Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.     

Nodule-specific glutamine synthetase is expressed before the onset of nitrogen fixation in Phaseolus vulgaris L.

Glutamine synthetase expression was studied in developing root-nodules of common bean with regard to the time-course of specific activity, antigen accumulation, polypeptide composition and in vitro translation products. This analysis shows that the nodule-specific GS polypeptide (GS-gamma) is detected prior to the nitrogenase acetylene-reducing activity, and that its accumulation together with that of the GS-alpha and GS-beta polypeptides vary with nodule age. GS-gamma is present in ineffective nodules, although in a lower ratio to GS-beta than in wild-type nodules. Comparisons of in vitro translated and in vivo synthesized GS polypeptides suggest no post-translational modifications. The possible factors and mechanisms involved in the regulation of expression of GS polypeptides are discussed.     

Purification and characterization of tubulin from mung bean (Vigna radiata)

Tubulin has been purified from mung bean seedling by Zn²⁺-induced polymerization. Both α- and β-subunits of mung bean tubulin are different from those of brain tubulin in electrophoretic mobility, colchicine binding and peptide map. Heterogeneity of mung bean tubulin has also been documented suggesting diversification of tubulin despite its conserved nature in general.     

Comparison of the conformation and stability of the native dimeric,monomelic, tetrameric and the desensitized forms of the nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings

A homogenous and crystalline form of nucleotide pyrophosphatase (EC 3.6.1.9) fromPhaseolus aureus (mung bean) seedlings was used for the study of the regulation of enzyme activity by adenine nucleotides. The native dimeric form of the enzyme had a helical content of about 65% which was reduced to almost zero values by the addition of AMP. In addition to this change in the helical content, AMP converted the native dimer to a tetramer. Desensitization of AMP regulation, without an alteration of the molecular weight, was achieved either by reversible denaturation with 6 M urea or by passage through a column of Blue Sepharose but additionofp-hydroxymercuribenzoate desensitized the enzyme by dissociating the native dimer to a monomer. The changes in the quaternary structure and conformation of the enzyme consequent to AMP interaction or desensitization were monitored by measuring the helical content, EDTA inactivation and Zn²⁺ reactivation, stability towards heat denaturation, profiles of urea denaturation and susceptibility towards proteolytic digestion. Based on these results and our earlier work on this enzyme, we propose a model for the regulation of the mung bean nucleotide pyrophosphatase by association-dissociation and conformational changes. The model emphasizes that multiple mechanisms are operative in the desensitization of regulatory proteins.     

Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean)

Herein, we report the purification and biochemical characterization of a novel bi-functional protein proteinase/amylase inhibitor from the dietary leguminous pulse Phaseolus aureus Roxb. (Vigna radiata L.) by means of acetic acid precipitation, salt fractionation, ion-exchange chromatography (DEAE-cellulose) and affinity chromatography on trypsin-sepharose column. P. aureus inhibitor is a bi-functional inhibitor since it exhibits inhibitory activity towards trypsin-like and alpha-chymotrypsin-like serine proteinases as well as against alpha-amylases. It is a helix-rich protein (Mr 13,600) containing approximately eight tyrosines, one tryptophan and two cystines. N-terminal sequence alignment reveals no homology to other proteinase inhibitors reported from Phaseolus sp. thereby confirming that it is a novel inhibitor. Inhibitory activity measurements show that the inhibitor is quite stable even at extremely high temperatures and is only slightly affected by pH changes. Circular dichroism (CD) conformational studies revealed some changes in its near- as well as far-ultraviolet spectrum at extremes of pH and temperature. Treatments with trypsin for varying time periods did not alter its proteolytic inhibitory activity but caused some reduction in its amylase inhibitory activity.     

NAD-dependent shikimate dehydrogenase from mung bean cells in suspension culture

Shikimate dehydrogenase in mung bean cell suspension culture was partially purified by chromatography on DEAE-cellulose. The NADP-dependent SDHase was eluted as a single peak, while the NAD-dependent one exhibited two peaks. Polyacrylamide gel electrophoresis revealed six NAD-dependent isozymes of SDHase, two of which corresponded to NADP-dependent ones.     

Studies onplant aspartate transcarbamylase: Regulatory properties of the enzyme from mung bean (Phaseolus aureus) seedlings

Aspartate transcarbamylase (EC 2·1·3·2) purified from mung bean seedlings was used as a model to understand the mechanism of allosteric regulation. The enzyme exhibited homotropic interactions with carbamyl phosphate. Preincubation of the enzyme with aspartate abolished the sigmoidicity of the carbamyl phosphate saturation curve. UMP was the most potent inhibitor of the reaction and was noncompetitive with respect to aspartate. The sigmoidicity of carbamyl phosphate saturation curves increased with increase in UMP concentration. These results were analysed by an iterative least squares procedure. There was no change inV _max values with increase in the UMP concentration, although theK _0·5 values (concentration of carbamyl phosphate required to reach half maximal velocity) increased. This implied that the effect of UMP was on the binding of carbamyl phosphate only and not on the catalytic function of the enzyme. The allosteric properties of the enzyme could be explained in terms ofK system of the symmetry model. The values of the allosteric constantsn, L andc calculated for mung bean enzyme, making use of the Monod equation accounted for all the observed properties. The enzyme appeared to be a tetramer (n=4) and in the absence of ligands was predominantly in theT form (L _o= 2·25). Carbamyl phosphate bound preferentially to theR form (c= 10_?3), while UMP bound preferentially to theT form and hence these two ligands exhibited the typical heterotropic interactions as expected of antagonistic ligands.     

Regulation of the activity of mung bean (Phaseolus aureus) glutamine synthetase by amino acids and nucleotides.

The activity of glutamine synthetase isolated from the germinated seedlings of Phaseolus aureus was regulated by feedback inhibition by alanine, glycine, histidine, AMP, and ADP. When glutamate was the varied substrate, alanine, histidine, and glycine were partial noncompetitive, competitive, and mixed-type inhibitors, respectively. The type of inhibition by these amino acids was confirmed by fractional inhibition analysis. The adenine nucleotides, AMP and ADP, completely inhibited the enzyme activity and were competitive with respect to ATP. Multiple inhibition analyses revealed the presence of separate and nonexclusive binding sites for the amino acids and mutually exclusive sites for adenine nucleotides. Cumulative inhibition was observed with these end products.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants

In this paper we have examined whether the four glutamine synthetase (gln) genes, expressed in roots and nodules of Phaseolus vulgaris are substrate-inducible by ammonium. Manipulation of the ammonium pool in roots, through addition and removal of exogenous ammonium, did not elicit any changes in the abundances of the four mRNAs thus suggesting that the gln genes in roots of this legume are neither substrate-inducible by ammonium nor derepressed during nitrogen starvation. In nodules the effect of the ammonium supply on expression of the gln genes has been examined by growing nodules under argon/oxygen atmospheres, or with a number of Fix^- Rhizobium mutants, and following addition of exogenous ammonium. The results of these experiments suggest that the expression of the gln- gene, which is strongly induced during nodule development, is primarily under a developmental control. However nitrogen fixation appears to have a quantitative effect on expression of gln- as the abundance of this mRNA is about 2 to 4-fold higher under nitrogen-fixing conditions. This effect could not be mimicked by addition of exogenous ammonium and moreover is not specific to the gln- gene as mRNA from a leghaemoglobin gene was similarly affected. Taken together these results have failed to find an effect of ammonium on specifically inducing the expression of glutamine synthetase genes in roots and nodules of P. vulgaris.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis)

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.     

Plant aspartate transcarbamylase: kinetic properties of the enzyme from mung bean (Phaseolus aureus) seedlings

Aspartate transcarbamylase (EC 2.1.3.2) catalyzes the bi substrate reaction—carbamyl phosphate+ L-aspartate ? carbamyl aspartate ? phosphate, The order of addition of substrates and release of products for the homogeneous aspartate transcarbamylase fromPhaseolus aureuss eedlings has been investigated by using the kinetic methods of analysis. p ]Initial velocity studies indicated that the mechanism might be a sequential one. Product inhibition studies showed that phosphate was a linear competitive inhibitor with respect to carbamyl phosphate and was anS (slope) andI (intercept) linear noncompetitive inhibitor with respect to aspartate. Carbamyl aspartate was a noncompetitive inhibitor with respect to both the substrates. These inhibition patterns agreed with an ordered mechanism of reaction with carbamyl phosphate as the leading substrate and phosphate as the last product to leave the enzyme surface. The presence of dead end complexes and the rapid equilibrium random mechanism were ruled out by the absence of inhibition by the substrate(s) and the linear replot slopevs. the inhibitor concentration. Acetyl phosphate, an analog ue of carbamyl phosphate was a non-competitive inhibitor with respect to aspartate. This result could be explained both in terms of an ordered as well as a random mechanism. On the other hand, succinate, an analog ue of aspartate was an uncompetitive inhibitor with respect to carbamyl phosphate, indicating that the mechanism was ordered. p ]The transition state analog ue, N-(phosphonoacetyl)-L-aspartate, binds much more tightly than either of the two substrates. This analog ue was a linear competitive inhibitor with respect to carbamyl phosphate and a linear noncompetitive inhibitor with respect to aspartate. These results are compatible with an ordered mechanism rather than a random one.     

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence,expression in different organs and uptake into isolated chloroplasts

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M _r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M _r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.     

Plant regeneration from seedling explants of green bean (Phaseolus vulgaris L) via organogenesis

Green bean (Phaseolus vulgaris L.) plants were regenerated from 3-day old seedling explants via organogenesis. The explants contained a cotyledon and a small portion (2–3 mm) of embryonic axis split in half. Explants were cultured on a defined medium containing glutamine as the sole nitrogen source. A ring of meristematic tissue was produced at the base of the axillary bud located at the cotyledonary node. The meristematic tissue was produced only if the axillary bud was present together with the cotyledon in the explant. Buds and shoots developed from the meristematic ring. Selected shoots produced roots when excised from the cluster of buds and transferred to root induction medium. Rooted shoots (plantlets) grew well and produced viable seeds when grown in the greenhouse. Histological studies revealed the origin of buds from the peripheral layers of the meristematic ring.Production of buds and shoots was a continuous process, so that new shoots could be removed from the explant for plantlet production every 10–14 days. With the cultivar Dark Red Kidney, an average of 49 buds and 8 shoots were regenerated per explant by 30 days after culture initiation. Sixty-seven percent of the shoots produced roots, and 90–95% of the plantlets survived greenhouse acclimatization to produce healthy plants.     

Cloning of the glutamine synthetase gene fromspirulina platensis

An 8 Kilobase-pair (Kbp) HindIII fragment containing the coding sequence forSpirulina platensis glutamine synthetase [EC 6.3.1.1.] has been identified utilizing a probe derived fromAnabaena 7120 and cloned in the vector pAT153.     

Studies onAspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grownAspergillus niger was increased 3–5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH ₄ ⁺ , and further, the enzyme is repressed by increasing concentrations of NH ₄ ⁺ . In contrast to other micro-organisms, theAspergillus niger enzyme was neither specifically inactivated by NH ₄ ⁺ or L-glutamine nor regulated by covalent modification. Glutamine synthetase fromAspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn²⁺ or Mg²⁺-dependent synthetase and Mn²⁺-dependent transferase activity. Aspergillusniger glutamine synthetase was completely inactivated by two mol of phenyl-glyoxal and one mol of N-ethylmaleimide with second order rate constants of 3.8 M^-1 min^-1 and 760 M^-1 min^-1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH ₄ ⁺ , Mn²⁺ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.     

Characterization ofVitis vinifera L. glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme

Grapevine (Vitis vinifera L.) glutamine synthetase (GS) was analysed into two distinct classes of isoforms; one of them was present in both leaf and root tissues while the other one showed leaf specificity. Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size and differential tissue specificity. Two structurally distinct cDNA clones, pGS1;1 and pGS1;2, encoding grapevine GS were isolated from a cell suspension library and characterized. Both clones contained open reading frames encoding for polypeptides of 356 amino acids with a predicted molecular mass of about 39 kDa. Although the coding sequences of pGS1;1 and pGS1;2 were 84% similar, their 5-and 3-untranslated sequences showed only 40% similarity. The coding sequences of the two clones and the derived amino acid sequences showed higher homology to cytosolic than to chloroplastic GSs of other higher plants indicating that the cDNAs isolated encode for cytosolic isoforms of grapevine GS. Southern blot analysis suggested the existence of more than two GS genes in the grapevine genome. In northern blots both clones were hybridized to mRNAs of about 1.4 kb that are differentially expressed in the various tissues. Supply of nitrate or ammonium in the cell suspension culture medium, as a sole nitrogen source, resulted in differential response of the pGS1;1-and pGS1;2-related genes.