Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Nucleotidases in plants : I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus)

The occurrence of an enzyme hydrolyzing flavine adenine dinucleotide (FAD) was demonstrated in a number of seed extracts. The enzyme from Phaseolus radiatus was purified 104-fold by fractionation with ammonium sulfate and ethanol and by negative adsorption on alumina Cγ gel. The enzyme cleaves the POP bond of FAD to yield flavine mononucleotide and adenosine monophosphate. When reduced glutathione is added to the enzyme, it cleaves FAD at the COP bond to yield riboflavine, adenosine, and pyrophosphate, Both the activities are optimal at a pH of 7.2 and at a temperature of 37 . The Km for both the activities is 1.65 × 10−5 M. The stoichiometry and the identity of the products of both the treated and untreated enzyme were established. The untreated enzyme was not inhibited by pCMB or arsenite, but the treated enzyme was sensitive to both these inhibitors. The inhibition by pCMB could be reversed by monothiols and the inhibition by arsenite by dithiols.

Biosynthesis of isoleucine and valine in mycobacterium tuberculosis H37Rv*1: II. Purification and properties of acetohydroxy acid isomerase

Acetohydroxy acid isomerase (AHA isomerase) was purified about 110-fold and separated from reductase and acetohydroxy acid isomeroreductase. The AHA isomerase was found to be homogeneous by agar and polyacrylamide gel electrophoreses at different pHs. The properties of AHA isomerase have been studied. The purified enzyme showed requirement for Image -ascorbic acid and sulfate ions for its activity. Synthetic ascorbic acid sulfate could replace Image -ascorbic acid and sulfate. α-Methyllactate and α-ketoisovalerate were found to inhibit AHA isomerase activity competitively whereas Image -valine and Image -isoleucine had no significant inhibitory effect. p-Hydroxymercuribenzoate inhibited AHA isomerase activity and the inhibition was reversed by β-mercaptoethanol.

Biosynthesis of isoleucine and valine in mycobacterium tuberculosis H37Rv. II. Purification and properties of acetohydroxy acid isomeras

Acetohydroxy acid isomerase (AHA isomerase) was purified about 110-fold and separated from reductase and acetohydroxy acid isomeroreductase. The AHA isomerase was found to be homogeneous by agar and polyacrylamide gel electrophoreses at different pHs. The properties of AHA isomerase have been studied. The purified enzyme showed requirement for l-ascorbic acid and sulfate ions for its activity. Synthetic ascorbic acid sulfate could replace l-ascorbic acid and sulfate. α-Methyllactate and α-ketoisovalerate were found to inhibit AHA isomerase activity competitively whereas l-valine and l-isoleucine had no significant inhibitory effect. p-Hydroxymercuribenzoate inhibited AHA isomerase activity and the inhibition was reversed by β-mercaptoethanol.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.

Studies on the enzyme hydrolysing flavin mononucleotide in plants: I. Partial purification and properties of the enzyme from Phaseolus radiatus

The partial purification of the enzyme hydrolysing FMN from extracts of greengram seeds (Phaseolus radiatus) is described. The procedures, which entailed precipitation of inert material by manganous sulfate and protamine sulfate treatment, fractional precipitation with alcohol and chromatography on CM-cellulose, afforded preparations whose specific activity was 200 times that of the initial crude extract. The preparation was comparatively specific for FMN. It also hydrolysed, to a much smaller extent, β-glycerophosphate, p-nitrophenyl phosphate and 5′-nucleotides. The differential effects of ions on the FMN and β-glycerophosphate hydrolysing activities are discussed.

Studies on the enzyme hydrolysing flavin mononucleotide in plants. I. Partial purification and properties of the enzyme from Phaseolus radiatus

The partial purification of the enzyme hydrolysing FMN from extracts of greengram seeds (Phaseolus radiatus) is described. The procedures, which entailed precipitation of inert material by manganous sulfate and protamine sulfate treatment, fractional precipitation with alcohol and chromatography on CM-cellulose, afforded preparations whose specific activity was 200 times that of the initial crude extract. The preparation was comparatively specific for FMN. It also hydrolysed, to a much smaller extent, β-glycerophosphate, p-nitrophenyl phosphate and 5′-nucleotides. The differential effects of ions on the FMN and β-glycerophosphate hydrolysing activities are discussed.

Studies on nucleotide pyrophosphatase I. Partial purification and properties of a sheep liver enzyme that catalyzes the hydrolysis of dinucleotides

A partially purified sheep liver enzyme that hydrolyzed dinucleotides at the pyrophosphate bond was obtained by solubilizing the 18,000g sediment with n-butanol and fractionating the solubilized enzyme with acetone. The enzyme activity when measured using FAD as substrate, (FAD → FMN + AMP), was optimal at pH 9.7 and temperatures between 30 °–36 ° and at 60 °. The rate of release of FMN with time occurred with an initial lag of 30 sec, a linear increase for 1 min, and a subsequent irregular rate. In the presence of orthophosphate (Pi; 10 μImage ), FMN was released at an uniformly continuous and enhanced rate. 32Pi was not incorporated into the substrate or products. Sodium arsenate counteracted the effects of Pi. The apparent Km and Vmax were 0.133 mImage and 100 units; and 0.133 mImage and 200 units, in the absence and presence of Pi, respectively. The temperature optimum was 42 ° in the presence of Pi.Negative cooperative interactions observed at low concentrations of FAD were abolished by the addition of Pi. The inhibition by AMP was sigmoid and Pi abolished this sigmoidal response. The enzyme hydrolyzed in addition to FAD, NAD+ and NADP+. Nucleoside triphosphates were potent inhibitors of the enzyme activity. The partial inhibition of the enzyme by o-phenanthroline and by p-hydroxymercuribenzoate could be reversed by Fe2+ ions and by reduced glutathione, respectively.

Oxidation of catechol in plants : III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The Km for catechol was determined as 4 × 10−4 Image . The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3

The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 angstrom. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V-M) of 2.3 angstrom(3) Da(-1), corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.

Purification and characterization of two cellobiohydrolases from Chaetomium thermophile var. coprophile

Cellobiohydrolases I and II were purified to homogeneity from culture filtrates of a thermophilic fungus, Chaetomium thermophile var. coprophile, by using a combination of ion-exchange and gel filtration chromatographic procedures. The molecular weights of cellobiohydrolase I and II were estimated to be 60000 and 40000 and the enzymes were found to be glycoproteins containing 17 and 22.8% carbohydrate, respectively. The two forms differed in their amino-acid composition mainly with respect to threonine, alanine, methionine and arginine. Antibodies produced against either form of cellobiohydrolases failed to cross-react with the other. The tryptic maps of the two enzymes were found to be different. The temperature optima for cellobiohydrolase I and II were 75 and 70°C, and they were optimally active at pH 5.8 and 6.4, respectively. Both enzymes were stable at higher temperatures and were able to degrade crystalline cellulosic materals.