돼지 간장 조직내 Protein Methylase Ⅱ 저해제의 정제 및 특성

권명희,정기경,이회영,이향우,홍성렬 ( Myung Hee Kwon,Ki Kyung Jung Hoi,Young Lee,Hyang Woo Lee,Sungyoul Hong ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.6

An inhibitor for protein methylase II (EC 2.1.1.24) was solubilized from porcine liver microsomal fraction by heat treatment, and purified by ultrafiltration, Sephadex G-25 chromatogrnphy, and HPLC using μ-Bondapak C_(18) column. The purified inhibitor was near homogeniety as judged by HPLC. The molecular weight of the inhibitor was estimated to be 1,676 Da by analysis of amino acid composition. And it was revealed that the inhibitor molecule is rich in alanine and glycine. The activity of the inhibitor was not affected by heat treatment up to 100℃ as well as hydrolytic enzymes. The K; value for the protein methylase II which has been purified from porcine spleen was measured to be 1.3 × 10^(-7) M. Inhibition studies showed that the inhibitor was noncompetitive with respect to S-adenosyl-L-methionine (SAM) and activities of several SAM-dependent methylases were also inhibited by the purified inhibitor.

돼지 간 및 정소에서 단백질 카르복실메칠화 현상

조재열(Jae Youl Cho),김성수(Hyang Woo Lee),이향우(Hyang Woo Lee),홍성렬(Sungyoul Hong) 대한약학회 2001 약학회지 Vol.45 No.1

Protein carboxy O-methylation is a kind of enzymatic reaction producing carboxy methylester catalyzed protein carboxyl O-methyltransferases at the carboxyl group of amino acid residues in polypeptide. Since the finding of carboxyl methylester, many studies have been focused on the understanding of biological functions in eukaryotes but still not clear except for roles in Ras attachment to membrane and protein repair. In this study, we investigated the protein carboxyl methylation in porcine liver and testis in repect of identification and characterization of carboxyl methylesters and natural proteinous substrates using pH stability of the esters and electrophoresis under acidic and bacis conditions. We detected several kinds of methyl esters, 3 kimds each in cytosolic fractions from liver and testis. Under the treatment of strong acid and base, the ratio between base- stable substrates and unstable ones in liver (4:6) was different from the ratio obtainde in testis (6:4). The methyl accepting capacities were affected by enzymatic proteolysis between the range of 55 to 65% in liver and of 35 to 45% in testis. Separation of the methylated proteins by acidic elelctrophoresis in the presesnce of urea and SDS revealed distinctively natural substrates of 26, 33 and 80kD in the cytosol from liver, and of 14, 25, 32 and 86kD from testis. Most of the labelling, however, were lost following electrophoreses under moderare alkaline condition, except fot molecules of newly detected 7 and 17kD in liver, and 15, 29, 40 and 80kD in testis. From these results, it was proposed that protein carboxyl O-methylation in each organs may be catalyzed by different classes of protein carboxyl O- methyltransferases. In addition, it is suggested that the protein carboxyl methylation in liver and testis may have different patterns in respect of natural substrates.

시중 유통 막걸리의 유기산 조성과 생리활성

이상진(Sang-Jin Lee),김지혜(Ji-Hye Kim),정용우(Yong-Woo Jung),박선영(Sunyoung Park),신우창(Woo-Chang Shin),박천석(Cheon-Seok Park),홍성렬(Sungyoul Hong),김계원(Gye-Won Kim) 한국식품과학회 2011 한국식품과학회지 Vol.43 No.2

막걸리는 고려시대 이후 제조된 우리나라 전통술이다. 본 연구는 막걸리의 유기산 분석과 생리 기능활성을 평가하고자 수행하였다. 막걸리의 유기산 분석 결과, 막걸리는 유기산 중에서 유산균에 의한 대사 산물인 lactic acid를 가장 많이 함유하고 있었으며 시판 막걸리에 따라 다양한 유기산 조성을 나타내고 있었다. 막걸리의 항비만 효과를 전지방세포인 3T3-L1 세포주를 사용하여 평가한 결과, 대조군 대비 막걸리는 전지방세포 분화를 저해하였으며, 특히 100 μg/mL의 농도에서 40-70% 수준으로 분화를 억제하였다. 또한 혈관신생 억제 효과와 항염증 활성을 관찰하기 위하여, 각각 CAM assay와 LPS를 처리하여 염증반응을 유발한 murine macrophage RAW264.7 세포주에서 NO를 정량하였다. 그 결과, 대부분의 막걸리는 50% 이상의 혈관신생 억제 효과를 확인하였다. 또한 농도의존적으로 NO의 생성량을 유의적 수준으로 저해하였다. 이런 결과를 종합할 때, 막걸리는 항비만, 혈관신생 억제, 그리고 항염증 활성을 가지고 있으며, 이는 막걸리의 누룩 미생물 균총의 2차 대사산물의 효과에 기인한다고 생각된다. Makgeolli is Korean traditional alcoholic beverage that has historically been brewed. In this study, we analyzed the profile of organic acids in makgeolli and also evaluated its physiological characteristics. Makgeolli contained excess lactic acid, which is produced by lactic acid bacteria (LAB). Anti-obesity effects of makgeolli were investigated in 3T3-L1 preadipocytes. Compared to the negative control, makgeolli inhibited the differentiation of preadipocyte as quantified by Oil red O dye. In particular, 100 μg/mL makgeolli reduced 40 to 70% of differentiation. To evaluate the anti-angiogenic and anti-inflammatory effects of makgeolli, we performed chorioallantoic membrane assay and measured nitric oxide production from lipopolysaccharide-induced RAW264.7 cells. Most makgeolli interrupted the formation of neo-vasculature and significantly inhibited NO production in a dose-dependent manner. Taken together, these findings suggest that commercial makgeolli has inhibitory activities against adipogenesis, neo-vascularization, and inflammation, and also they are influenced by second metabolites from nuruk microflora containing fungi and LAB.

Expression, Purification and Characterization of Protein Carboxyl O-Methyltransferase from Porcine Brain

Shim, Kishuk,Koh, Eunjin,Hong, Sungyoul 성균관대학교 생명공학연구소 2000 生命工學硏究 Vol.6 No.1

Protein carboxyl D-aspartyl and L-isoaspartyl methyltransferase (E.C.2.1.1.77) is suggested to play a role in the repair of aged protein spontaneously incorporated with isoaspartyl residues. Protein carboxyl L-isoaspartyl-O-methyltransferase which was transformed into E.coli BL32 cell carrying pET32b was expressed, purified by Nickel-nitrilotriacetic acid column, Superose 12HR chromatography and characterized its properties. Protein carboxyl L-isoaspartyl-O-methyl transferase was purified with a specific activity of 464 pmoles of ^14C-methyl per minute per mg of protein, representing 1.47 fold purification with a yield of 78.82% total activity. The molecular weight of the enzyme was 28,000 Dalton estimated by SDS-polyacrylamide gel electrophoresis. The K_m value was 0.599x10^-5 M for Histone and 0.111 x10^-6 M for S-adenosyl-L-methionine (AdoMet). S-adnosyl-L-homocysteine (AdoHcy) was showned to a competitive type of inhibitor and the K_i value was 1.38 x10^-4 M. The enzyme had optimal activity in the pH 6.0 and 37℃. This result indicated that the expressed enzyme was functionally similar to the natu+ral protein and suggests that may be a suitable model to further understanding the function of the mammalian enzyme.

The roles of neurons on the nitric oxide production of microglia stimulated by LPS and IFN-γ

Kim, Minsun,Lee, Sangjin,Jung, Kikyung,Hong, Sungyoul 성균관대학교 생명공학연구소 2001 生命工學硏究 Vol.7 No.1

Activation of glial cells often occurs at neuronal injury or death. But interaction between neuronal cells and microglia on the production of nitric oxide (NO) are not fully understood. This study aimed to elucidate whether neurons is important in modulating the production of NO in microglia stimulated by the lipopolysaccharide (LPS) and interferon-γ (IFN-γ). When PC12 cells were cocultured with microglia through direct or indirect contact, PC12 cells enhanced nitrite production in particularly LPS and IFN-γ-stimulated microglia. The NO production was decreased by a neutralizing anti-neural cell adhesion molecule (NCAM) and anti-tumor necrosis factor alpha (TNF-α) antibodies in LPS and IFN-γ-treated coculture, while neuraminidase, MMP-1 inhibitor had no effects on the cells. When TNF-α was added to PC12 cells or microglia in absence or presence of LPS and IFN-γ, results of immunoblotting and RT-PCR showed that levels of iNOS mRNA and iNOS expression were upregulated. But, level of TNF-α mRNA made no difference in untreated or treated cells. The PC12-conditioned medium (PC12CM) enhanced the nitrite production in microglia treated with LPS and IFN-γ. Blockade of the MAP kinase signal transduction pathway with either PD98059 (an inhibitor of ERK kinase) or SP203580 (an inhibitor of p38 kinase) did not inhibit the action of PC12CM. However, TPCK (an inhibitor of NF_-k B) decreased the effect of PC12CM in dose-dependent manner. These results suggest that PC12 cells induce nitrite production in microglia treated with LPS and IFN_-γ through release of soluble factors. And the soluble factors are postulated to be TNF_-α or NCAM.

Effects of Curcumin on the Modulation of Microglial Activation(Ⅱ)

Jung, ki Kyung,Lee, Jin Ho,Han, Hyung Mee,Kim, Tae Gyun,Kang, Ju Hye,Jou, Ilo,Hong, Sungyoul,Kim, Seung Hee,Kang, Seog Youn 식품의약품안전청 2000 식품의약품안전청 연보 Vol.4 No.-

뇌손상 및 Alzheiirler's disease등 만성 염증을 동반하는 뇌질환을 악화시키는 원인이 된다고 알려진 microglia의 활성화를 카레의 주성분인 curcumin이 억제하는지 알아보고자 하였다. 쟁후 0일에서 2일된 rat의 대뇌피질로부터 rrlicroglial cell을 2~3주간 배양하여 얻은 다음 LPS와 fFN-r로 활성화를 유도하고 curcumin을 I~SrM 범위로 처리했을 패, NO와 PGEr 생성에 관여하는 iNOS꽈 COX-2발현이 용량의존적으로 억제되었으며 iNOS의 경우 세포단계에서도 용량의존적으로 억제되었다. 뿐만 아니라 curcumin은 I~SrM 범위에서 염증반응을 매개하는 IL-1 및 U-6의 생성을 용량의존적으로 억제하였다. 전사주즐인자인 NF-rB는 ploinffarnmatory cytokine인 TNF-α, a-1, IL-6 및 iNOS와 COX-2등의 발현을 조절하는 것으로 알려져 있는데, curcumin이 NF-rB의 활성화를 억제하는 것으로 보아 LPS 와 IFN-γ로 활성화된 microglia 모델에서 curcumin의 항염증효과는 NF-rB pathu~·ay를 통하여 나타나는 겄으로 사료된다. -MicrogEa, braf resident macrophages, play a central role in the inflammatory responses of the brain and are activated in brain injuries and several neurodegenerative6seases such as Alzhehner's and Huntington's disease, thereby aggravating the course o(these diseases. We previ()usly repofed that curcunBin (1 ~8#M) inhibited tlte release ofinflammatory mediators such as nitric oxide (NO), prostaglandin Ef (PGEa), Tumor necrosisfactor- α (TNF-o) in Ipopolysacchafde (LPS) plus interferon- γ (HN-γ)-stimulatedmicroglia. h thiE sttdy, we examined whether curcuBCn can inhibit the production ofFfoinflarnmatdry mecators by suppressing the activation of Nuclear factor- f B (NP~ rE).Curcunfn inRbited other inflammatory cytokines, InterBeukin-1 (TL-1) and Interleukin-6 (t-6)in a dose-dependent manner, and also suppressed the induction of both inducible nitricoxide synthase fHOS) and cyclooxygenase-2 (COX-2), which catalyses the conversion ofarachidonic acid to prostagtandins. In addition, curcumin inhibited the activation of NF- f B,which controls the expresslon of a wide variety of genes active in inflammation that includecytokines (e.g., U-1, T.fF- f , IL-5), enzymes (e.g., iNOS, COX-2). Antiinflammatory effectof curcumin in cuthlred m:icroglial model seems to be mediated through NF- f B pathway,suggesting that may havc a significant impact in the prevention of immune-mediatedneurodeg enerativ e disordors.

Effects of Curcumin on the Modulation of Microglial Activation(Ⅲ)

Jung, ki Kyung,Lee, Hae Sung,Kim, Tae Gyun,Kang, Ju Hye,Cho, Mi Young,Kim, Seung Hee,Hong, Sungyoul,Kang, Seog Youn 식품의약품안전청 2001 식품의약품안전청 연보 Vol.5 No.-

노인성 치때의 원인물질로 추정되는 Af뀁타이드와 IFN- r로 운도된 microglia의 활성화를 카레의 주성분인 curcumin이 이를 억제하는지 알아보았으며, 또한 도파민에 의해 부분적으로 손상된 신경세포가 활성화된 micro잃.ia회부터 생성된 NO에 의해 손상정도가 증가하는지를 밝히고, curcumin이 NO 캥성을 막제하여 손긍된 신경세포를 보호할 수 있는지를 연구하였다. 생후 0일에서 』일된 rat의 데뇌피질로부터 microgtial cell을 2~3주간 culture하여 얻은 다음 Aβ(25-35)와 IFN-γ로 활성죡를 유도하고 curcLlmin을 2.j~20rH1 범위로 처리했을 패, NO, TNf-o, IL-lr, IL-6의 생성이 용량의존걱으로 억제되었다. 특히 ,4.Ji'(2i-35)와 IFN-r에 의해 활성화된 micro앙la 세포와 도파민에 의해 부분짙으조 손상된 신경세포(CATH.a cell)와의 coculture를 통하여 활성화된 rrllcroglia로부터 생성된 NO가 도과민에 의한 신경세포의 손상을 더욱 더 증가시켰다. 또한 이러한 신경세포의 손상이 J,fl포사멸 (apoptosis)의 형태로 나타남을 밝혔다. 이러한 모델에 curcumin을 적응한 결과 신경세포의 손상이 감소됨을 알 수 있었다. 따라서, 이러한 결과는 curcumin이 Aβ(23-3i)와 IFh- r로 microglia의 확성화를 유도했을 때 생성되는 NO를 억제함으로서 부분적으로 신경J,11포를 보호할 수 있음을 시사한다. Microglia, brain resident macrophages, play a central role in the inflammatory responses of the brain and are activated in brain injuries and several neurodegenerative diseases such as Alzheimer's and Huntigton's disease, thereby aggravating the course of these diseases. In this study we investigated whether curcumin, which is a major component of turmeric (Curcuma longa) and is known as antiinflammatory and antioxidant material, can inhibit production of inflammatory factors in amyloid β(Aβ(25-35)) and interferon-γ-stimulated microglia. Curcumin inhibited production of these factors in a dose dependent manner and also dexamethasone (DEX) inhibited production of them. Continuously, we examined whether curcumin can protect the partially dopamine-damaged neuron through suppression of the production of NO in Aβ(25-35) and interferon-γ-stimulated microglia using potentiation of CATH.a cell death induced by dopamine. Curcumin reduced CATH.a cell death by dopamine level in co-culture with immunostimulated microglia. iNOS inhibitor, NMMA and DEX also reduced CATH.a cell death. Furthermore, both curcumin and NMMA reduced apoptotic death of CATH.a cells in DAPI staining and DNA fragmentation experiments. This result suggest that curcumin can protect neuron in part through suppression of NO produced from Aβ(25-35) plus IFN-γ-stimulated microglia.