Browsing by Author "Chen, Jiann - Shin"
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- Identification and characterization of a receptor for Clostridium Difficile enterotoxinKrivan, Howard C. (Virginia Polytechnic Institute and State University, 1986)Clostridium difficile and its toxins are implicated as the cause of pseudomembranous colitis in patients undergoing antibiotic therapy. Very little information is known about how these toxins bind to cells and cause disease. In an attempt to understand how these toxins work this dissertation research was undertaken to determine whether a receptor for C. difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. Erythrocytes from various animal species were also examined for binding activity. Only rabbit erythrocytes could bind the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMS, rabbit erythrocytes, and to BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin binding frequency: rabbit erythrocytes > hamster BBMs > rat BBMs. Binding of toxin A to BBMS from hamsters at 37°C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4°C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal α-linked galactose. Treatment of both membrane types with α-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. An efficient, single-step method for isolation of highly purified toxin A from C. difficile culture filtrate has been developed based on toxin association with calf thyroglobulin. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes could bind the toxin. This indicated that toxin A was likely binding to Ga1α1-3Ga1ß1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate binding site for toxin A that has at least a Ga1α1—3Ga1ßl-4GlcNAc nonreducing terminal sequence.
- The pathway of electron transfer within the nitrogenase complexPeters, John W. (Virginia Tech, 1995)Site-directed mutagenesis and gene replacement were used to probe the pathway of electron transfer in nitrogenase by substituting single or groups of amino acid residues that, within the current view of component protein docking and nitrogenase catalysis, are likely to be involved in inter- or intra-molecular electron transfer. Intermolecular electron transfer was probed by substituting charged residues that, within the model for component protein docking proposed by Rees and Howard (Kim and Rees. 1992. Nature 360:553-60; Howard. 1993. In Molybdenum Enzymes, Cofactors and Model Systems, eds. EI Stiefel, D Coucouvanis, and WE Newton, pp.271-89. Washington DC: Am. Chem. Soc. 387 pp), are likely to be involved in electrostatic interactions that facilitate component protein association or dissociation. Intramolecular electron transfer was probed by substituting residues which are located in the polypeptide matrix that separates the P cluster and the iron-molybdenum cofactor based on the generally accepted view that the P cluster is an intermediate in electron transfer from the Fe protein to the iron-molybdenum cofactor at the substrate reduction site. The results of the biochemical characterization of a hybrid Azotobacter vinelandii-Clostridium pasteurianum Fe protein indicate that the region of the A. vinelandii Fe protein defined by residues 59 through 67 is involved in Fe protein-MoFe protein interaction. The rationale for construction of this hybrid Fe protein was based partially on the observation that the Fe protein from C. pasteurianum forms a tight-binding inactive complex with the MoFe protein from A. vinelandii. Detailed studies involving NaCl sensitivity and component protein ratio titrations suggest that this region may have a specific role in component protein dissociation. Further studies involving substitution of individual residues of the MoFe protein indicate that α-Asp¹⁶¹ is involved in component protein interaction. MoFe protein intramolecular electron transfer was probed by placing amino acid substitutions at β-Tyr⁹⁸, which is located directly between the P cluster and the iron-molybdenum cofactor. The results of the biochemical characterization of an altered MoFe with β-Tyr⁹⁸ substituted by His, support the generally accepted view that electron transfer from the Fe protein to the substrate reduction site involves the P cluster as an intermediate electron acceptor. It was also indicated that the P cluster may be able to accept more than one electron, which is consistent with the mechanism of P cluster reduction suggested by Rees (Rees DC, Chan MK, Kim J. 1993. Adv. Inorg. Chem. 40:89-119).
- Purification and characterization of Clostridium perfringens iota toxinStiles, Bradley G. (Virginia Polytechnic Institute and State University, 1987)Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (ib), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (ia) antigen, had little to no biological activity. When fractions containing ia and ib were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with ia or ib, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of ia and ib, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of ia and ib were mixed. Monospecific antisera against purified ia and ib neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The ia and ib proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The ia protein is heat stable (85° C/15 min) while ib lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified ia, but not ib, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor.
- Purification and characterization of Clostridium sordellii toxins HT and LT and comparison to toxins A and B of Clostridium difficileMartinez, Ramon D. (Virginia Polytechnic Institute and State University, 1989)Clostridium sordellii cause gas gangrene in man and animals, and more recently it has been implicated as a causal agent of diarrhea and enterotoxemia in domestic animals. This organism was once believed to cause pseudomembranous colitis (PMC) in humans, however, Clostridium difficile, not C. sordellii, was found to be the causative agent of this disease. It is now known that C. difficile produces two toxins, designated A and B, that are implicated in the pathogenesis of the disease. C. sordellii produces two toxins, designated HT (Hemorrhagic Toxin) and LT (Lethal Toxin), that are similar to toxins A and B of C. difficile. The goal of my research was to purify and characterize the two toxins of C. sordellii, and compare their properties to those of C. difficile. Toxin HT was purified from C. sordellii (VPI strain 9048) culture filtrate by ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography using a monoclonal antibody to toxin A of C. difficile as the ligand. Toxin LT was purified to 80% homogeneity by ultrafiltration on an XM-100 membrane filter and ion-exchange chromatography. Toxin HT migrated as a major band with molecular weight of 525,000 and a minor band at 450,000 on non-denaturing PAGE. By SDS-PAGE the molecular weight was estimated at 300,000. Isoelectric focusing indicated a pI of 6.1. Like toxin A, toxin HT was cytotoxic to cultured cells, lethal for mice, and elicited an accumulation of hemorrhagic fluid in rabbit ileal loops. Toxin LT exhibited properties similar to toxin B, although LT was about a 1000-fold less cytotoxic than toxin B. By SDS-PAGE the molecular weight was estimated at 260,000. Immunodiffusion analysis revealed a reaction of partial identity between these toxins and their amino-terminal sequences were very similar. Toxins HT and LT of C. sordellii have retained remarkable immunological similarities as well as physicochemical and biological properties with toxins A and B of Q. difficult however the toxins are not identical.
- Purification and characterization of malate dehydrogenase and 6- phosphogluconate dehydrogenase from Haemophilus influenzaeYoon, Heejeong (Virginia Polytechnic Institute and State University, 1988)Haemophilus influenzae, the primary causative factor in bacterial meningitis, displays a unique growth requirement for intact NAD. Selective inhibitors of the pyridine nucleotide-requiring enzymes from H. influenzae could have a pronounced effect on growth of the organism. Haemophilus malate dehydrogenase was purified 109-fold with a 26% recovery through a 4-step procedure involving salt fractionation, and hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of M,= 61,000. Initial velocity, product, and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme. Several NAD analogs structurally altered in either the pyridine or purine moiety functioned as coenzymes in the reaction catalyzed. Selective interactions occurring at the coenzyme binding sites were investigated. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate moiety of the coenzyme. Positive chain length effects in the coenzyme-competitive inhibition by aliphatic carboxylic acids indicated the presence of a hydrophobic region close to the pyrophosphate region at the coenzyme binding site. Several structural analogs of NAD and malate were evaluated as selective inhibitors of the enzyme. The enzyme was inactivated by incubation with diethylpyrocarbonate whereas no inactivation was observed with sulfhydryl reagents. Haemophilus influenzae 6-phosphogluconate dehydrogenase was purified 308-fold with a 16% recovery through a 4-step chromatographic procedure involving a PhenylSepharose hydrophobic column, and affinity chromatography on Matrex gel Green A, Matrex gel Red A, and 2',5’ADP-Sepharose resin. The purified enzyme was demonstrated to be a dimer of M,= 70,000. Initial velocity studies of 6-phosphogluconate oxidation indicated a sequential reaction mechanism. Although certain product and dead-end inhibition studies were consistent with an ordered mechanism, the direct binding of 6-phosphogluconate in protection experiments did not support a strictly ordered reaction sequence. Inhibition by adenosine derivatives indicated that the 2’-phosphate is important in binding to the coenzyme binding site of the enzyme. The 3-acetylpyridine analogs of NAD and NADP which support growth of H. influenzae were demonstrated to function as coenzymes with the two dehydrogenases studied. The most effective inhibitors of the purified malate dehydrogenase and 6-phosphogluconate dehydrogenase were observed to inhibit the growth of Haemophilus influenzae. However, the most potent inhibition of growth by 3-aminopyridine analogs of NAD and NADP could not be explained on the basis of interactions of these analogs with the two dehydrogenases studied.
- Roles of MoFe protein α-274-histidine, α-276-tyrosine and α-277-arginine residues in Azotobacter vinelandii nitrogenase catalysisShen, Joan (Virginia Tech, 1994)Previous studies revealed that α-275-Cys provides an essential ligand to one of the Fe atoms on the FeMo-cofactor, and its substitutions resulted in inactive nitrogenase. In order to study the structural-functional relationship of the protein environment in this region with respect to the FeMo-cofactor, subtle changes were introduced through substitutions using a site-directed mutagenesis and gene-replacement method at α-274-His, α-276-Tyr and α-277-Arg in Azotobacter vinelandii nitrogenase. Characterization of mutants strains resulting from amino acid substitutions at residues, α-274-His, α-276-Tyr or α-277-Arg, using activity assays, resulted in mixed Nif phenotypes. Therefore, none of these residues is absolutely required for nitrogenase activity. However, the changed EPR spectra of the altered MoFe proteins from some strains with substitutions at either α-276-Tyr or α-277-Arg indicated that the FeMo-cofactor environment had been perturbed by these substitutions. Together with its changed EPR spectrum, substituting α-277-Arg with His showed some extraordinary catalytic features, such as its inability to reduce N₂ while retaining respectable C₂H₂- and H⁺-reduction activities. It was also found that this altered protein used a higher percentage of total electron flux for H₂ evolution under an C₂H₂/Ar atmosphere than did wild type. Further characterization of the purified α-277his MoFe protein in parallel with its wild type counterpart revealed that the alteration in the α-277his MoFe protein caused a lower affinity for C₂H₂ binding, whereas it did not affect the CO binding. Interestingly, CO-induced cooperativity during C₂H₂ reduction was observed in this altered MoFe protein clearly indicating two sites for C₂H₄ evolution, one of which might be in the vicinity of this residue. Furthermore, the α-277his MoFe protein does not bind or reduce N₂ leading to the proposal of a nonexistent E₄ redox state in the MoFe protein catalytic cycle which was supported by stopped-flow spectrophotometric evidence. This altered α-277his MoFe protein showed comparable physical stabilities to that of the wild-type protein, and its ATP hydrolysis rates remained constant under a number of substrates assayed. Therefore, the substitution has not affected the overall protein structure, rather, it has changed the local FeMo-cofactor environment. When we studied the purified α-276his and α-274gin/α-276his MoFe proteins and compared the results with the data from the α-277his and wild-type MoFe protein, we found that there is no direct correlation between the additional set of EPR signals observed in these altered MoFe proteins and their catalytic activities. The current understanding concerning the functionality of these residues is that they are involved in maintaining a proper environment for FeMoco to bind and in stabilizing the different redox states of the enzyme during catalysis.
- Studies on the structure and function of various nif and nif- associated gene products encoded within the Azotobacter vinelandii nif gene clusterBrigle, Kevin Eugene (Virginia Polytechnic Institute and State University, 1989)The present study investigates the structural and functional roles of the metalloclusters present within the MoFe protein of nitrogenase from Azotobacter vinelandii. A gene replacement strategy was developed for oligonucleotide-directed mutagenesis of these proteins and the resulting biological and biochemical effects of these changes were examined. Identification of structurally important regions in the MoFe protein subunits and assignment of specific amino acid residues as potential metal cluster ligands were based upon several criteria: i. metallocluster extrusion requirements; spectroscopic properties of the MoFe protein; interspecies and intersubunit comparisons; iv. comparison of the MoFe protein subunit sequences to iron-molybdenum cofactor biosynthetic gene products. This mutagenesis strategy has permitted the construction of thirty-three mutant strains having specific amino acid substitutions within the MoFe protein subunits. Based on the diazotrophic growth characteristics and substrate reduction capabilities of these mutant strains, a model is presented in which potential metallocluster binding sites within the MoFe protein subunits are defined. In addition to analysis of the MoFe protein subunits, this site-directed mutagenesis and gene replacement strategy can be used to place specific mutations into any gene product encoded within the A. vinelandii nif gene cluster. Finally, nucleotide sequence analysis of the regions flanking the nifEN genes revealed the presence of three nif genes (nifT, nifY, and nifX) and four open reading frames (ORF1, ORF2, ORF3, and ORF4). Two of these genes, nifX and ORF3, were shown to be under nif control and synthesis of their products elevated in response to a demand for fixed nitrogen. Mutant strains with deletions in ORF3 appeared to accumulate an excess amount of MoFe protein when compared to wild type. The ORF3 gene product has been overproduced in E. coli. This provides an important step toward characterizing the protein and elucidating the molecular basis for its control of nifDK gene expression.