Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolus aureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 104 times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with nH values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme. H4folate; (±)-L-tetrahydrofolate

Studies on Plant Aspartate Transcarbamylase Purification and Properties of the Enzyme from Mung-Bean (Phaseolus aureus) Seedlings

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE-cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex-G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis-Menten kinetics with l-aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p-hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2-mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources.

Studies on Plant Aspartate Transcarbamylase Purification and Properties of the Enzyme from Mung-Bean (Phaseolus aureus) Seedlings

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE-cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex-G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis-Menten kinetics with l-aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p-hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2-mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources.

Studies on nucleotidases in plants: Fluorescence and kinetic properties of nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings

Nucleotide pyrophosphatase of mung bean seedlings has earlier been isolated in our laboratory in a dimeric form (Mr 65,000) and has been shown to be converted to a tetramer by AMP and to a monomer by p-hydroxymercuribenzoate. All the molecular forms were enzymatically active with different kinetic properties. By a modified procedure using blue-Sepharose affinity chromatography, we have now obtained a dimeric form of the enzyme which is desensitized to AMP interaction. The molecular weight of the desensitized form of the enzyme was found to be the same as that of the native dimeric enzyme. However, the desensitized enzyme functioned with a linear time course, contrary to the biphasic time course exhibited by the native enzyme. In addition, it was not converted to a tetramer on the addition of AMP, had only one binding site for adenine nucleotides, and p-hydroxy-mercuribenzoate had no effect on the time course of the reaction or on the molecular weight of the enzyme. The temperature optimum of the desensitized enzyme was found to be 67 °C in contrast to the optimum of 49 °C for the native dimer. Fifty percent of the tryptophan residues of the desensitized enzyme were not accessible for quenching by iodide. Fluorescence studies gave Kd values of 0.34, 2.2, and 0.8 mImage for AMP, ADP, and ATP, which were close to the Ki values of 0.12, 2.2, and 0.9 mImage , respectively, for these nucleotides. The binding and inhibition studies with AMP and its analogs showed that the 6-amino group and the 5′-phosphate group were essential for the inhibition of the enzyme activity.

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.

Affinity-Chromatographic Procedure For The Purification Of The Enzyme From Mung-Bean (Phaseolus, Aureus) Seeds And Conformational-Changes On Its Interaction With Ortho-Phosphate

Flavokinase was purified, for the first time from a plant source [mung bean (Phaseolus aureus)] by affinity chromatography in the presence of orthophosphate and by using C-8 ATP-agarose (ATP linked through the C-8 position to beaded agarose), Cibacron Blue and riboflavin--Sepharoses. An altered substrates-saturation pattern was observed in the presence of K2HPO4. The conformational changes of the enzyme in the presence of K2HPO4 were monitored by fluorescence spectroscopy. These results highlight the regulatory nature of this enzyme.

Plant aspartate transcarbamylase: An affinity chromatographic method for the purification of the enzyme from germinated seedlings

A simple and rapid affinity chromatographic method for the isolation of aspartate transcarbamylase from germinated seedlings of mung bean (Phaseolus aureus) was developed. A partially purified preparation of the enzyme was chromatographed on an affinity column containing aspartate linked to CNBr-activated Sepharose 4B. Aspartate transcarbamylase was specifically eluted from the column with 10 mImage aspartate or 0.5 Image KCl. The enzyme migrated as a single sharp band during disc electrophoresis at pH 8.6 on polyacrylamide gels. Electrophoresis of the sodium dodecyl sulfate-treated enzyme showed two distinct protein bands, suggesting that the mung bean aspartate transcarbamylase was made up of nonidentical subunits. Like the enzyme purified by conventional procedures, this enzyme preparation also exhibited positive homotropic interactions with carbamyl phosphate and negative heterotropic interactions with UMP. This method was extended to the purification of aspartate transcarbamylase from Lathyrus sativus, Eleucine coracona, and Trigonella foenum graecum.

Allelopathic plants. 7. Sunflower (Helianthus annuus L.)

Sunflower is an annual dicotyledonous plant, herbaceous, erect and native of North America. It is thermo- and photo-insensitive, hence, can be grown round the year in sub-tropical and tropical countries. Only two spp. H. annuus and H. tuberosum are cultivated for food, remaining spp. are ornamentals, weeds and wild plants. However, H. annuus is allelopathic and inhibit the growth and development of other plants thus reducing their productivity. Much information is available about the allelopathic effects of sunflower crop on following crops in crop rotations. Although it is harmful to all crops, but, is less harmful to crops of Graminae family than other families. It seems that the harmful effects of sunflower in crop rotations are due to release and accumulation of root exudates during crop growth in soil. Soil incorporation of its fresh (green manure) or dry biomass in soil is inhibitory to both crops and weed spp. Several allelochemicals have been characterized from the H. annuus, which inhibit the seed germination and seedling growth of A. albus, A. viridis, Agropyron repens (Elymus repens), Ambrosia artemsiifolia, Avena fatua, Celosia crustata, Chenopodium album, Chloris barbara, Cynodon dactylon, D. sanguinalis, Dactyloctenium ageyptium, Digitaria ciliaris, Echinochloa crus-galli, Flaveria australasica, Parthenium hysterophorus, Portulaca oleracea, Sida spinosa, Trianthema portulacastrum, Veronica perisca the inhibitory effects of this crop may be used for weed management with less herbicides for sustainable agriculture.

Two Isoforms of NADPH:Cytochrome P450 Reductase in Arabidopsis thaliana : Gene Structure, Heterologous Expression in Insect Cells, and Differential Regulation

We have investigated two NADPH-cytochrome (Cyt) P450 reductase isoforms encoded by separate genes (AR1 and AR2) in Arabidopsis thaliana. We isolated AR1 and AR2 cDNAs using a mung bean (Phaseolus aureus L.) NADPH-Cyt P450 reductase cDNA as a probe. The recombinant AR1 and AR2 proteins produced using a baculovirus expression system showed similar Km values for Cyt c and NADPH, respectively. In the reconstitution system with a recombinant cinnamate 4-hydroxylase (CYP73A5), the recombinant AR1 and AR2 proteins gave the same level of cinnamate 4-hydroxylase activity (about 70 nmol min−1 nmol−1 P450). The AR2 gene expression was transiently induced by 4- and 3-fold within 1 h of wounding and light treatments, respectively, and the induction time course preceded those of CYP73A5 and a phenylalanine ammonia-lyase (PAL1) gene. On the contrary, the AR1 expression level did not change during the treatments. Analysis of the AR1 and AR2 gene structure revealed that only the AR2 promoter contained three putative sequence motifs (boxes P, A, and L), which are involved in the coordinated expression of CYP73A5 and other phenylpropanoid pathway genes. These results suggest the possibility that AR2 transcription may be functionally linked to the induced levels of phenylpropanoid pathway enzymes.

myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate

1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).

An investigation of contact transmission of methicillin-resistant staphylococcus aureus

Hand hygiene is critical in the healthcare setting and it is believed that methicillin-resistant Staphylococcus aureus (MRSA), for example, is transmitted from patient to patient largely via the hands of health professionals. A study has been carried out at a large teaching hospital to estimate how often the gloves of a healthcare worker are contaminated with MRSA after contact with a colonized patient. The effectiveness of handwashing procedures to decontaminate the health professionals' hands was also investigated, together with how well different healthcare professional groups complied with handwashing procedures. The study showed that about 17% (9–25%) of contacts between a healthcare worker and a MRSA-colonized patient results in transmission of MRSA from a patient to the gloves of a healthcare worker. Different health professional groups have different rates of compliance with infection control procedures. Non-contact staff (cleaners, food services) had the shortest handwashing times. In this study, glove use compliance rates were 75% or above in all healthcare worker groups except doctors whose compliance was only 27%.