A hemagglutinin from Pongamia pinnata (L.) Pierre seed

Lectins are diverse proteins that bind to carbohydrates and are involved in many important physiological processes. They are highly specific to the sugar molecules they bind and therefore, have many therapeutic and diagnostic applications. There are several lectins that display antiviral, antibacterial antifungal and antitumour activities. Characterization of new lectins paves way for comprehension of their diverse biological roles and mechanism of action, thus aiding in further exploration of lectins in various domains of biology. Here, we endeavoured to purify and characterize lectin from the seed of the legume,Pongamia. Pongamia pinnata (L.) Pierre (syn. Millettia pinnata), seed lectin (PPSL) was purified conventionally by ammonium-sulfate precipitation followed by size exclusion chromatography. The further lectin was physicochemically characterized by CD, fluorescence spectroscopy, mass spectroscopy and isothermal calorimetry. Hemagglutination studies with various mono and disaccharides showed specificity towards galactose. This specificity was reaffirmed by isothermal studies with appreciable thermodynamic parameters. Lectins have tremendous diagnostic applications. They are used as second-generation drug delivery systems.

Methylenetetrahydrofolate reductase gene polymorphisms and risk of acute lymphoblastic leukemia in children.

Introduction: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and is involved in DNA synthesis, DNA repair and DNA methylation. Genetic polymorphisms of this enzyme have been shown to impact several diseases, including cancer. Leukemias are malignancies arising from rapidly proliferating hematopoietic cells having great requirement of DNA synthesis. This case-control study was undertaken to analyze the association of the MTHFR gene polymorphisms 677 C"T and 1298 A"C and the risk of acute lymphoblastic leukemia in children. Materials and Methods: Eighty-six patients aged below 15 years with a confirmed diagnosis of acute lymphoblastic leukemia (ALL) and 99 matched controls were taken for this study. Analysis of the polymorphisms was done using the polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) method. Results: Frequency of MTHFR 677 CC and CT were 85.9% and 14.1% in the controls, and 84.9% and 15.1% in the cases. The 'T' allele frequency was 7% and 7.5% in cases and controls respectively. The frequency of MTHFR 1298 AA, AC, and CC were 28.3%, 55.6% and 16.1% for controls and 23.3%, 59.3% and 17.4% for cases respectively. The 'C' allele frequency for 1298 A→C was 43.9% and 47% respectively for controls and cases. The odds ratio (OR) for C677T was 1.08 (95% CI 0.48- 2.45, p = 0.851) and OR for A1298C was 1.29(95% CI 0.65-2.29, p = 0.46) and OR for 1298 CC was 1.31 (95% CI 0.53-3.26, p =0.56). The OR for the combined heterozygous status (677 CT and 1298 AC) was 1.94 (95% CI 0.58 -6.52, p = 0.286). Conclusion: The prevalence of 'T' allele for 677 MTHFR polymorphism was low in the population studied. There was no association between MTHFR 677 C→T and 1298 A→C gene polymorphisms and risk of ALL, which may be due to the small sample size.

Assembly of recombinant coat protein of sugarcane streak mosaic virus into potyvirus-like particles.

Coat protein (CP) gene of sugarcane streak mosaic virus-AP isolate (SCSMV-AP) was expressed in E. coli and recombinant CP (SCSMV-AP rCP) was purified by linear sucrose density gradient centrifugation. Observation of purified SCSMV-AP rCP under electron microscope revealed the presence of potyvirus-like particles (PVLPs). The assembled particles were shown to encapsidate CP gene transcripts by slot-blot hybridization.

Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate.

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.

Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus.

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5' -phosphate (PLP) dependent enzyme catalyzes the interconversion of L-Ser and Gly using tetrahydrofolate as a substrate. The gene encoding for SHMT was amplified by PCR from genomic DNA of Bacillus stearothermophilus and the PCR product was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme was isolated as a mixture of dimer (90%) and tetramer (10%). This is the first report demonstrating the existence of SHMT as a dimer and tetramer in the same organism. The specific activities at 37 C of the dimeric and tetrameric forms were 6 7 U/mg and 4 1 U/mg, respectively. The purified dimer was extremely thermostable with a T(m) of 85 degrees C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80 degrees C with a specific activity of 32 4 U/mg at this temperature. The enzyme catalyzed tetrahydrofolate-independent reactions at a slower rate compared to the tetrahydrofolate-dependent retro-aldol cleavage of L-Ser. The interaction with substrates and their analogues indicated that the orientation of PLP ring of B. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT).

Affinity properties of phosvitin: interaction of phosvitin with serine hydroxymethyl transferase.

The affinity of phosvitin with serine hydroxymethyl transferase (SHMT), an acidic multi-subunit protein, was evaluated by measurements of enzyme activity, sedimentation velocity, steady-state fluorescence, circular dichroism and kinetic thermal stability. While the presence of phosvitin had no effect on the SHMT activity, the sedimentation coefficient of SHMT increased from 8.7 S to 12.5 S suggesting the formation of a complex at a SHMT:phosvitin molar ratio of 2:1. Based on steady-state fluorescence quenching measurements an association constant of 2.4 +/- 0.2 x 10(5) M-1 at 25 degrees C was obtained for the interaction of phosvitin with SHMT. The temperature dependency of the association constant in the range 15-35 degrees C suggests the involvement of ionic forces in the interaction. The thermal inactivation of SHMT followed first order kinetics. In the presence of phosvitin the rate constant decreased and half time increased. The circular dichroism measurements suggest that phosvitin interaction does not involve pyridoxal phosphate binding domain of the enzyme. Although minor changes in the secondary structure of the enzyme were observed, the environment around aromatic amino acids did not change significantly.

Interactions of 3' terminal and 5' terminal regions of physalis mottle virus genomic RNA with its replication complex.

Physalis mottle virus (PhMV) belongs to the tymogroup of positive-strand RNA viruses with a genome size of 6 kb. Crude membrane preparations from PhMV-infected Nicotiana glutinosa plants catalyzed the synthesis of PhMV genomic RNA from endogenously bound template. Addition of exogenous gnomic RNA enhanced the synthesis which was specifically inhibited by the addition of sense and antisense transcripts corresponding to 3' terminal 242 nucleotides as well as the 5' terminal 458 nucleotides of PhMV genomic RNA while yeast tRNA or ribosomal RNA failed to inhibit the synthesis. This specific inhibition suggested that the 5' and 3' non-coding regions of PhMV RNA might play an important role in viral replication.

An unusual distribution of glucose-6-phosphate dehydrogenase deficiency of south Indian newborn population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is seen at a higher frequency in many national and ethnic groups in areas of current or former malaria endemicity. A screening programme undertaken to evaluate the gene frequencies for this deficiency in the highly inbred South Indian population of Karnataka revealed that of the 5140 neonates screened, 7.8% were G6PD deficient with no correlation between the reported level of inbreeding and enzyme deficiency. An interesting finding was the equal number of male (198) and female (207) individuals, with G6PD activity of less than 3 IU. The possible implications of this finding with regard to the expression of G6PD gene is discussed.

Characterization of a variant glucose-6-phosphate dehydrogenase from the south Indian population.

Glucose-6-phosphate dehydrogenase (G6PD) is coded by a gene on the X-chromosome. Earlier studies have shown that the South Indian population has a high incidence of this enzyme deficiency. The electrophoretic mobility, pH optimum and the Km values for G6PD from normal and variant individuals were identical. However, the specific activity of the variant enzyme was 8 times less compared to the value of the normal enzyme. Western blot analysis of partially purified G6PD from normal and variant individuals performed using equal amounts of total protein showed that the variant protein was 3 times less in concentration. Similar analysis performed using protein corresponding to equal enzyme activity units in the normal and variant samples showed that the variant enzyme was 2.25 times less efficient compared to the normal enzyme. RNA dot blot analysis using full length G6PD cDNA probe (PGDT5B, a kind gift from Prof. L Luzzatto) revealed that lymphocytes from normal and variant individuals had equal amounts of G6PD specific mRNA.

Primary structure of sesbania mosaic virus coat protein: its implications to the assembly and architecture of the virus.

Sesbania mosaic virus (SMV) is a plant virus that infects Sesbania grandiflora plants in Andhra Pradesh, India. The amino acid sequence of the coat protein of SMV was determined using purified peptides generated by cleavage with trypsin, chymotrypsin, V8 protease and clostripain. The 230 residues so far determined were compared to the corresponding residues of southern bean mosaic virus (SBMV), the type member of sobemoviruses. The overall identity between the sequences is 61.7%. The amino terminal 64 residues, which constitute an independent domain (R-domain) known to interact with RNA, are conserved to a lower extent (52.5%). Comparison of the positively charged residues in this domain suggests that the RNA-protein interactions are considerably weaker in SMV. The residues that constitute the major domain of the coat protein, the surface domain (S-domain, residues 65-260), are better conserved (66.5%). The positively charged residues of this domain that face the nucleic acid are well conserved. The longest conserved stretch of residues (131- 142) corresponds to the loop involved in intersubunit interactions between subunits related by the quasi 3-fold symmetry. A unique cation binding site located on the quasi 3-fold axis contributes to the stability of SMV. These differences are reflected in the increased stability of the SMV coat protein and its ability to be reconstituted with RNA at pH 7.5. A major epitope was identified using monoclonal antibodies to SMV in the segment 201-223 which contains an exposed helix in the capsid structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of poly- and monoclonal antibodies to physalis mottle (tymo) virus.

Polyclonal antibodies were raised against the Physalis mottle virus (PhMV) and its denatured coat protein (PhMV-P). Analysis of the reactivity of the polyclonal antibodies with tryptic peptides of PhMV-P in dot-blot assays revealed that many of the epitopes were common to intact virus and denatured coat protein. Five monoclonal antibodies to the intact virus were obtained using hybridoma technology. These monoclonal antibodies reacted well with the denatured coat protein. Epitope analysis suggested that probably these monoclonal antibodies recognize overlapping epitopes. This was substantiated by epitope mapping using the CNBr digest of PhMV-P in western blots. All the five monoclonals recognized the N-terminal 15 K fragment. Attempts to further delineate the specific region recognized by the monoclonals by various enzymatic cleavages resulted in the loss of reactivity in all the cases. The results indicate that these monoclonals probably recognize epitopes within the N-terminal 15 K fragment of the coat protein.

Partial amino acid sequence of sheep liver serine hydroxymethyltransferase and comparison of peptide maps of the enzyme from human, ox livers and Escherichia coli.

A comparison of the tryptic peptide maps of serine hydroxymethyltransferase from sheep, human, ox livers and Escherichia coli revealed that the mammalian enzymes were similar, while the bacterial enzyme exhibited differences in the primary structure. N-terminus of the reduced carboxymethylated sheep liver enzyme was acetylated. Serine hydroxymethyltransferase was hydrolyzed with trypsin and fragments of peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase and ion-pair chromatography. The peptides were sequenced manually using the 4-N,N'-dimethyl aminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double coupling method. The tryptic peptides with 80% homology or above were aligned on the rabbit liver enzyme sequence.

Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase.

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.

In vitro expression of belladonna mottle virus genome.

In vitro translation of belladonna mottle virus BDMV(I) genomic RNA in a rabbit reticulocyte lysate system produced proteins of Mr 210,000, 150,000 and 78,000 which form the non-structural proteins. The coat protein, on the other hand, was expressed from a subgenomic RNA which was found to be encapsidated in the empty capsids forming the top component viral particles. The implications of subgenomic RNA encapsidation in viral replication and assembly are discussed.

Isolation and characterization of a naturally occurring inhibitor from mung bean (Vigna radiata) seedlings for serine hydroxymethyltransferase.

A naturally occurring inhibitor of serine hydroxymethyltransferase (EC 2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98 degrees C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.

Interaction of Cibacron Blue F3G-A and Procion Red HE-3B with sheep liver 5,10-methylenetetrahydrofolate reductase.

Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5, 10-methylenetetrahydrofolate reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. The Ki values obtained by kinetic methods and the Kd value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0·9-1·2 μM. Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of the enzyme by binding at a hydrophobic region.

Similarities in the genomic sequence and coat protein structure of plant virsuses.

The genomic sequences of several RNA plant viruses including cucumber mosaic virus, brome mosaic virus, alfalfa mosaic virus and tobacco mosaic virus have become available recently. The former two viruses are icosahedral while the latter two are bullet and rod shaped, respectively in particle morphology. The non-structural 3a proteins of cucumber mosaic virus and brome mosaic virus have an amino acid sequence homology of 35% and hence are evolutionarily related. In contrast, the coat proteins exhibit little homology, although the circular dichroism spectrum of these viruses are similar. The non-coding regions of the genome also exhibit variable but extensive homology. Comparison of the brome mosaic virus and alfalfa mosaic virus sequences reveals that they are probably related although with a much larger evolutionary distance. The polypeptide folds of the coat protein of three biologically distinct isometric plant viruses, tomato bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus have been shown to display a striking resemblance. All of them consist of a topologically similar 8-standard β-barrel. The implications of these studies to the understanding of the evolution of plant viruses will be discussed.

Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver.

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH4)2 SO4 fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphatepolyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate. K0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 μΜ, 2.45 μΜ and 16 μΜ. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus.

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses – cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.