Vitreoscilla Catalase의 분리 및 특성에 관한 연구

박기인,박충웅 全北大學校 基礎科學硏究所 1994 基礎科學 Vol.17 No.-

Vitreoscilla는 헤모글로빈을 갖고 있는 그람 음성 미생물체로써, 이 헤모글로빈을 통한 산소호흡으로부터 파생된 과산화수소를 많은 양의 catalase를 합성하여 효과적으로 분해한다. 이 효소의 분자량은 약 250,000 daltons이며, subunit의 구조는 모든 다른 미생물체에서 보여주는 A_4와는 달리 A_2B_2(A:64,000 daltons, B:58,000 daltons)를 보였다. 최적 pH는 7.0∼8.0이었고, oxidized form의 absorption spectra에서 Soret peak는 406㎚에서 나타났으며 reducing agent인 sodium dithionite로부터 야기된 reduced form의 Soret peak는 442㎚로 이동되었다. 또한 이 효소는 열에 불안정 했고, K_m값은 0.016M hydrogen peroxide를 나타냈으며, tumover number는 25,000mol이었다. 0.25mM potassium cyanide는 경쟁적 저해로써 이 효소의 활성을 저해하였으며, 효소-저해제 복합체의 dissociation constant는 0.67mM을 보였다. 두 subunits의 N-말단 아미노산 서열이 다음 실험을 위한 oligonucleotide 합성을 위하여 결정되었다. Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin and grows very well under the condition of low oxygen concentration. It also contain a bacterial catalase to be not correspond with another species on genus Beggiatoa. The primary function of Vitreoscilla catalase may be to remove hydrogen peroxide produced by VitHb oxidation. The molecular size of the catalase was estimated to be approximately 250,000 Da. The subunit structure of this enzyme may be A_2B_2 (A: MW 64,000 Da, B: MW 58,000 Da) but is not clear in the research reported here. Optimum pH is 7.0∼8.0 for catalase activity and Soret peak on absorption spectra of oxidized catalase is represented in 406㎚ and Soret peak of reduced form from sodium dithionite moved at 442㎚. Vitreoscilla catalase is unstable a high temperature, and its Michealis constant, K_m, was 0.016 M hydrogen peroxide. The tumover number of the enzyme was 25,000mol. The 0.25mM potassium cyanide was competitive inhibitor and the dissociation constant of the enzyme-inhibitor complex was 0.67mM. N-terminal amino acid sequences of two subunits are determined for the probe synthesis using to the cloning of Vitreoscilla catalase gene.

Colchicine과 EMS 돌연변이 처리가 고추냉이 식물체의 단백질 Pattern에 미치는 영향

박기인,은종선,최선영,권성환,이강수,소상섭,윤명자 韓國藥用作物學會 1997 한국약용작물학회지 Vol.5 No.1

고추냉이의 배수체를 유도하기 위하여 EMS와 colchicine을 처리하였을 때 변화되는 단백질과 과산화효소 활성은 다음과 같이 나타났다. 발아율은 무주산이 울릉도산 보다 높았으며, GA3 100 ppm+ EMS 10 ppm 혼용처리시 무처리구에 비하여 모두 6배 이상 증가되었다. Mutagen 처리후 생존율도 울릉도산 보다 무주산에서 더 높게 나타났으며, 과산화효소 활성과 와사비의 초장은 감소되어 나타난 반면, 줄기와 뿌리는 비대되는 경향을 보였다. 무주산과 울릉도산의 단백질 양상이 비슷하고, mutagen을 처리했을 때 polypeptide spot수가 증가되었으나 단백질 pattern은 다르게 나타났고, 특히 EMS 처리는 무처리구에 비하여 현저하게 다른 양상을 보였다. This study was investigated on the effects of colchicine and EMS. induced mutation, plant on the germination and polypeptide pattern and peroxidase activity monitored by two dimensional gel analysis in Wasabia japonica Matsum (wasabi). Germination rate of Muju was higher than that of Ulrung-Do and optimum concentration for germination was appeared 100 ppm GA3 containing with 10 ppm BAP in these cultivars. Survived plants rate of Muju was higher than that of Ulrung-Do after colchicine and EMS treatment. Peroxidase activity and plant height were decreased by mutagen treatments, while incresed in root and stem thickness. The number of protein spots and pattern showed difference between Muju and Ulrung-Do . The plants treated mutagen increased polypeptide spots, especially EMS treatment showed more different polypeptide pattern compared to control.

Colchicine과 EMS 처리에 의한 고추냉이의 변이체 유도

박기인,윤명자,권성환,최선영,이강수 한국약용작물학회 1996 한국약용작물학회지 Vol.4 No.1

Wasabia japonica종자의 생장조절물질에 의한 발아율 돌연변이 유도, 유도체의 핵형 분석과 isozyme pattern을 조사한 결과는 다음과 같다. 1) 고추냉이의 종자 발아는 GA3 100ppm 처리 시 36%, BAP 10ppm 처리 시 32%, GA3 100ppm+BAP 10ppm 조합 처리 시 65%의 높은 발아율을 나타냈다. 2) colchicine과 EMS처리로 인한 돌연변이 유도에 따른 생존율은 발아전 처리의 경우 colchicine 0.5%일때 62% 생존율을 보이며, 발아 후 처리는 colchicine 0.5% 일때 85%, EMS 0.06% 일때 55%의 생존율을 보였다. 3) colchicine과 EMS처리로 유도된 식물체의 핵형 분석은 colchicine 0.5%처리로 유도된 식물체에서 다수의 4배체를 관찰할 수 있있으며, EMS 0.06% 처리의 유도체에서는 정상 배수체만이 관찰되었다. 4) 돌연변이원으로 유도된 식물체의 peroxidase pattern은 colchicine 처리 식물체가 정상체와 다른 양상 (1 band)을 보이는 반면, EMS처리 식물체는 정상체와 같은 양상 (3 band)을 보였다. This study was investigated to improve the rate of the seed germination of Wasabia japonica with GA3 and BAP. The germination rate was 36% with 100ppm GA3 only and 32% with 10ppm BAP only, while that was 65% with combination of 100ppm GA3 and 10ppm BAP compared to that of 7.5% in control. This results show us that the most effect of germination rate was with 100ppm GA3 and 10ppm BAP. Mutagenesis was induced with mutagen, EMS and colchicine. Before the germination, the survival rate was 62% with 0.5% colchicine compared to that of 7% in control. After the germination, the 85% of developed seed was alive with treatment of 0.5% colchicine and 55% of those was alive with 0.06% EMS compared to that of 8% in control. We analyzed the karyotype and isozyme pattern of Wasabia japonica induced with colchicine and EMS. The tetratype of Wasabia japonica was observed from Wasabia japonica treated with 0.5% colchicine. whereas it was not observed with 0.06% EMS. The peroxidase pattern of colchicine treated Wasabia japonica was different from nontreatment. but that of EMS treated Wasabia japonica was the same with normal one.

Vitreoscilla Catalase 의 분리 및 특성에 관한 연구

박기인,박충웅 ( Kie In Park,Chung Ung Park ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.6

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin and grows very well under the condition of low oxygen concentration. It also contain a bacterial catalase to be not correspond with another species on genus Beggiatoa. The primary function of Vitreoscilla catalase may be to remove hydrogen peroxide produced by ViWb oxidation. The molecular size of the catalase was estimated to be approximately 250,000 Da. The subunit structure of this enzyme may be A₂B₂ (A : MW 64,000 Da, B : MW 58,000 Da) but is not clear in the research reported here. Optimum pH is 7.0∼8.0 for catalase activity and Soret peak on absorption spectra of oxidized catalase is represented in 406 ㎚ and Soret peak of reduced form from sodium dithionite moved at 442 ㎚. Vitreoscilla catalase is unstable a high tempernture, and its Michealis constant, K_m was 0.016 M hydrogen peroxide. The turnover number of the enzyme was 25,000 mol. The 0.25 mM potassium cyanide was competitive inhibitor and the dissociation constant of the enzyme-inhibitor complex was 0.67 mM. N-terminal amino acid sequences of two subunits are determined for the probe synthesis using to the cloning of Vitreoscilla catalase gene.

月南 李商在의 民族主義思想 小考

朴基仁 경희사학회 1982 경희사학 Vol.9ㆍ10 No.-

RANKL에 의해 유도되는 뼈파괴세포 분화에 맥문동의 효과

박기인(Kie-In Park),오재민(Jaemin Oh) 대한체질인류학회 2011 해부·생물인류학 (Anat Biol Anthropol) Vol.24 No.4

뼈파괴세포의 생성이나 활성 증가는 원하지 않는 뼈 흡수를 야기한다. 뼈파괴세포의 분화와 기능을 억제하는 천연물에 대한 연구가 뼈엉성증성 뼈질환의 치료와 예방을 위해 진행중이다. 맥문동은 동양의학에서 진해, 거담작용, 구갈 작용을 억제하는 작용 및 강장작용, 혈당 조절 작용, 이뇨작용, 변비를 조절하는 데 사용되어 왔다. 또한 최근에는 항염증 작용과 항혈전작용, 항유착작용이 있는 것으로 보고되고 있다. 본 연구의 목적은 맥문동이 뼈파괴세포 분화에 미치는 영향과 그 기전을 알아보기 위한 것이다. 맥문동 물 추출물은 RANKL에 의해 유도되는 큰포식세포의 뼈파괴세포로의 분화를 농도 의존적으로 억제하였다. 그러나 맥문동 추출물은 세포 독성은 나타내지 않았다. c-Fos, NFATc1, tartrate resistant-acid phosphatase (TRAP), cathepsin K의 mRNA의 발현이 맥문동 추출물에 의해 억제되었다. 또한 RANKL에 의해 유도되는 c-Fos, NFATc1의 단백질 발현도 저해되었다. 신호 경로 중에서 맥문동 추출물은 RANKL에 의해 유도되는 p38의 인산화를 저해하였다. 그러므로, 맥문동은 뼈파괴세포 분화에 필수 유전자인 c-Fos, NFATc1의 발현을 억제함으로 뼈파괴세포 분화를 억제한다. 결론적으로 이러한 결과는 맥문동이 특이한 독성 없이 뼈엉성증을 치료하는 유용한 후보물질이 될 수 있음을 암시하는 것이다. Increased formation and activation of osteoclast lead to unwanted bone resorption. Several natural products which have inhibitory effects on osteoclast differentiation and function are under investigation to prevent and treat the osteoporotic bone disease. Liriopis tuber has been used in Oriental medicine for the suppression of cough, expectoration, thirst, and has been used for sthenia, diuresis, blood glucose regulation, treatment of xerostomia, and constipation. Also, recently it has been reported that Liriopsis tuber has anti-inflammatory, anti-thrombic, anti-adhesive activities. The purpose of this study was to evaluate the effects of Liriopis tuber on osteoclast differentiation and was to evaluate of its mechanism. Water extract of Liriopis tuber significantly inhibited receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages (BMMs) in a dose dependent manner. However, water extract of Liriopis tuber did not affect cytotoxicity when compared with control. The mRNA expression of c-Fos, NFATc1, tartrate resistant-acid phosphatase (TRAP), and cathepsin K induced by RANKL was inhibited by water extract of Liriopis tuber treatment. Also, water extract of Liriopis tuber inhibited the protein expression of c-Fos and NFATc1 expression in BMMs treated with RANKL. Among the signal pathways, water extract of Liriopis tuber suppressed the phosphorylation of p38 induced by RANKL. In summary, Liriopis tuber exerted inhibitory effects on osteoclast diffentiation via suppression of c-Fos and NFATc1 which are essential gene to osteoclastogenesis. Taken together, these results suggest that Liriopis tuber may be a useful candidate in the treatment of osteoporosis without special toxicity.

Vitreoscilla C₁에서 Paraquat와 Iron에 의한 Iron Superoxide Dismutase의 유도

박기인(Kie-In Park) 한국생물공학회 2003 KSBB Journal Vol.18 No.6

Vitreoscilla Hemoglobin 유전자가 재조합 대장균의 성장조건에 미치는 영향

박충웅,박기인 한국유전학회 1994 Genes & Genomics Vol.16 No.4

In this rearch plasmid pMK79 was engineered by pMK57 which contain Bacillus stearothermophilus α-amylase gene into pUC8 and pUC8: 15 which has Vitreoscilla hemoglobin gene (VtHb). Both pMK57 and pMK79 were transformed into Escherichia coli. The presence of Vitreoscilla hemoglobin expressed VtHb improves the growth of MK79 (pMK79 transformant) in an oxygen limited environment compared to strain MK57 (pMK57 trasformant) as using efficiently oxygen. Expression of the amylase gene in both systems was also measured. The result showed that amylase production in MK79 was also significantly higher in mid log and late log phases than in MK57. It was also found that α-amylase gene transcription can occur from the B. stearothermophilus promoter as well, at induced levels, from the lac promoter in pUC8.

PC12 세포 신경전달물질 방출 저해제 S9940이 신경세포 돌기신장에 미치는 영향

이윤식,박기인,Lee, Yun-Sik,Park, Kie-In 한국독성학회 1998 Toxicological Research Vol.14 No.3

We identified S9940, a novel microbial metabolite from Streptomyces spp., to inhibit the release of neurotransmitter from PC12 cells. S9940 is an inhibitor of trifiated norepinephrine ([$^{3}H$]-NE) release in high $K^+$ buffer solution containing ionomycin, indicating that S9940 inhibits neurotransmitter release after the influx of $Ca^{2+}$ ions. We also examined the effect of S9940 on $\beta-glucuronidase$ release from guinea pig neurophils and the effect on the neurite extension of PC12 cells and rat hippocampal neurons. As a result, S9940 inhibited $\beta-glucuronidase$ release: when treated with $5{\mu}g/ml$ of S9940, which prevented [$^{3}H$]-NE release, the inhibition of neurite extension for both PC12 cells and rat hippocampal neurons was observed.

PC12 세포에서 신경전달물질 방출을 저해하는 물질 S9940 물질의 탐색

이윤식,박기인,Lee, Yun-Sik,Park, Kie-In 한국독성학회 1998 Toxicological Research Vol.14 No.3

We established an in vitro experimental system using the following procedure. We first introduced tritium-labelled norepinephrine ([3H]-NE)into PC12 cells. The [3H]-NE incorporated into PC12 cells were then stimulated by a high concentration (60 mM) of $K^+$ during 12 minutes. Then, we counted the amount of [3H]-NE release from PC12 cells with the scintillation counter. After screening fungal, Streptomyces or bacterial product using this experimental system, we obtained S9940 from Streptomyces spp. which inhibited [3H]-NE release from PC12 cells. S9940 also inhibits the release of ATP as a neurotransmitter of PC12 cells and rat cortical neurons. The inhibitory effect was seen even when the PC12 cells were treated with low $K^+$ buffer containing ionomycin $(1\muM)$ as an ionopore. This result suggests that the inhibitory action of S9940 on neurotransmitter release appeared after the influx of $Ca^{2+}$.