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Yu. V. Mezentsev A. A. Molnar O. V. Gnedenko Yu. V. Krasotkina N. N. Sokolov A. S. Ivanov 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2007,1(1):58-67
Bacterial L-asparaginases catalyzing hydrolysis of L-asparagine to L-aspartate and ammonia, are used in medical practice for treatment of acute lymphoblastic leukemia. The long-term therapy with these preparations is accompanied by a number of side effects, which are attributed to glutaminase activity of L-asparaginase. Substrate specificity and activity of L-asparaginases are directly associated with the oligomerization process of this enzyme, which is active only as the tetramer because its active sites are located in the contact areas between monomers. The present work is devoted to homology modeling of spatial structure of L-asparaginase from Erwinia carotovora, the comparative molecular-graphic analysis of subunit interfaces, and the development of a new experimental approach for studies of enzyme oligomerization. L-Asparaginase was immobilized on a surface of CM5 optical chip of biosensor Biacore 3000, which is based on the surface plasmon resonance technology. The dissociation process of enzyme tetrameric complexes up to monomers and subsequent oligomerization process have been registered. 相似文献
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Catechols are co-planar cis-diols known to form stable, isolable complexes with borate under weakly basic conditions. We exploited this chemistry and developed a boronate affinity chromatography for isolating catechol siderophores. The method was applied to the isolation of chrysobactin, enterobactin, and an unknown catechol siderophore produce by Erwinia carotovora subsp. carotovora W3C105. Yields of chrysobactin and enterobactin purified by boronate affinity chromatography were at least two-fold greater than those achieved through alternate methods. The unknown catechol produced by E. carotovora subsp. carotovora W3C105 was isolated by boronate affinity chromatography and shown to be identical to chrysobactin. Boronate affinity chromatography enabled separation of catechol from its rust-colored decomposition products, and simultaneous isolation of catechol and hydroxamate siderophores. Boronate affinity chromatography is a rapid and efficient method for purifying catechol siderophores from bacterial culture supernatants 相似文献
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Yomiko Kegoya Masato Setoguchi Keiichi Yokohiki Chitoshi Hatanaka 《Bioscience, biotechnology, and biochemistry》2013,77(4):1055-1060
An exopolygalacturonate lyase (exo-PGL) was rapidly purified from the cells of E. carotovora subsp. carotovora with a modified cross-linked pectate (mdCLPA); the material CLPA was partially degraded by an endopolygalacturonase to increase its adsorption capacity, followed by reduction with sodium borohydride. The Erwinia strain used here produced no pectolytic enzyme other than the exo-PGL under the present culture conditions. Since the mdCLPA was scarcely affected by the exo-PGL, the adsorbent can be repeatedly used for this enzyme purification with no risk of decomposition. The yield of the purified enzyme, which gave a single protein band on polyacryl-amide gel electrophoresis, was about 43%. The apparent molecular weight was about 76,000. 相似文献
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Specificity factor of yeast mitochondrial RNA polymerase. Purification and interaction with core RNA polymerase 总被引:17,自引:0,他引:17
A H Schinkel M J Koerkamp E P Touw H F Tabak 《The Journal of biological chemistry》1987,262(26):12785-12791
We have identified a mitochondrial protein from Saccharomyces cerevisiae which confers the ability to recognize mitochondrial promoters onto a nonspecifically transcribing mitochondrial core RNA polymerase and we have purified this specificity factor 10,700-fold from a whole cell extract. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fraction followed by elution and renaturation of protein activity shows that the specificity factor is a 43-kDa polypeptide which directs mitochondrial core RNA polymerase to promoters belonging to rRNA-, tRNA-, and protein-encoding genes, as well as to mitochondrial replication origins. Gel filtration and glycerol gradient sedimentation studies indicate that the specificity factor shows little association with core RNA polymerase in the absence of DNA, and that it behaves like a monomeric 43-kDa protein. 相似文献
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Polygalacturonase of Erwinia carotovora 总被引:22,自引:0,他引:22
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Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica 总被引:10,自引:0,他引:10
Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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B. Alarcon M. M. Lopez M. Cambra M. T. Gorris J. Guerri 《Journal of applied microbiology》1990,69(1):17-24
Electrotransfer of protein bands from a polyacrylamide gel to a hydrophobic poly-vinylidene difluoride (PVDF) membrane (Western blot) and their serological determination by indirect ELISA (immunoblotting) were used to differentiate Erwinia carotovora subsp. carotovora (Ecc) from Erwinia carotovora subsp. atroseptica (Eca). Ninety strains: 69 Ecc, 19 Eca and two Erwinia chrysanthemi (Echr) were examined. Eight polyclonal antisera against whole cells, glutaraldehyde fixed cells, glycopro-teins, and somatic antigens were prepared. Antisera produced with glutaraldehyde fixed cells did not recognize any band of the protein pattern. The remaining antisera recognized a limited number of bands. Two protein bands allowed differentiation of the two subspecies by the antisera against glycoproteins. One band with an estimated molecular weight of 36000 Da was present in the 19 Eca strains tested and another band with an estimated molecular weight of 35 000 Da was present in the 69 Ecc strains, except for three cases. The strains of Echr showed a band with an estimated weight of 33 000 Da. 相似文献
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S. T. SEO N. FURUYA C. K. LIM Y. TAKANAMI K. TSUCHIYA 《Journal of Phytopathology》2002,150(3):120-127
A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia. 相似文献
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Jay C.D. Hinton Michel C.M. Pérombelon George P.C. Salmond 《FEMS microbiology letters》1985,28(1):103-106
Abstract A promiscuous plasmid (pLM2) carrying amber mutations in two antibiotic-resistance genes was transferred to a derivative of Erwinia carotovora subsp. carotovora strain SCRI193. Following mutagenesis, two putative amber-suppressing mutants of this strain were isolated. The genotype of these mutants was confirmed by use of rep am plasmid-specific phage. This constitutes the first isolation of amber-suppressing mutants in Erwinia spp. 相似文献
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A novel approach is proposed for the study of the macromolecular bacteriocins of Erwinia carotovora (MCTVs). The approach lies in that the bacteriocinogeny of pectolytic erwinia is studied using a lawn of a bacterial mutant resistant to nalidixic acid, an inducer of MCTVs. The high efficiency of this approach was demonstrated by studying carotovoricins in 104 different E. carotovora strains, 88% of which bear MCTVs, distinguished by the morphology of zones of induced lysis on a lawn of susceptible cells, the lysis pattern, and some other characteristics. Preliminary studies by this approach showed that there is no correlation between the occurrence of MCTVs in particular E. carotovora strains and the habitat of the host plants from which these strains were isolated. There are grounds to believe that the approach proposed can also be used for investigating bacterial lysogeny. 相似文献
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A plasmid clone expressing a beta(1,4)-glucan glucanohydrolase (EC 3.2.1.4; endoglucanase) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subsp. carotovora. The DNA segment carrying the corresponding structural gene, named celS, contained an open reading frame encoding a 264-amino acid (aa) polypeptide. The N-terminal aa sequence of CelS showed that the protein was synthesized with a 32-aa cleavable signal peptide. The mature 232-aa CelS had a calculated Mr of 26,228 and pI of 5.5. The pH optimum was about 6.8 and the temperature optimum was between 45 and 55 degrees C. Comparison of the aa sequence of CelS by hydrophobic cluster analysis with a range of cellulases and other quasi-isofunctional enzymes revealed only very limited sequence similarities, suggesting that the CelS protein may represent the first member of an additional cellulase family. 相似文献
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Sodium fluoride induces filamentous growth of Erwinia carotovora when it is grown in liquid media containing aspartic acid only as the sole source of nitrogen. It is proposed that a stable complex of F(-)-Mg2+-enzyme and PO2(4) resulting in Mg2+ deficiency and consequent inability of E. carotovora cells to oxidize aspartic acid normally, is responsible for the formation of filaments in the presence of fluoride ions. 相似文献
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Oligogalacturonide trans-eliminase of Erwinia carotovora 总被引:10,自引:0,他引:10
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Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia. 相似文献