Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.

Chemical Modification Studies On Ricinus-Communis (Castor Bean) Agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha- conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N- dansylgalactosamine and the lectin are consistent with a simple one- step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(- 2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.

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 U M P 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.

Individual-based modelling of the efficacy of fumigation tactics to control lesser grain borer (Rhyzopertha dominica) in stored grain

Increasing resistance to phosphine (PH 3) in insect pests, including lesser grain borer (Rhyzopertha dominica) has become a critical issue, and development of effective and sustainable strategies to manage resistance is crucial. In practice, the same grain store may be fumigated multiple times, but usually for the same exposure period and concentration. Simulating a single fumigation allows us to look more closely at the effects of this standard treatment.We used an individual-based, two-locus model to investigate three key questions about the use of phosphine fumigant in relation to the development of PH 3 resistance. First, which is more effective for insect control; long exposure time with a low concentration or short exposure period with a high concentration? Our results showed that extending exposure duration is a much more efficient control tactic than increasing the phosphine concentration. Second, how long should the fumigation period be extended to deal with higher frequencies of resistant insects in the grain? Our results indicated that if the original frequency of resistant insects is increased n times, then the fumigation needs to be extended, at most, n days to achieve the same level of insect control. The third question is how does the presence of varying numbers of insects inside grain storages impact the effectiveness of phosphine fumigation? We found that, for a given fumigation, as the initial population number was increased, the final survival of resistant insects increased proportionally. To control initial populations of insects that were n times larger, it was necessary to increase the fumigation time by about n days. Our results indicate that, in a 2-gene mediated resistance where dilution of resistance gene frequencies through immigration of susceptibles has greater effect, extending fumigation times to reduce survival of homozygous resistant insects will have a significant impact on delaying the development of resistance. © 2012 Elsevier Ltd.

Constructing a new individual-based model of phosphine resistance in lesser grain borer (Rhyzopertha dominica): do we need to include two loci rather than one?

In this article, we describe and compare two individual-based models constructed to investigate how genetic factors influence the development of phosphine resistance in lesser grain borer (R. dominica). One model is based on the simplifying assumption that resistance is conferred by alleles at a single locus, while the other is based on the more realistic assumption that resistance is conferred by alleles at two separate loci. We simulated the population dynamic of R. dominica in the absence of phosphine fumigation, and under high and low dose phosphine treatments, and found important differences between the predictions of the two models in all three cases. In the absence of fumigation, starting from the same initial frequencies of genotypes, the two models tended to different stable frequencies, although both reached Hardy-Weinberg equilibrium. The one-locus model exaggerated the equilibrium proportion of strongly resistant beetles by 3.6 times, compared to the aggregated predictions of the two-locus model. Under a low dose treatment the one-locus model overestimated the proportion of strongly resistant individuals within the population and underestimated the total population numbers compared to the two-locus model. These results show the importance of basing resistance evolution models on realistic genetics and that using oversimplified one-locus models to develop pest control strategies runs the risk of not correctly identifying tactics to minimise the incidence of pest infestation.

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

Corymbia phloem phenolics, tannins and terpenoids: interactions with a cerambycid borer

Plant secondary chemistry mediates the ability of herbivores to locate, accept and survive on potential host plants. We examined the relationship between attack by the cerambycid beetle Phoracantha solida and the chemistry of the secondary phloem (inner bark) of two differentially attacked plantation forestry taxa, Corymbia variegata and its hybrid with C. torelliana. We hypothesised that this differential rate of attack may have to do with differences in secondary chemistry between the taxa. We found differences in the bark chemistry of the taxa, both with respect to phenolic compounds and terpenoids. We could detect no difference between bored and non-bored C. variegata trees (the less preferred, but co-evolved host). Hybrid trees were not different in levels of total polyphenols, flavanols or terpenes according to attack status, but acetone extracts were significantly different between bored and non-bored trees. We propose that variations in the bark chemistry explain the differential attack rate between C. variegata and the hybrid hosts.

Studies on nucleotidases in plants : Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect. The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The Km (0.2 mImage ) and V (3.2) of the native enzyme increased on renaturation to 1.8 mImage and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn2+.

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.

Infection of mungbean seed by Macrophomina phaseolina is more likely to result from localized pod infection than from systemic plant infection

The ubiquitous fungal pathogen Macrophomina phaseolina is best known as causing charcoal rot and premature death when host plants are subject to post-flowering stress. Overseas reports of M.phaseolina causing a rapid rot during the sprouting of Australian mungbean seed resulted in an investigation of the possible modes of infection of seed. Isolations from serial portions of 10 mungbean plants naturally infected with the pathogen revealed that on most plants there were discrete portions of infected tissue separated by apparently healthy tissue. The results from these studies, together with molecular analysis of isolates collected from infected tissue on two of the plants, suggested that aerial infection of aboveground parts by different isolates is common. Inoculations of roots and aboveground parts of mungbean plants at nine temperaturexsoil moisture incubation combinations and of detached green pods strongly supported the concept that seed infection results from infection of pods by microsclerotia, rather than from hyphae growing systemically through the plant after root or stem infection. This proposal is reinforced by anecdotal evidence that high levels of seed infection are common when rainfall occurs during pod fill, and by the isolation of M.phaseolina from soil peds collected on pods of mungbean plants in the field. However, other experiments showed that when inoculum was placed within 130mm of a green developing pod and a herbicide containing paraquat and diquat was sprayed on the inoculated plants, M.phaseolina was capable of some systemic growth from vegetative tissue into the pods and seeds.

Studies on nucleotidases in plants - Reversible denaturation of the crystalline mung bean nucleotide pyrophosphatase and the effect of adenylates on the native and renatured enzyme

The crystalline mung bean nucleotide pyrophosphatase was inhibited nonlinearly by AMP, one of the products of the reaction. The partially inactive enzyme was specifically reactivated by ADP, and V at maximal activation was the same as that of the native enzyme. ATP was a linear, noncompetitive inhibitor. The kinetic evidence suggested that ADP and ATP might not be reacting at the same site as AMP. The electrophoretic mobility of the enzyme was increased by AMP, whereas ADP and ATP were without effect. The enzyme was denatured on treatment with urea or guanidine hydrochloride. The renatured and the native enzyme had the same pH (9.4) and temperature (49 °C) optimum. The Km (0.2 m ) and V (3.2) of the native enzyme increased on renaturation to 1.8 m and 8.0, respectively. In addition, renaturation resulted in desensitization of the enzyme to inhibition by low concentrations of AMP. Renaturation did not affect the reactivation of the apoenzyme by Zn2+.

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.