Comparison of the conformation and stability of the native dimeric, monomeric, 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) from Phaseolus 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 additionof phydroxymercuribenzoate 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

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

Decensitization of allosteric sites: consequences on the conformation and catalytic properties of regulatory enzymes

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Influence of kynurenines in pathogenesis of cataract formation in tryptophan-deficient regimen in Wistar rats

543-548L-Tryptophan (Trp) is an essential amino acid and its deficiency is involved in various pathologies. In this present investigation an attempt was made to study the role of tryptophan and its metabolites in cataract formation in wistar rats. Rats were divided and maintained in 3 groups, Group A- control; Group B-marginal-tryptophan and Group C- Tryptophan-deficient diet for 3 months. Slit lamp microscope observations indicated lenticular opacities in Group-C (tryptophan-deficient) rats. In the rats that were maintained on tryptophan deficient diet, a decrease in protein content, kynurenines, reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione-s-tranferase (GSTs) and tryptophan-fluorescence intensities and an increase in lipid peroxidation indicative of oxidative stress have been observed. The above changes were normalized in the rats on supplementation of 0.05% tryptophan (Group-B) in their diets. These results suggest that tryptophan-deficiency in the diet leads to an overall significant decrease in kynurenines and levels of antioxidant enzymes (except SOD) in ocular tissue with a concomitant lenticular opacification. The results suggest that diet with adequate tryptophan has protective influence and is of immense benefit in mitigating the changes that may otherwise contribute to the lenticular opacities