Purification and properties of benzoate-4-hydroxylase from a soil pseudomonad

Benzoate-4-hydroxylase from a soil pseudomonad was isolated and purified about 50-fold. Polyacrylamide gel electrophoresis of this enzyme preparation showed one major band and one minor band. The approximate molecular weight of the enzyme was found to be 120,000. Benzoate-4-hydroxylase was most active around pH 7.2. The enzyme showed requirements for tetrahydropteridine as the cofactor and molecular oxygen as the electron acceptor. NADPH, NADH, dithiothreitol, β-mercaptoethanol, and ascorbic acid when added alone to the reaction mixture did not support the hydroxylation reaction to any significant extent. However, when these compounds were added together with tetrahydropteridine, they stimulated the hydroxylation. This stimulation is probably due to the reduction of the oxidized pteridine back to the reduced form. This enzyme was activated by Fe2+ and benzoate. It was observed that benzoate-4-hydroxylase could catalyze the oxidation of NADPH in the presence of benzoate,p-aminobenzoate, p-nitrobenzoate, p-chlorobenzoate, and p-methylbenzoate, with only benzoate showing maximum hydroxylation. Inhibition studies with substrate analogs and their kinetic analysis revealed that the carboxyl group is involved in binding the substrate to the enzyme at the active center. The enzyme catalyzed the conversion of 1 mol of benzoate to 1 mol of p-hydroxybenzoate with the consumption of slightly more than 1 mol of NADPH and oxygen.

Properties of acetylcholinesterase from Pisum sativum

Acetylcholinesterase (AChE) from Pisum sativum purified 28 fold showed two closely moving protein bands on polyacrylamide gel electrophoresis, both of which have AChE activity. AChE activity occurs in roots, stem and leaves, that in roots varying with age. Activity is optimal at pH 9 and at 30”. The energy of activation is 9.82 x lo3 J per mol and MW is greater than 200000. Although the enzyme can hydrolyze both choline and non-choline esters, it has greater affinity for acetylthiocholine (ATCh) and acetylcholine (ACh). ATCh inhibits the enzyme at higher concentrations and the K, is 0.2 mM with this as substrate. The enzyme is not as sensitive to Eserine as it is to Neostigmine. It is also inhibited by organophosphorus pesticides such as Fensulfothion, Parathion and Dimethoate. Treatment of the seeds with Fensulfothion [O, O-diethyl (p-methylsulfinylphenyl) phosphorothioate] affects growth and secondary root development. This might be explained by its inhibition of AChE and the consequent increase of endogenous levels of ACh.

Preparation and x-ray characterization of four new crystal forms of Jacalin, a lectin from Artocarpus integrifolia

Four new crystal forms of the anti-T lectin from jackfruit (Artocarpus integrifolia) have been prepared and characterized. Three of them, two monoclinic (P21, A = 59·4 Å, B = 83·3 Å, C = 63·5 Å, β = 107·7°; C2, A = 106·1,Å, B = 53·9 Å, C = 128·0 Å, β = 95·0 Å) and one orthorhombic (C2221, A = 98·1 Å, B = 67·3 Å, C = 95·1 Å) were grown with 2-methylpentan-2,4-diol (MPD) as the precipitant while the fourth, an hexagonal from (P6122, A = b = 129·6 Å, C = 157·9 Å), was obtained in the presence of methyl-ga-Image -galactopyranoside with polyethylene glycol 4000 as the precipitant. The reported relative molecular mass (Mr) of the lectin was found to be inconsistent with the solvent content of the crystals estimated using measured densities. The Mr was redetermined using size-exclusion chromatography in the presence of methyl-α-Image -galactopyranoside and Ferguson-plot analysis of mobilities in polyacrylamide gel electrophoresis. The redetermined Mr (66,000) is consistent with the measured crystal densities. The orthorhombic and the hexagonal forms, which have one half molecule and one molecule, respectively, in the asymmetric unit, are suitable for high-resolution X-ray analysis.

Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs

An analysis of the recently reported cDNA derived amino acid sequences of mouse (Kleene and Flynn, J. Biol. Chem. , 17272–17277, 1987) and rat (Luersson Image ,Nucl. Acids Res. Image , 3585, 1989). TP2 has revealed the presence of two potential zinc finger motifs involving cysteine and histidine residues. TP2, as purified from rat elongating spermatids, is shown here to contain 0.2 atoms of zinc bound per molecule of the protein by atomic absorption spectroscopy. On incubation with 10 μM ZnCl2, Image , and subsequent exhaustive dialysis, TP2 had 2 atoms of zinc bound per molecule. The involvement of cysteine residues of TP2 in coordination with zinc was also suggested by the observation that TP2 could be labeled, Image , with iodoacetamidofluorescein only after preincubation of spermatid nuclei with EDTA. The zinc finger domains of TP2 may play an important role in initiation of chromatin condensation and /or cessation of transcriptional activity during mammalian spermiogenesis. DTT, Dithiothreitol; IAF, Iodoacetamido-fluorescein; SDS, Sodium dodecyl sulfate; PAGE, Polyacrylamide gel electrophoresis; PMSF, Phynyl methyl sulfonyl fluoride

Synthesis and Evaluation of a Novel Class of G-Quadruplex-Stabilizing Small Molecules Based on the 1,3-Phenylene-Bis(piperazinyl benzimidazole) System

Achieving stabilization of telomeric DNA in G-quadruplex conformation by Various organic compounds has been an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology, of the preformed quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T(2)G(4))(4) have been investigated using high-resolution techniques Such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li+, Na+, or K+), indicate that all the new compounds have a high affinity for G-quadruplex DNA, and the strength of the binding with G-quadruplex depends on (1) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further Suggest that these compounds are able to abet the conversion of the Intramolecular quadruplex into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded quadruplex from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes Induced by these compounds could be followed by recording the changes in the CD signal as a function of time. The implications of the findings mentioned above are discussed in this paper.

Detection of alpha(2u)-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha 2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha 2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha 2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats.This novel alpha 2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha 2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha 2u. The present investigation confirms the presence of alpha 2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.Copyright (C) 2010 John Wiley & Sons, Ltd.

Endo-polygalacturonate lyase of Cytophaga johnsonii

An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca++ ions for its maximum activity. The apparent Kmfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.

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.

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.

Isolation Of A Cytokinin-Binding Protein From Cucumis-Sativus Cotyledons

A cytokinin-binding protein which exists as monomer and dimer was isolated from cucumber (Cucumis sativus L. var Guntur) cotyledons by affinity chromatography on AH-Sepharose-pi6 Ap /s> column. The protein bound to [3H]-N6-(Δ2-isopentenyl) adenosine trialcohol. On Sephadex G-50 chromatography it gave 2 peaks corresponding to molecular weight 4000 and 8000 daltons. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, it gave only one band with an apparent molecular weight of 4000 daltons.

Ribosomal phosphoproteins in Microsporum canis

Ribosomal phosphoproteins of Microsporum canis labelled in vivo were characterised by two-dimensional and SDS polyacrylamide gel electrophoresis. A small subunit protein, S6, was the only phosphoprotein identified in 40S and 80S in basic-acidic two-dimensional gels. Three different forms of phosphorylated S6 were also observed in 40S subunit. On SDS gels five phosphoproteins were identified in 80S; of these three were present in 40S and two in 60S. S6 was the only basic phosphoprotein, while the other four were acidic.

Isolation and characterization of heat-stable allergens from shrimp (Penaeus indicus)

Shrimp are among the more common causes of immediate hypersensitivity reactions to food. To characterize better the allergenic substances within shrimp, extracts from heated shrimp were systematically examined with solid-phase radioimmunoassay and sera from patients clinically sensitive to shrimp. Two heat-stable protein allergens, designated as Sa-I and Sa-II, were identified from boiled shrimp (Penaeus indicus) extracts. Sa-I was isolated by ultrafiltration, Sephadex G-25, and diethylaminoethyl-Sephacel chromatography, whereas Sa-II, the major allergen, was purified by successive chromatography on diethylaminoethyl-Sephacel, Bio-Gel P-200, and Sepharose 4B columns. Sa-I, which was homogeneous by polyacrylamide gel electrophoresis (PAGE), elicited a single band on sodium dodecyl sulfate-PAGE corresponding to a molecular weight of 8.2 kd. Sa-II was also found to be homogeneous by PAGE, crossed immunoelectrophoresis, and immunoblotting. On sodium dodecyl sulfate-PAGE, it elicited a single band with a molecular weight of 34 kd. Sa-II was found to contain 301 amino acid residues and was particularly rich in glutamate/glutamine and aspartate/asparagine. Solid-phase radioimmunoassay-inhibition studies revealed that Sa-I and Sa-II share 54% of the allergenic epitopes, suggesting that Sa-I may be a fragment of Sa-II.SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MW, Molecular weight; BSA, Bovine serum albumin; DEAE, Diethylaminoethyl; SPRIA, Solid-phase radioimmunoassay; CIE, Crossed immunoelectrophoresis .

A possible role for a low molecular weight peptide in regulation of testosterone production by rat Leydig cells

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Percoll purified Leydig cell proteins from 20- and 120-day-old rats revealed a significant decrease in a low molecular weight peptide in the adult rats. Administration of human chorionic gonadotropin to immature rats resulted in a decrease in the low molecular weight peptide along with increase in testosterone production. Modulation of the peptide by human chorionic gonadotropin could be confirmed by Western blotting. The presence of a similar peptide could be detected by Western blotting in testes of immature mouse, hamster, guinea pig but not in adrenal, placenta and corpus luteum. Administration of testosterone propionate which is known to inhibit the pituitary luteinizing hormone levels in adult rats resulted in an increase in the low molecular weight peptide, as checked by Western blotting. It is suggested that this peptide may have a role in regulation of acquisition of responsiveness to luteinizing hormone by immature rat Leydig cells.

Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms−1 at a 0.63 nImage concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.

Isolation and characterization of thiamin-binding protein from chicken egg white

A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.