• 미생물학회지
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• 제32권1호
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• pp.84-90
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• 1994

Adenosine deaminase (ADA)의 여러 기질과 억제물질의 반응 속도론적 상수가 Aspergillusoryzae의 세포내 효소인 ADA의 활성자리에 어떻게 부착하고 어떤 요인을 필요로 하는지를 알기위해 측적되어졌다. kcat/$K_m$값에 의하면 조사된 기질로 작용하는 화합물 중에 3'-deoxyadenosine이 가장 좋은 기질로 작용하는 것으로 밝혀졌다. 몇 개의 유사체가 억제물질로 조사되었는데 purine riboside가 $3.7{\times}10^{-5}$M의 값 $K_i$값을 가지고 가장 강한 억베물질로 나타났다. Adenine은 기질로도 억제물질로도 작용을 못하므로 adenine의 N-9 위치의 ribose가 효소에 부착하는데 중요하다는 것을 시사하고 있다. 또 ADA는 6-chloropurine riboside(6-CPR)의 dechlorination을 촉매화하여 inosine과 Cl이온을 생성한다. 6-CPR의 ADA에 대한 기질 특이성은 정상 기질인 adenine의 0.86%로 측정되었다. ADA의 경쟁적 억제물질인 purine riboside는 비슷한 $K_i$값을 가지고 dechlorination도 억제하므로 deamination과 dechlorination 반응은 효소의 부착자리를 공유하고 있다고 생각되어진다. SH기에 작용하는 화합물중 수은제인 p-chloromercuribenzoate(PCMB), mersalyl acid, $HgCl_2$는 효소의 deamination 반응을 억제했다. Mersalyl acid에 의해 활성이 억제된 ADA는 thiol reagent인 dithiothreitol이나 2-mercaptoethanol에 의해 활성이 회복되지만 PCMB에 의해 억제된 효소는 회복되지 않았다. 각 수은제들이 억제물질로 작용할 때 $K_i$값과 억제양상이 측정되었는데 모두 경쟁적 방해를 보였다. $K_i$값은 10mM 인산완충용액에서 측정한 것이 100 mM 인산완충용액에서 측정한 것보다 훨씬 낮아 인산기가 기질이 아니어도 효소의 부착에 큰 영향을 미치는 것을 보여주고 있다.

• 한국의학물리학회지:의학물리
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• 제15권4호
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• pp.220-227
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• 2004

N형 칼슘통로의 비활성화기전에 관하여는 아직까지도 막전압의존성 기전과 칼슘의존성 기전간에 논란이 계속되고 있다. 2003년에 의학물리에 발표한 논문1)에서 본 연구자는 N형 칼슘통로의 비활성화 기전은 2가지 성분 -빠른 성분과 느린 성분을 가지고 있고 빠른 성분은 칼슘의존적이 아니며 오직 느린 성분만이 칼슘의존적일 가능성을 제시하였다. 본 논문에서는 막전압의존성 기전이 옳건 칼슘의존성 기전이 옳건 간에 세포 신호전달 체계로서 비활성화와 연계된 기전이 필요하므로 이러한 맥락에서 인산화 기전을 연구하였다. 흰쥐 경동맥 결절뉴론을 단일 세포로 얻은 후 whole cell patch clamp technique를 사용하여 N형 칼슘전류를 기록하고 대조 세포내액을 사용하였을 때와 phosphatase inhibitor인 okadaic acid를 포함한 세포내액을 사용하였을 때의 차이를 비교하였다. Okadaic acid에 의하여 비활성화정도가 증가되었고 이러한 okadaic acid 효과는 주로 N형 통로를 통하여 영향을 미침을 N형 칼슘통로 억제제인 $\omega$-conotoxin GVIA를 사용함으로써 확인하였다. Okadaic acid에 의한 비활성화 증가 효과는 protein kinase를 비특이적으로 억제하는 staurosporine에 의하여 억제되었고 또한 calmodulin dependent protein kinase의 특이적 억제제인 lavendustin C에 의하여 억제되었으므로 인산화과정이 N형 칼슘통로 비활성화와 관련되어 있고 특히 calmodulin을 통한 인산화과정이 주로 관여함을 확인하였다. 본 연구자가 발표한 선행논문1)에 의해 외부의 2가 양이온에 의해 빠른 비활성화가 진행되며, 본 논문에 의하여 인산화과정에 의해 빠른 비활성화가 촉진된다는 사실이 확인되었다. 그러나 본 연구결과만으로는 인산화과정이 비활성화 자체라고는 볼 수 없으며 단지 인산화과정에 의해 비활성화가 가속되었다고 해석할 수 밖에 없다. 인산화과정이 비활성화자 체인지 여부는 2가 양이온이 칼슘통로에 작용하는 결합부위에 관한 연구 및 인산화 부위가 칼슘통로인지 아니면 다른 조절 부위인지 여부를 확인할 수 있는 연구가 진행되어야 확실히 알 수 있을 것이다.

• BMB Reports
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• 제34권2호
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• pp.109-113
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• 2001

The Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). The three-dimensional structure of SmaPI was calculated by computer modeling using the structure complex between SMP and the Erwinia chrysanthemi inhibitor as a template. Based on this model structure, the substitution of the amino acid residues, Ala4, Leu-5, Pro-6, and Thr-7, were located at the hinge region of the N-terminal segment by site-directed mutagenesis. Although the A4R and T7A mutant SmaPIs showed a nearly full inhibitory activity, the inhibitory activity of SmaPI decreased significantly when the Leu-5 was converted to Ala, Gly, Ile, or Val. Surprisingly, the L5I and L5V mutant SmaPIs showed less inhibitory activities than the L5A mutant. From these results, we suggested that the orientations and positions of respective aliphatic groups in the side-chain of position 5 mainly affected the inhibitory activity of SmaPI. The overall side-chain hydrophobicity was only slightly affected. The side-chain of the Leu-5 residue contributed approximately 0.79 kcal/mol out of 8.44 kcal/mol to the binding of SmaPI with SMP The inhibitory activities of P6A and F6G were also severely decreased. The Pro-6 may have a critical role in maintaining the strict conformation of the N-terminal portion that may be important in the inhibitory activity of SmaPI. In conclusion, Leu-5 and Pro-6 have crucial roles in the inhibitory function of SmaPI toward SMP.

• 한국발생생물학회지:발생과생식
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• 제22권3호
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• pp.235-244
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• 2018

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) $E+10{\mu}M$ Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, $100{\mu}M$) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

• Biomolecules & Therapeutics
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• 제27권3호
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• pp.302-310
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• 2019

Melanoma cells have been shown to respond to BRAF inhibitors; however, intrinsic and acquired resistance limits their clinical application. In this study, we performed RNA-Seq analysis with BRAF inhibitor-sensitive (A375P) and -resistant (A375P/Mdr with acquired resistance and SK-MEL-2 with intrinsic resistance) melanoma cell lines, to reveal the genes and pathways potentially involved in intrinsic and acquired resistance to BRAF inhibitors. A total of 546 differentially expressed genes (DEGs), including 239 up-regulated and 307 down-regulated genes, were identified in both intrinsic and acquired resistant cells. Gene ontology (GO) analysis revealed that the top 10 biological processes associated with these genes included angiogenesis, immune response, cell adhesion, antigen processing and presentation, extracellular matrix organization, osteoblast differentiation, collagen catabolic process, viral entry into host cell, cell migration, and positive regulation of protein kinase B signaling. In addition, using the PAN-THER GO classification system, we showed that the highest enriched GOs targeted by the 546 DEGs were responses to cellular processes (ontology: biological process), binding (ontology: molecular function), and cell subcellular localization (ontology: cellular component). Ingenuity pathway analysis (IPA) network analysis showed a network that was common to two BRAF inhibitorresistant cells. Taken together, the present study may provide a useful platform to further reveal biological processes associated with BRAF inhibitor resistance, and present areas for therapeutic tool development to overcome BRAF inhibitor resistance.

• 한국발생생물학회지:발생과생식
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• 제25권1호
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• pp.43-53
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• 2021

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 µM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

• Journal of Photoscience
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• 제9권2호
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• pp.373-375
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• 2002

Lhcb genes encoding light-harvesting chlorophyll-a/b binding (LHC) proteins of photosystem (PS) II were comprehensively characterized using the expressed sequence tag (EST) databases in the green alga, Chlamydomonas reinhardtii. The gene family was composed of eight Lhcb genes including four new genes, which were isolated and sequenced. The effects of light intensity on the levels of mRNAs accumulation of multiple Lhcb genes were studied under various conditions. The results indicate that Lhcb genes are coordinately regulated in response to light conditions, and repressed when the input light energy exceeded the requirement for $CO_2$ assimilation. The effects of high light on the expression of the Lhcb genes observed in the presence of an electron transport inhibitor, DCMU, and in mutants deficient in photosynthetic reaction centers suggest the presence of two alternative mechanisms for regulating the genes expression under high-light conditions.

• Animal cells and systems
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• 제7권4호
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• pp.337-343
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• 2003

Methyl jasmonate (MeJA) is a signal molecule in the activation of defense responses in plants. In this study, we isolated 15 MeJA-inducible genes by subtractive hybridization. These genes encode two myrosinase-binding proteins, five lipase-like proteins, a polygalacturonase inhibitor, a putative chlorophyll-associated protein, a terpene synthase, a dehydroascorbate reductase, an ascorbate oxidase, a cysteine protease, an O-methyltransferase, and an epithiospecifier protein. Northern analysis showed that most of the Chinese cabbage genes are barely expressed in healthy leaves, but are strongly induced by MeJA treatment. We also examined whether these MeJA-inducible genes were activated by ethethon, BTH, and Pseudomonas syringae pv. tomato (Pst), a nonhost pathogen of Chinese cabbage. The results showed that none of the MeJA-inducible genes was strongly induced by ethephon or by BTH. The genes encoding lipase-like proteins and a myrosinase-binding protein were weakly induced by Pst. Other MeJA-inducible genes were not activated at all by the pathogen.

• 한국응용과학기술학회지
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• 제24권4호
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• pp.362-368
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• 2007

To study the relationship between elementary biochemical states and structural states of the actomyosin crossbridges in muscle, the effects of binding of MgADP to myosin heads in the rigor muscle were examined by X-ray diffraction using synchrotron radiation. X-ray diffraction studies have been made to investigate the effects of binding of ADP on the structure of glycerinated rabbit skeletal muscle in the rigor state. The intensity increase was accompanied by a slight but distinct decrease in the 5.9 am layer-line intensity close to the meridian. These results strongly suggest that myosin heads altered their attached conformation in the proximal end toward the plane perpendicular to the fiber axis when MgADP was bound to them. We found that the intensity of the 14.5 nm-based meridional reflections increase by 20-50% when MgADP was added to the rigor muscle in the presence of hexokinase and myokinase inhibitor.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2002년도 제9회 학술 발표회 프로그램과 논문초록
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• pp.18-18
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• 2002

Nitrogenase, comprised of the MoFe and Fe proteins, catalyzes the reduction of dinitrogen to ammonia at ambient temperature and pressure. The MoFe protein contains two metal centers, the P-cluster (Fe8S7-8) and the FeMo-cofactor (Fe7S9：homocitrate), the substrate binding site. Despite the availability of the crystal structure of the MoFe protein, suprisingly little is known about the molecular details of catalysis at the active site, and no small-molecule substrate or inhibitor had ever been shown to directly interact with a protein-bound cluster of the functioning enzyme, until our electron-nuclear double resonance(ENDOR) study of CO-inhibited nitrogenase.(omitted)