• Journal of Oral Medicine and Pain
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• 제30권3호
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• pp.329-336
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• 2005

본 연구는 superoxide의 제거물질로 알려진 pyrroloquinoline quinone (PQQ)이 파골세포의 분화 및 성숙한 파골세포의 활성에 미치는 영향을 알아보고자 시행하였다. Superoxide를 인지하는 방법인 nitroblue tetrazolium (NBT) 염색방법을 이용하여 PQQ가 HD-11 세포가 생성한 superoxide를 제거하는지 확인하였다. 본 연구에서 이용된 HD-11세포는 닭 myelomonocytic 세포주로써 1,25-dihydroxyvitamin $D_3\;[1,25(OH)_2D_3]$ 처리시 tartrate-저항성 산성인산분해효소 (tartrate-resistant acid phosphatase, TRAP)의 활성을 나타내는 등 파골세포의 특성을 지니는 세포로 알려져 있다. HD-11세포에서 TRAP 활성을 확인하기 위하여 조직화학적 염색을 시행하였다. PQQ는 NBT의 환원을 감소시켰으며 1,25(OH)2D3에 의해 유도된 TRAP 활성을 억제하였다. 또한 PQQ가 닭 골수세포에서 TRAP 양성 다핵세포의 형성에 미치는 영향도 관찰한 결과 20 ${\mu}M$의 PQQ는 TRAP 양성 다핵세포의 형성을 현저히 억제하였다. 닭 파골세포를 상아질 절편에서 배양하면서 20 ${\mu}M$의 PQQ를 처치한 경우 파골세포에 의한 상아질 흡수가 현저히 억제되었다. 따라서 본 연구결과 PQQ가 superoxide의 제거물질로 작용하여 파골세포의 분화 및 활성도에 영향을 미칠 것으로 사료되며, 이는 생리적 혹은 병적 골흡수에 억제적인 작용을 할 물질로의 가능성을 시사한다.

• Bulletin of the Korean Chemical Society
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• 제31권6호
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• pp.1711-1714
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• 2010

• 전기화학회지
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• 제2권1호
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• pp.27-30
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• 1999

Self-Assembled monolayers of carboxyl-terminated alkanethiols, which is negatively charged in pH 7.0, were usually used to facilitate the electron transfer between the positively charged protein and the electrode. In case of L-cysteine, as it has both positive and negative group, it can be a candidate for a new modifier to facilitate positively charged protein or negatively charged protein. Our investigation of L-cysteine shows that the electron transfer occurs successfully to both cytochrome c (cyt. c) and pyrroloquinoline quinone (PQQ). By using 1-ethyl-3-(3-dime-thlyaminopropyl) carbodiimide (EDC), we made a covalent bond between cyt. c and monolayer. Then PQQ was electrostatically adsorbed to the same monolayer. Cyclic voltammograms show that both molecules do not interfere each other and electron transfer is appreciable.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.95-98
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• 2000

An immunochemical method has been develooped as the most sensitive tool for studying the expression of quinoproteins containing pyrroloquinoline qinone(PQQ) in E. coli. The PQQ was conjugated to bovine serum albumin (BSA), and the conjugant was purified by using a $KwikSep^{TM}$ dextran desalting column chromatography. The PQQ-BSA conjugant was immunized to rabbits, and the IgG fractions of the antisera were purified. The most sensitive antibody against PQQ-BSA conjugant recognized some nanogram quantity of the antigen on the blot, but had little cross reactivity with BSA. Using this batch of the antibody, all the immunochemical assays of quinoproteins in E. coli were preformed. Some six different PQQ-specfic spots were detected by Western blot analysis of the soluble proteins in E. coli were performed. Some six different PQQ-specific spots were detected by Western blot analysis of the soluble proteins in E. coli after two-dimensional gel electrophoresis. Their molecular weights on the blot were estimated to be about 100-, 90-, 72-, 58-, 52-, and 50kDa. Their pI values fell in the range from 4.8 to 5.5. These results stronly suggest that quinoproteins are present in E. coli, and that the protein moieties were covalently bound to PQQ.

• The Plant Pathology Journal
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• 제22권4호
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• pp.323-328
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• 2006

Serratia marcescens W1, isolated from cucumber-cultivated soil in Suwon, Korea, evidenced profound antifungal activity and produced the extracellular hydrolytic enzymes, chitinase and protease. In order to isolate the antifungal genes from S. marcescens W1, a cosmid genomic library was constructed and expressed in Escherichia coli. Transformants exhibiting chitinase and protease expression were selected, as well as those transformants evidencing antifungal effects against the rice blast fungus, Magnaporthe grisea, and the cucumber leaf spot fungus, Cercospora citrullina. Cosmid clones expressing chitinase or protease exerted no inhibitory effects against the growth of fungal pathogens. However, two cosmid clones evidencing profound antifungal activities were selected for further characterization. An 8.2 kb HindIII fragment from these clones conditioned the expression of antagonistic activity, and harbored seven predicted complete open reading frames(ORFs) and two incomplete ORFs. The deduced amino acid sequences indicated that six ORFs were highly homologous with genes from S. marcescens generating pyrroloquinoline quinone(PQQ). Only subclones harboring the full set of pqq genes were shown to solubilize insoluble phosphate and inhibit fungal pathogen growth. The results of this study indicate that the functional expression of the pqq genes of S. marcescens W1 in E. coli may be involved in antifungal activity, via as-yet unknown mechanisms.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1261-1271
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• 2020

Cytochrome c_L (Cytc_L) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between Cytc_L and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing Cytc_L from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MP^T at 2.13 Å resolution. The crystal structure of Ma-Cytc_L revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na⁺, Ca⁺, and Fe²⁺ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-Cytc_L, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to Cytc_L within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

• Journal of Microbiology
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• 제35권3호
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• pp.200-205
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• 1997

Bothl $Ca^{2+}$ and Sr$^{2+}$-containing methanol dehydrogenases (MDH) were purified to homogeneity with yields of 48% and 42%, respectively, from Methylabacillus methanolovorus sp. strain SK5. Most of the biochemical and structural properties were similar to each other. However, some differences were found: (1) although the overall shape of the absorption spectrum of Sr$^{2+}$-MDH was very similar to that of $Ca^{2+}$-MDH, the absorption intensity originating from the cofactor in Sr$^{2+}$. MDH was higher than that in $Ca^{2+}$-MDH. Small blue shift of the maximum was also observed. These are probably due to a difference in redox state of the cofactors in $Ca^{2+}$ and Sr$^{2+}$-MDH; (2) Sr$^{2+}$-MDH was more heat-stable than $Ca^{2+}$-MDH above 56$^{\circ}C$; (3) the V$_{max}$ values for the methanol-dependent activities of Sr$^{2+}$- and $Ca^{2+}$-MDH in the presence of 3 mM KCN were 2.038 and 808 nmol/mg protein/min, respectively. In addition, the $K_{m}$ values of Sr$^{2+}$ and $Ca^{2+}$ MDH for methanol were 12 and 21 $\mu$M, respectively; (4) the endogenous activity of $Ca^{2+}$-MDH was more sensitive than that of Sr$^{2+}$-MDH in the presence of cyanide; (5) Diethyl pyrocarbonate treatment increased the enzyme activities of $Ca^{2+}$- and Sr$^{2+}$-MDH 4.2- and 1.4-folds, respectively. These results indicate that Sr$^{2+}$ stabilizes the structural conformation and enhances the activity of MDH more than $Ca^{2+}$.