules were used to study the subcellular location of the enzyme and its relative distribution between infected, interstitial and cortical cells of soybean (cv. Lincoln) nodules. Post-embedding immunogold labelling was carried out on nodules harvested 5, 12, 19 and 25 days after the first sign of nodulation. Labelling for glyceraldehyde-3-phosphate dehydrogenase was observed over the cytoplasm and nuclei of infected and uninfected cells, as well as over the nucleoid regions of bacteroids. In 5-day-old nodules, label also bound adjacent to the peribacteroid membranes. Statistical analysis of the number of gold particles per cell area indicated that in 5-day-old nodules, glyceraldehyde-3-phosphate dehydrogenase was distributed equally between infected, interstitial and cortical cells, but in older nodules the enzyme was more prominent in the interstitial and cortical cells than in infected cells.     

Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.     

Aldrin epoxidase from soybean root nodules

Soybean (Glycine max cv Forrest) root nodule homogenates oxidized aldrin to its epoxide, dieldrin. In crude tissue brei, addition of an NADPH-generating system was inhibitory to epoxidation. However, anaerobic gel filtration and sucrose density separation removed factors required for inhibition by NADPH, allowing a normal stimulation by the NADPH-generating system. In fractions from sucrose density gradients, activity was found predominantly at a density containing rough microsomes, with additional activities in the soluble and other fractions. Epoxidase activity was 2–4-times greater in the nitrogen-fixing nodules than in roots. This demonstration of active epoxidation indicates the capacity of nodules to detoxify other pesticides and xenobiotics.     

Aspartate aminotransferase activity is required for aspartate catabolism and symbiotic nitrogen fixation in Rhizobium meliloti.

A mutant of Rhizobium meliloti, 4R3, which is unable to grow on aspartate has been isolated. The defect is specific to aspartate utilization, since 4R3 is not an auxotroph and grows as well as its parent strain on other carbon and nitrogen sources. The defect was correlated with an inability to fix nitrogen within nodules formed on alfalfa. Transport of aspartate into the mutant cells was found to be normal. Analysis of enzymes involved in aspartate catabolism showed a significantly lower level of aspartate aminotransferase activity in cell extracts of 4R3 than in the wild type. Two unrelated regions identified from a genomic cosmid bank each complemented the aspartate catabolism and symbiotic defects in 4R3. One of the cosmids was found to encode an aspartate aminotransferase enzyme and resulted in restoration of aspartate aminotransferase activity in the mutant. Analysis of the region cloned in this cosmid by transposon mutagenesis showed that mutations within this region generate the original mutant phenotypes. The second type of cosmid was found to encode an aromatic aminotransferase enzyme and resulted in highly elevated levels of aromatic aminotransferase activity. This enzyme apparently compensated for the mutation by its ability to partially utilize aspartate as a substrate. These findings demonstrate that R. meliloti contains an aspartate aminotransferase activity required for symbiotic nitrogen fixation and implicate aspartate as an essential substrate for bacteria in the nodule.     

Aspartate aminotransferase in alfalfa root nodules : I. Purification and partial characterization

Aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1 [AAT]), a key enzyme in the assimilation of C and N compounds, was purified from the cytosol of alfalfa (Medicago sativa L.) root nodules. Isoforms that increased during nodule development, AAT-2a, AAT-2b, and AAT-2c, were purified greater than 447-fold to apparent homogeneity, and high titer polyclonal antibodies were produced. The native molecular weight of the AAT-2 isoforms was approximately 80 kilodatons with a subunit molecular weight of 40 kilodatons, indicating that the holoenzymes are dimers. The AAT-2 isoforms comprised approximately 0.4% of the total soluble nodule protein. The AAT specific activity was measured in leaf, stem, root, and nodule organs, and zymograms of each were compared. Enzyme activity was 4- to 37-fold greater in effective (nitrogen fixing) nodules than in leaves, stems, and roots. Effective nodule AAT-specific activity was 3- to 8-fold greater than that of plant-controlled ineffective nodules. No differences in K_m were observed between AAT-1 and AAT-2. Antibodies raised against AAT-2 were more selective against AAT-2 than AAT-1. Evidence obtained from zymograms suggests that the expression of alfalfa nodule AAT is controlled at two different gene loci, AAT-1 and AAT-2, resulting in different dimeric isoforms.