Biosynthesis of galactan by a particulate enzyme preparation from Phaseolus aureus seedlings

A particulate cell-free enzyme system was prepared from Phaseolus aureus shoots. This preparation was able to incorporate [(14)C]galactose from UDP-[(14)C]galactose into a water-soluble polysaccharide, which has a probable molecular weight of at least 4600. The only labelled component detectable in the polymer was shown to be [(14)C]galactose; two labelled oligosaccharides containing only [(14)C]galactose were isolated by partial hydrolysis. The galactan-synthesizing activity of this particulate preparation is maximal at 30 degrees and pH7.1 in the presence of 5.0mm-magnesium chloride and 0.2m-sucrose. Although 3-day-old seedlings were used as a source of enzyme, it appears that 4- or 5-day-old beans contain greater synthetase activity. The enzyme system has an apparent Michaelis constant of 5.8x10(-6)m, and will catalyse the polymerization of galactose residues at the rate of 7.5mmumoles/mg. of protein/min. at a substrate concentration of 9.6mm.     

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the P_fr/P ratio present at the beginning of the assay, such that far-red was more efficient at high P_fr/P and red at low P_fr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe Fluram (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.Abbreviations F far-red light - P_r and P_fr phytochrome in the red and far-red absorbing forms - P_tot total phytochrome - R red light - RNP ribonucleoprotein     

Further characterization of phytase from Phaseolus aureus

Phytase purified to homogeneity from germinated mungbean cotyledons was inhibited by EDTA although it did not show any absolute requirement for divalent cations. Sodium fluoride, sodium citrate, mercaptoethanol and pCMB also inhibit the phytase activity but l-phenylalanine has no effect on activity. The phytase has a low affinity for inositol monophosphate. The relative rate of dephosphorylation of myo-inositol-1 -phosphate and myo-inositol-5 phosphate by phytase is 6 and 18% respectively of that of myo-inositol-hexaphosphate. Mungbean phytase cannot cleave myo-inositol-2-phosphate, 1,2-cyclic inositol phosphate, Na-β-glycerophosphate or p-nitrophenylphosphate. The relative rates of hydrolysis of different isomers of inositol hexaphosphate are in the following order: myo-IP₆, > neo-IP₆ > scyllo-IP₆ = d-chiro-IP₆, > l-chiro-IP₆. This enzyme seems to be most active with myo-inositol hexaphosphate.     

Utilization of Selenocysteine by a Cysteinyl-tRNA Synthetase from Phaseolus aureus

An l-cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus has been purified approximately 200-fold. The enzyme uses selenocysteine as substrate in the ATP-PPi exchange assay; other cysteine analogs were inactive. The molecular weight as determined by Sephadex G-200 column chromatography is about 61,000; sodium dodecyl sulfate and 8 m urea acrylamide gel electrophoresis indicate that the enzyme is a dimer consisting of two identical monomers of molecular weight 30,000. A method for the preparation of selenocysteine from selenocystine is described.     

Large-scale isolation of prolyl-tRNA synthetase from Phaseolus aureus

Some of the problems involved in the scale-up of enzyme isolation procedures have been examined. In particular the isolation of prolyl-tRNA synthetase has been investigated. Using a modified procedure (500 × laboratory scale) the enzyme has been separated from 25 kg. of Phaseolus aureus (mung bean) and Partially purified by acid precipitation followed by fractional precipitation with ammonium sulfate. The yield of enzyme was 42 g. (10.5% recovery).     

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.     

Inositol hexaphosphate guanosine diphosphate phosphotransferase from Phaseolus aureus

Inositol hexaphosphate guanosine diphosphate phosphotransferase which transfers phosphate from inositol hexaphosphate to guanosine diphosphate, synthesizing guanosine triphosphate, has been isolated from germinating mung bean. A purification of 86-fold with 33% recovery has been obtained and the protein was made homogeneous after polyacrylamide gel electrophoresis. The MW of this enzyme was ca 92000. The optimal pH was 7·0 and Mn²⁺ was stimulatory. Inositol hexaphosphate was the most active donor of the phosphoryl group (P) to GDP. Inositol penta- or tetra-phosphate (mixed) was partially active, but inositol pentaphosphate produced in this reaction did not act further as phosphate donor. The transfer of P from inositol hexaphosphate was mediated through a phosphoprotein. Polyphosphate (poly Pi), pyrophosphate (PPi) and orthophosphate (Pi) were inactive in this reaction. ADP, CDP and UDP could not substitute for GDP, neither could dGDP nor GMP accept P from inositolphosphate. GTP inhibited the reaction, but ATP did not interfere with the reaction. The products have been shown to be [GMP- ³²P] and inositol pentaphosphate by several criteria. The reaction is practically irreversible. K_m values for GDP and inositol hexaphosphate were 1·1 × 10⁻⁴ M and 1·6 × 10⁻⁶ M respectively.     

Squalene synthetase. Solubilization from yeast microsomes of a phospholipid-requiring enzyme.

Squalene synthetase was solubilized from yeast microsomal membranes with deoxycholate. Solubilized enzyme was associated with one or more proteins with s20, w = 3.3 S, Stokes' radius = 40 A, and computed molecular weight = 54,500. In the presence of detergent the enzyme was catalytically inactive and unstable to heat. When detergent was removed with cholestyramine resin, both phases of squalene synthesis (farnesyl pyrophosphate leads to presqualene pyrophosphate leads to squalene) were recovered, and the enzyme was reaggregated to form sedimentable particles with a density of approximately 1.16 g/ml. Both activities were lost to variable extent upon chromatography over Sephadex G-200 in the presence of 0.2% deoxycholate, but could be recovered if phosphatidylcholine or phosphatidylethanolamine (but not phosphatidylserine or phosphatidylinositol) were added to fractions before removal of detergent. There was an apparently absolute requirement for phospholipid by the enzyme. The proteins catalyzing the two phases of squalene synthesis could not be resolved from one another and behaved in an identical fashion throughout a variety of manipulations.     

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.     

Methionyl-tRNA Synthetase from Phaseolus aureus: Purification and Properties

l-Methionyl-tRNA synthetase (EC 6.1.1.10) from seeds of Phaseolus aureus has been purified approximately 290-fold. Optimum assay conditions were determined by using the ATP-pyrophosphate exchange assay and the aminoacylation assay. The enzyme catalyzes both selenomethionine- and selenoethionine-dependent ATP-pyrophosphate exchange in addition to catalyzing the formation of selenomethionyl-tRNA at a rate comparable to the rate of formation of methionyl-tRNA. Competition experiments were conducted to investigate further the substrate specificity of the purified enzyme. Two peaks of methionyl-tRNA synthetase were detected by using Sephadex G-200 gel filtration; the molecular weights of the two enzymes as determined by Sephadex G-200 column chromatography were 340,000 and 85,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a tetramer consisting of four identical monomers with molecular weights of 85,000.     

Cysteinyl-tRNA Synthetase from Phaseolus aureus: Purification and Properties

l-Cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus was purified approximately 300-fold and was free of contaminating aminoacyl-tRNA synthetases. Optimum assay conditions were determined and substrate specificity and inhibitor properties were investigated using the ATP-PPi exchange reaction. The Km values for l-cysteine, ATP, and PPi were 6.20 x 10(-5)m, 1.15 x 10(-3)m, and 1 x 10(-3)m, respectively. Both l-selenocysteine (Km = 5 x 10(-5)m) and alpha-l-aminobutyric acid (Km = 1 x 10(-2)m) acted as alternative substrates of the purified cysteinyl-tRNA synthetase. The enzyme was sensitive to sulfhydryl group reagents; it was inhibited by sulfide, 0-acetylserine, and reduced glutathione.     

Purification and mode of action of phytase from Phaseolus aureus

Phytase isolated from germinated mung bean cotyledons was further purified and migrated as a single protein band in polyacrylamide gel electrophoresis.     

Solubilization of Acid-swollen cellulose by an enzyme system from a species of alternaria

An unknown species of Alternaria, when grown on a medium containing carboxymethylcellulose as a carbon source produced a mixture of extracellular enzymes which solubilized acid-swollen cellulose. The product of the hydrolysis was a 1:2 molar mixture of cellobiose and glucose. The organism apparently produced no cellobiase. It is suggested that the mixture of cellulolytic enzymes contains at least two different enzymes which degrade cellulose in an endwise manner.     

Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.     

Evidence for the existence of a novel enzyme system. myo-Inositol-1-phosphate dehydrogenase in Phaseolus aureus.

A novel enzyme system, myo-inositol-1-phosphate dehydrogenase, has been isolated from germinating mung bean seeds. The dehydrogenation and cleavage of myo-inositol 1-phosphate by this enzyme leads to the synthesis of a pentose phosphate which appears to be ribulose 5-phosphate. The pH optimum of the enzyme is 8.6; NAD+ is required as coenzyme and no other nucleotides can replace NAD+. Mono- or divalent cations are not essential for the enzyme activity. Stoichiometry of the reaction suggests that 2 mol of NAD+ are reduced per mol of ribulose-5-P generated.