• 생명과학회지
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• 제10권2호
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• pp.210-217
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• 2000

A kinetic profile of the catalytic domain of stromelysin-1 (SCD) using the fluorescent peptide substrate has been determined by the stopped-flow technique. The pH profile has a pH optimum of about 5.5 with an extended shoulder above pH 7. Three pKa values, 5.0, 5.7, and 9.8 are found for the free enzyme state and two pH independent Kcat/Km values of 4.1$\times$104 M-1 s-1 and 1.4$\times$104 M-1 s-1 at low and high pH, respectively. The profile is quite different in shape with other MMP family which has been reported, having broad pH optimum with two pKa values. The substrate specificity of SCD towards fluorescent heptapeptide substrates has been also examined by thin layer chromatography. The cleavage sites of the substrates have been identified using reverse-phase HPLC method.SCD cleaves Dns-PLA↓L↓WAR and Dns-PLA↓L↓FAR at two positions. However, the Dns-PLA↓LRAR, Dns-PLE↓LFAR, adn Dns-PLSar↓LFAR are cleaved exclusively at one bond. The double cleavages of Dns-PLALWAR and Dns-PLALFAR by SCD are in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis agrees with the structural data that the S1' pocket of SCD is deeper than that of matriysin. The differences observed between SCD and matrilysin may form the basis of understanding the structural relationships and substrate specificities of the MMP family in vivo.

• 대한수의학회지
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• 제43권3호
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• pp.439-448
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• 2003

The VP2 gene of infectious bursal disease virus (IBDV) Chinju which was previously detected in Chinju, Korea was cloned and sequenced to establish the information for the development of genetically engineered vaccines and diagnostic reagents against IBDV. The nucleotide sequence of the entire Chinju VP2 gene consisted of 1,356 bases long encoding 452 amino acids in a single open reading frame (ORF). It consisted of 368 adenine (27.1%), 363 cytosine (26.8%), 339 guanine (25.0%) and 286 thymine (21.1%) residues. The predicted $M_r$ of the Chinju VP2 protein was 48 kDa, and the protein contained 13 phosphorylation sites by protein kinase C, casein kinase II or tyrosine kinase, whereas 3 asparagine-linked glycosylation sites were recognized. The nucleotide sequence of Chinju VP2 ORF had a very close phylogenetic relationship with 98-99% homology to that of the very virulent IBDVs (vvIBDVs) HK46, OKYM, D6948, UK661, UPM97/61 and BD3/99. Also, the Chinju VP2 protein revealed a very close phylogenetic relationship with 99-100% homology to that of these vvIBDVs. The Chinju VP2 protein had 100% amino acid identity in the variable region of residues 206-360 with that of the D6948, HK46, OKYM and UK661, as well as 100% identity in two hypervariable regions of residues 212-224 and 314-324 with those of the D6948, HK46, OKYM, UK661, UPM97/61 and BD3/99. The amino acid sequence of the chinju VP2 protein contained a serine-rich heptapeptide of SWSASGS as in these vvIBDVs.

• 한국자기공명학회논문지
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• 제19권2호
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• pp.67-73
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• 2015

The microcystin is a cyclic heptapeptide from metabolites of cyanobacteria in the genera mycrocystis, anabaeba as a result of eutrophication. It has been known that microcystin-LR is a potent inhibitor of the catalytic subunits of protein phosphatase-1 (PP-1) as well as powerful tumor promoter. The active site of microcystin actually has two metal ions $Fe^{2+}/Zn^{2+}$ close to the nucleophilic portion of PP-1-microcystin complex. We report the isolation and purification of this microcystin-LR from cyanobacteria (blue-green algae) obtained from Daechung Dam in Chung-cheong Do, Korea. Microcystin-LR was extracted from solid-phase extraction (SPE) sample preparation using a CN cartridge. The cyanobacteria extract was purified to obtain microcystin-LR by HPLC method and identified by LC/MS. The detail structural studies that can elucidate the possible role of monovalent and divalent metal ions in PP-1-microcystin complexation were carried out by utilizing molecular dynamics. Conformational changes in metal binding for ligands were monitored by molecular dynamic computation and potential of mean force (PMF) using the method of the free energy perturbation. The microcystin-metal binding PMF simulation results exhibit that microcystin can have very stable binding free energy of -10.95 kcal/mol by adopting the $Mg^{2+}$ ion at broad geometrical distribution of $0.5{\sim}4.5{\AA}$, and show that the $K^+$ ion can form a stable metal complex rather than other monovalent alkali metal ions.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.859-865
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• 2003

Surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a ${\beta}-hydroxy$ fatty acid produced by several strains of Bacillus sp. Surfactin isoforms produced by endophytic Bacillus sp. CY22 from a balloon flower were isolated and characterized. It was found that the purified surfactin had three isoforms with protonated masses of m/z 1,008, 1,022, and 1,036, and different structures in combination with Na, K, Ca ions using MALDI-TOF MS, ESI-MS/MS, and ICP MS, respectively. In the MS/MS analysis, the isolated surfactin had the identical amino acid sequence (LLVDLL) and hydroxy fatty acids (with 13 to 15 carbons in length), even though isolated from different Bacillus strains. The sfp22 gene, required for producing the surfactin, consisted of an open reading frame (ORF) of 675 bp encoding 224 amino acid residues with a signal peptide of 20 amino acids. The predicted amino acid sequence of sfp22 was very similar to that of Ipa-8.

• Journal of Microbiology and Biotechnology
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• 제13권1호
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• pp.50-56
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• 2003

A freshwater bloom-forming cyanobacterium, Microcystis aeruginosa, and local soil isolate Scytonema sp. strain BT 23 were demonstrated to contain biotoxic secondary metabolites with pesticidal and mosquito larvicidal activities. A purified toxic constituent from M aeruginosa showed an absorption maximum at 230 nm and its toxicity symptoms, Rf value on TLC, and retention time observed ill an HPLC analysis were similar to those of the hepatotoxic heptapeptide microcystin-LR. The bioactive constituent of the Scytonema sp. was less polar in nature and exhibited two peaks at 240 and 285 m. When applied to two cruciffrous pests, Pieris brassicae and Plutella flostella, the crude extracts and toxic principles from the two cyanobacteria showed significant antifeedant activity in a no-choice bioassay, and at higher concenuations exhibited contact toxicity to the insect larvae. The purified toxin from M. aeruginosa was found to be more effective and produced 97.5 and $92.8\%$ larval mortality in the two pests, fo11owing 2 h of toxin treatment at a concentration of $25{\mu}g$ Per leaf disc (2.5 cm dia.). Meanwhile, similar treatment with the purified toxin from Sytonema sp. stain BT 23 only produced 73 and $78\%$ mortality in the two pests. The cyanobacterial constituents also showed significant activity against Culex and Anopheles larvae. The M. aeruginosa toxin ($20{\mu}g\;ml^-1$) caused 98.2 and $88.1\%$ mortality in the Culex and Anopheles larvae, respectively, while the purified toxin from the Sytonema sp. was less toxic and only produced a 96.3 and $91.2\%$ mortality, respectively, at a much higher concentration ($40{\mu}g\;ml^-1$). Accordingly, the current results point to certain hitherto unknown biological properties of cyanobacterial biotoxins.

• Environmental Engineering Research
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• 제14권4호
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• pp.250-254
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• 2009

Contamination of microcystins, a family of heptapeptide hepatotoxins, in eutrophic water bodies is a worldwide problem. Due to their poisoning effects on animals and humans, there is a requirement to characterize and quantify all microcystins present in a sample. As microcystins are, for most part, intracellular toxins produced by some genera of cyanobacteria, lysing cyanobacterial cells to release all microcystins is considered an important step. To date, although many cell lysis methods have been used, little work has been conducted comparing the results of those different methods. In this study, various methods for cell lysis and toxin extraction from the cell lysates were investigated, including sonication, bead beating, freeze/thaw, lyophilization and lysing with TritonX-100 surfactant. It was found that lyophilization, followed by extraction with 75% methanol, was the most effective for extracting toxins from Microcystis aeruginosa cells. Another important step prior to the analysis is removing impurities and concentrating the target analyte. For these purposes, a C18 Sep-Pak solid phase extraction cartridge was used, with the percentage of the eluent methanol also evaluated. As a result, methanol percentages higher than 75% appeared to be the best eluting solvent in terms of microcystin-leucine-arginine (MC-LR) recovery efficiency for the further chromatographic and mass spectrometric analyses.

• 대한수의학회지
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• 제46권3호
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• pp.235-248
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• 2006

The VP2 full gene of Korean infectious bursal disease virus(IBDV) strain, SH/92, three attenuated vaccine strains, Bur706, Bursine-2 and CEV/AC strains, were amplified by reverse transcriptase-polymerase chain reaction and sequenced and compared with published VP2 gene sequences of IBDVs. The VP2 nucleotide sequence similarity between SH/92 and three vaccine stains was 95.6~96.5% whereas the nucleic acid similarity among three vaccine strains was 97.5~98.5%. The amino acid sequence similarity of VP2 of SH/92 compared with three vaccine strains was between 94.4 and 97.6% while the amino acid similarity among three vaccine strains was between 97.4 and 98.4%. The amino acid similarity between SH/92 and classical virulent strain, 52/70 and STC strain was 96.4 and 96.5%, respectively. The serine-rich heptapeptide was conserved in CEVAC and Bursine-2 as well as SH/92 but not in Bur706. The phylogenetic tree developed from amino acid sequences showed that SH/92 was categorized with vv IBDVs(HK46, OKYM, KKI, UPM94/273, SH95) in one branch while three vaccine strains were catagorized with STC strain in the other branch.

• Journal of Microbiology
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• 제43권6호
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• pp.516-522
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• 2005

The phosphorylation of C-terminal domain (CTD) of Rpb1p, the largest subunit of RNA polymerase II plays an important role in transcription and the coupling of various cellular events to transcription. In this study, its role in DNA damage response is closely examined in Saccharomyces cerevisiae, focusing specifically on several transcription factors that mediate or respond to the phosphorylation of the CTD. CTDK-1, the pol II CTD kinase, FCP1, the CTD phosphatase, ESS1, the CTD phosphorylation dependent cis-trans isomerase, and RSP5, the phosphorylation dependent pol II ubiquitinating enzyme, were chosen for the study. We determined that the CTD phosphorylation of CTD, which occurred predominantly at serine 2 within a heptapeptide repeat, was enhanced in response to a variety of sources of DNA damage. This modification was shown to be mediated by CTDK-1. Although mutations in ESS1 or FCP1 caused cells to become quite sensitive to DNA damage, the characteristic pattern of CTD phosphorylation remained unaltered, thereby implying that ESS1 and FCP1 play roles downstream of CTD phosphorylation in response to DNA damage. Our data suggest that the location or extent of CTD phosphorylation might be altered in response to DNA damage, and that the modified CTD, ESS1, and FCP1 all contribute to cellular survival in such conditions.