Studies on glutamine synthetase: purification of the enzyme from mung bean (Phaseolus aureus) seedlings and modulation of the enzyme-antibody reaction by the substrates

Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2) from Phaseolus aureus (mung bean) seedlings was purified to homogeneity by ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and affinity chromatography on histidine-Sepharose. The enzyme had a molecular weight of 775,000 ± 25,000. The enzyme consisted of identical subunits with an approximate subunit molecular weight of 50,000. Hyperbolic saturation curves were obtained with the substrates, glutamate, ATP and hydroxylamine. Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely inhibited the activity of the enzyme. Preincubation of the enzyme with glutamate and ATP, prior to the addition of the antibody, partially protected the enzyme against inhibition. The Km values of this enzyme-antibody complex and the native enzyme were identical (glutamate, 2.5mM; ATP, 1 mM; hydroxylamine, 0.5 mM). The Km values of the partially inhibited enzyme (the enzyme pretreated with antibody prior to the addition of substrates) were 2-fold higher than those of the native enzyme. These results suggested that the substrate-induced conformational changes in the enzyme were responsible for the protection against inhibition of the enzyme activity by the antibody

Purification and regulation of aspartate transcarbamylase from germinated mung bean (Vigna radiata) seedlings

Aspartate transcarbamylase (EC 2.1.3.2) was purified to homogeniety from germinated mung bean seedlings by treatment with carbamyl phosphate. The purified enzyme was a hexamer with a subunit molecular weight of 20,600. The enzyme exhibited multiple activity bands on Polyacrylamide gel electrophoresis, which could be altered by treatment with carbamyl phosphate or UMP indicating that the enzyme was probably undergoing reversible association or dissociation in the presence of these effectors. The carbamyl phosphate stabilized enzyme did not exhibit positive homotropic interactions with carbamyl phosphate and hysteresis. The enzyme which had not been exposed to carbamyl phosphate showed a decrease in specific activity with a change in the concentration of both carbamyl phosphate and protein. The carbamyl phosphate saturation and UMP inhibition patterns were complex with a maximum and a plateau region. The partially purified enzyme also exhibited hysteresis and the hysteretic response, a function of protein concentration, was abolished by preincubation with carbamyl phosphate and enhanced by preincubation with UMP. All these observations are compatible with a postulation that the enzyme activity may be regulated by slow reversible association-dissociation dependent on the interaction with allosteric ligands

Water stress induced alterations in ornithine aminotransferase of ragi (Eleusine coracana): protection by proline against heat inactivation and denaturation by urea and guanidinium chloride

Water stress resulted in a specific response leading to a large and significant increase (80-fold) in free proline content of ragi (Eleusine coracana) leaves and seedlings. L-Proline protected ornithine aminotransferase, an enzyme in the pathway for proline biosynthesis, isolated from normal and stressed ragi leaves against heat inactivation and denaturation by urea and guanidinium chloride. The protection of the stressed enzyme by L-proline was much more complete than that of the enzyme isolated from normal leaves. While L-ornithine, one of the substrates, protected the stressed enzyme against inactivation, it enhanced the rate of inactivation of the normal enzyme. α-Ketoglutarate protected both the normal and stressed enzyme against inactivation and denaturation. These results support the suggestion that ornithine aminotransferase has undergone a structural alteration during water stress. In view of the causal relationship between elevated temperature and water stress of plants under natural conditions, the protection afforded by proline against inactivation and denaturation of the enzyme from stressed leaves assumes significance. These results provide an explanation for a possible functional importance of proline accumulation during water stress

Transport of water-soluble vitamins in pregnancy

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Interaction of Rose bengal with Mung bean Aspartate transcarbamylase

The fluorescein dye, rose bengal in the dark: (i) inhibited the activity of mung bean aspartate transcarbamylase (EC 2.1.3.2) in a non-competitive manner, when aspartate was the varied substrate; (ii) induced a lag in the time course of reaction and this hysteresis was abolished upon preincubation with carbamyl phosphate; and (iii) converted the multiple bands observed on polyacrylamide gel electrophoresis of enzyme into a single band. The binding of the dye to the enzyme induced a red shift in the visible spectrum of dye suggesting that it was probably interacting at a hydrophobic region in the enzyme. The dye, in the presence of light, inactivated the enzyme and the inactivation was not dependent on pH. All the effects of the dye could be reversed by UMP, an allosteric inhibitor of the enzyme. The loss of enzyme activity on photoinactivation and the partial protection afforded by Nphosphonoacetyl- L-aspartate, a transition state analog and carbamyl phosphate plus succinate, a competitive inhibitor for aspartate, as well as the reversal of the dye difference spectrum by N-phosphonoacetyl-L-aspartate suggested that in the mung bean aspartate transcarbamylase, unlike in the case of Escherichia coli enzyme, the active and allosteric sites may be located close to each other

Purification, physicochemical and regulatory properties of serine hydroxymethyltransferase from sheep liver

Serine hydroxymethyltransferase (EC 2.1.2.1) was purified from the cytosolic fraction of sheep liver by ammonium sulphate fractionation, CM-Sephadex chromatography, gel filtration using Ultrogel ACA 34 and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was rigorously established by Polyacrylamide gel and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, isoelectrofocusing, ultracentrifugation, immunodiffusion and Immunoelectrophoresis. The enzyme was a homotetramer with a molecular weight of 210,000 ± 5000. The enzyme showed homotropic cooperative interactions with tetrahydrofolate (nH = 2.8) and a hyperbolic saturation pattern with L-serine. At the lowest concentration of tetrahydrofolate used (0.2 mM), only 5% of the added folate was oxidized during preincubation and assay. ThenH value was independent of the time of preincubation. Preincubation of the enzyme with serine resulted in a partial loss of the cooperative interactions (nH =1.6) with tetrahydrofolate. The enzyme was regulated allosterically by interaction with nicotinamide nucleotides; NADH was a positive effector while NAD+ was a negative allosteric effector. The subunit interactions were retained even at the temperature optimum of 60‡C unlike in the case of the monkey liver enzyme, where these interactions were absent at higher temperatures. D-Cycloserine, a structural analogue of serine caused a sigmoid pattern of inhibition, in contrast with the observations on the monkey liver enzyme. Cibacron blue F3GA completely inhibited the enzyme and this inhibition could be reversed by tetrahydrofolate. Unlike in the monkey liver enzyme, NAD+ and NADH gave considerable protection against this inhibition. The sheep liver enzyme differs significantly in its kinetic and regulatory properties from the serine hydroxymethyltransferases isolated from other sources

Studies on Aspergillus 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 grown Aspergillus 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 NH4+ , and further, the enzyme is repressed by increasing concentrations of NH4+. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4+ or L-glutamine nor regulated by covalent modification. Glutamine synthetase from Aspergillus 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. Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal 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 NH4+, 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

Interaction of cibacron blue F3G-A and procion red HE-3B with sheep liver 5,10-methylenetetrahydrofolate reductase

Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5,10-methylenetetrahydrofolate reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. The Ki values obtained by kinetic methods and the Kd value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0.9-1.2 μM. Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of the enzyme by binding at a hydrophobic region

Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH4)2 SO4 fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate. K 0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 MM, 2.45 MM and 16 MM. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong

Arginine residues involved in binding of tetrahydrofolate to sheep liver serine hydroxymethyltransferase

The arginine residue(s) necessary for tetrahydrofolate binding to sheep liver serineh ydroxymethyltransferase were located by phenylglyoxal modification. The incorporation of [7-14C]phenylglyoxal indicated that 2 arginine residues were modified per subunit of the enzyme and the modification of these residues was prevented by tetrahydrofolate. In order to locate the sites of phenylglyoxal modification, the enzyme was reacted in the presence and absence of tetrahydrofolate using unlabeled and radioactive phenylglyoxal, respectively. The labeled phenylglyoxal-treated enzyme was digested with trypsin, and the radiolabeled peptides were purified by high-performance liquid chromatography on reversed-phase columns. Sequencing the tryptic peptides indicated that Arg-269 and Arg-462 were the sites of phenylglyoxal modification. Neither a spectrally discernible 495-nm intermediate (characteristic of the native enzyme when substrates are added) nor its enhancement by the addition of tetrahydrofolate, was observed with the phenylglyoxalmodified enzyme. There was no enhancement of the rate of the exchange of the α-proton of glycine upon addition of tetrahydrofolate to the modified enzyme as was observed with the native enzyme. These results demonstrate the requirement of specific arginine residues for the interaction of tetrahydrofolate with sheep liver serine hydroxymethyltransferase