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.

Understanding Human Drug Conjugating Enzymes; Regio- and Stereoselectivity in Sulfotransferase 1A3 and the UDP-Glucuronosyltransferases

Sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs) are important detoxification enzymes and they contribute to bioavailability and elimination of many drugs. SULT1A3 is an extrahepatic enzyme responsible for the sulfonation of dopamine, which is often used as its probe substrate. A new method for analyzing dopamine-3-O-sulfate and dopamine-4-O-sulfate by high-performance liquid chromatography was developed and the enzyme kinetic parameters for their formation were determined using purified recombinant human SULT1A3. The results show that SULT1A3 strongly favors the 3-hydroxy group of dopamine, which indicates that it may be the major enzyme responsible for the difference between the circulating levels of dopamine sulfates in human blood. All 19 known human UGTs were expressed as recombinant enzymes in baculovirus infected insect cells and their activities toward dopamine and estradiol were studied. UGT1A10 was identified as the only UGT capable of dopamine glucuronidation at a substantial level. The results were supported by studies with human intestinal and liver microsomes. The affinity was low indicating that UGT1A10 is not an important enzyme in dopamine metabolism in vivo. Despite the low affinity, dopamine is a potential new probe substrate for UGT1A10 due to its selectivity. Dopamine was used to study the importance of phenylalanines 90 and 93 in UGT1A10. The results revealed distinct effects that are dependent on differences in the size of the side chain and on the differences in their position within the protein. Examination of twelve mutants revealed lower activity in all of them. However, the enzyme kinetic studies of four mutants showed that their affinities were similar to that of UGT1A10 suggesting that F90 and F93 are not directly involved in dopamine binding in the active site. The glucuronidation of β-estradiol and epiestradiol (α-estradiol) was studied to elucidate how the orientation of the 17-OH group affects conjugation at the 3-OH or the 17-OH of either diastereomer. The results show that there are clear differences in the regio- and stereoselectivities of UGTs. The most active isoforms were UGT1A10 and UGT2B7 demonstrating opposite regioselectivity. The stereoselectivities of UGT2Bs were more complex than those of UGT1As. The amino acid sequences of the human UGTs 1A9 and 1A10 are 93% identical, yet there are large differences in their activity and substrate selectivity. Several mutants were constructed to identify the residues responsible for the activity differences. The results revealed that the residues between Leu86 and Tyr176 of UGT1A9 determine the differences between UGT1A9 and UGT1A10. Phe117 of UGT1A9 participated in 1-naphthol binding and the residues at positions 152 and 169 contributed to the higher glucuronidation rates of UGT1A10. In summary, the results emphasize that the substrate selectivities, including regio- and stereoselectivities, of UGTs are complex and they are controlled by many amino acids rather than one critical residue.

A study of the purification and properties of tryptophan synthetase of Bengal gram (Cicer arietinum)

Active preparations of tryptophan synthetase were obtained from Bengal gram (Cicer arietinum) by the following procedure: (1) precipitation of inactive materials by manganous sulfate, (2) Adsorption of impurities on Alumina Cγ, (3) Adsorption of tryptophan synthetase on tricalcium phosphate gel, removal of inert protein from the gel by treatment with phosphate buffer (pH 7.2), and selective elution of the enzyme by 0.15 M phosphate buffer pH 7.2 containing 10% ammonium sulfate and 10−3 M serine. A 220-fold purification of the enzyme with 44% recovery of the activity was achieved. The pH optimum, effect of temperature, and substrate concentration and other properties of the purified enzyme have been studied in detail. Only the Image -isomer of serine takes part in the reaction. The Km values for indole, Image -serine, and Image -serine were calculated to be 0.66, 4.1, and 8.6 × 10−4 M, respectively. A kinetic study of the inhibition of tryptophan synthetase by indole-propionic acid has shown that it is of a competitive type. It has been demonstrated for the first time that 4-nitro-salicylaldehyde can replace pyridoxal phosphate as a coenzyme for the tryptophan synthetase reaction.

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.

Proton magnetic resonance study of the structure of two tutton's salts

Proton magnetic resonance spectra of single crystals of two Tutton's salts, K2Zn (SO4)2.6H22O and K2Mg (SO4)2.6H2O, have been studied and the orientations of the water molecules in the structure have been determined. Using the heavy-atom structure of (NH4) 2Mgt(SO4)2.6H2O as determined by x-ray diffraction, a system of hydrogen bonds between the water and sulfate oxygens in Tutton's salts has been proposed. It appears that the x-ray structure needs considerable refinement.

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.

Synthesis, Structure, and Magnetic Properties of Amine-Templated Transition-Metal Phosphites

Transition-metal phosphites of cobalt and vanadium, [C4N2H12][Co(HPO3)(2)] (I), [C4N2H14][Co(HPO3)(2)] (II), [Co[C4H8N12)(H2PO3)(2)] (III),[C4N2H14][(VF)-F-III(HPO3)(2)]center dot H2O (IV), and[C3N2H5](2)[V-4(III)(H2O)(3)(HPO3)(4)(HPO4)(3)] (V), have been synthesized and characterized. Organophosphorus esters were employed to stabilize cobalt in tetrahedral coordination and also to prepare the low-dimensional structures, which are otherwise difficult to synthesize. The structures have one- (I, II, IV), two- (III) and three-dimensionally (V) extended networks built up by the linking of metal polyhedra and phosphite units. Another vanadyl phosphite, [C2N2H10][((VO)-O-IV)(3)(H2O) (HPO3)(4)]center dot H2O,([15]) was also prepared and investigated extensively by ESR, magnetic susceptibility, and other studies. All the compounds in the present study exhibit antiferromagnetic interactions. Well-established magnetic models have been used to fit the experimental data. The compounds havealso been characterized in detail by using UV/Vis spectroscopic studies.

Potassium phosphate as a reagent for the gravimetric estimation of lithium

Previous attempts for the quantitative estimation of lithium as orthophosphate, employing an alkali metal phosphate, have not been successful. A method, is described for the estimation of lithium as trilithium phosphate from 60% ethyl alcohol solution at 65° to 70° C., employing potassium phosphate reagent, at pH 9.5. The method is applicable in the presence of varying amounts of sodium and/or potassium cations and chloride, sulfate, nitrate, and phosphate anions.

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.

Synthesis, structure, transformation studies and catalytic properties of open-framework cadmium thiosulfate compounds

Five new thiosulfate based inorganic-organic hybrid open-framework compounds have been synthesized employing mild reaction conditions. Of the five compounds, [Na-2(H2O)(8)][Cd(C10H8N2)( S2O3)(2)]center dot 2H(2)O, I and [Cd-2(C10H8N2)(2)(HS2O3)(2)(S2O3)(2)][(C10H9N2)(2)(C10H8N2)(2)]center dot 8H(2)O, II have one-dimensional (1D) structures and [Cd(C10H8N2)(H2O)(2)(S2O3)]center dot 2H(2)O, III, [Cd-2(C10H8N2)(3)(S2O3)(2)], IV and [Cd-2(C10H8N2)(2.5)(S2O3)(2)], V have three- dimensional (3D) structures. The 1D structures are somewhat related, formed by the bonding between tetrahedral Cd centers (CdN2S2) and 4,4'-bipyridine (bpy) units. The inter-chain spaces are occupied by the hanging thiosulfate units in both the cases along with Na(H2O)(6) chains in I and free bpy units in II. The three 3D structures have one-dimensional cadmium thiosulfate chains linked by bpy units. Interpenetration has been observed in all the 3D structures. The 3D structures appear to be related and can be derived from fgs net. Transformation studies on the 1D compound, [Na-2(H2O)(8)][Cd(C10H8N2)(S2O3)(2)]center dot 2H(2)O, I, indicated a facile formation of [Cd(C10H8N2)(H2O)(2)(S2O3)]center dot 2H(2)O, III. Prolonged heating of I gave rise to a 3D cadmium sulfate phase, [Cd-2(C10H8N2)(2)(H2O)(3)(SO4)(2)]center dot 2H(2)O, VI. Compound VI has one-dimensional cadmium sulfate chains formed by six-membered rings connected by bpy units to form a 3D structure, which appears to resemble the topological arrangement of III. Transformation studies of III indicates the formation of IV and V, and at a higher temperature a new 3D cadmium sulfate, [Cd(C10H8N2)(SO4)], VII. Compound VII has a 4 x 4 grid cadmium sulfate layers pillared by bpy units. All the compounds were characterized by PXRD, TGA, IR and UV-visible studies. Preliminary studies on the possible use of the 3D compounds (III-VII) in heterogeneous cyanosilylation of imines appear to be promising.

Characterization of viral proteins ofOryctes baculovirus and comparison between two geographical isolates

BacilliformOryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infectedOryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reportedOryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins stained positive for glycosylation. Comparison between the viral proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

Redistribution of subcellular calcium in rat liver on administration of vanadate

Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca2+ — stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to a-adrenergic receptors, effectively inhibited vanadate-induced Ca2+ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking agr-adrenergic agonists in vivo.