• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 2003년도 정기총회 및 학술발표회
• /
• pp.45-45
• /
• 2003

We isolated a novel ankyrin-repeat containing protein, rSIAP (rSlo Interacting Ankyrin-repeat Protein), as an interacting protein to the cytosolic domain of the alpha-subunit of rat large-conductance Ca$\^$2+/-activated K$\^$+/ channel (rSlo) by yeast two-hybrid screening. Affinity pull-down assay showed the direct and specific interaction between rSIAP and rSlo domain. The channel-binding proteins can be classified into several categories according to their functional effects on the channel proteins, i.e. signaling adaptors, scaffolding net, molecular tuners, molecular chaperones, etc. To obtain initial clues on its functional roles, we investigated the cellular localization of rSIAP using immunofluorescent staining. The results showed the possible co-localization of rSlo and rSIAP protein near the plasma membrane, when co-expressed in CHO cells. We then investigated the functional effects of rSIAP on the rSlo channel using electrophysiological means. The co-expression of rSIAP accelerated the activation of rSlo channel. These effects were initiated at the micromolar [Ca$\^$2+/]$\_$i/ and gradually increased as [Ca$\^$2+/]$\_$i/ raised. Interestingly, rSIAP decreased the inactivation kinetics of rSlo channel at micromolar [Ca$\^$2+/]$\_$i/, while the rate was accelerated at sub-micromolar [Ca$\^$2+/]$\_$i/. These results suggest that rSIAP may modulate the activity of native BK$\_$Ca/ channel by altering its gating kinetics depending on [Ca$\^$2+/]$\_$i/. To localize critical regions involved in protein-protein interaction between rSlo and rSIAP, a series of sub-domain constructs were generated. We are currently investigating sub-domain interaction using both of yeast two-hybrid method and in vitro binding assay.

• 약학회지
• /
• 제33권1호
• /
• pp.64-71
• /
• 1989

Since time-dependent inactivation of MAO was found to be complete in a few minutes when high concentration ratios of tranylcypromine to MAO were used, a method to obtain kinetic parameters was sought suitable to the conditions in which concentrations of tranylcypromine analogs did not exceed that of MAO. For the purpose, kinetic equations were derived and the method applied to the kinetic studies of tranylcypromine enantiomers. It was found that (E)-(+)-2-phenylcyclopropylamine inhibited MAO by the mechanism following bimolecular reaction scheme with $\tilde{K}_i$ of $2.0\;{\times}\;10^6M^{-1}min^{-1}$. Whereas, MAO-inhibitory pattern of the (-)-enantiomer was to be interpreted by suicide inhibition scheme and measured $k_{in}\;and\;\tilde{K}'$ were $0.457\;min^{-1}\;and\;$5.4{\mu}M$, respectively.

• 한국식품과학회지
• /
• 제34권6호
• /
• pp.1067-1072
• /
• 2002

팽이 및 만가닥버섯의 가공 기술 개발의 일환으로 수행되진 본 연구는 가공 전 처리 공정으로서의 blanching 조건을 확립하기 위해 두 버섯 peroxidase의 열적 특성 결과를 기초 자료로 삼았다. 효소 활성 측정은 Gorin과 Heidema의 방법을 변형하여 사용하였고, 생 버섯으로부터 추출한 조효소핵을 열처리했을 경우 팽이버섯은 $75^{\circ}C$, 3분, 만가닥버섯의 경우 $80^{\circ}C$, 1분 이상의 온도, 시간 조건에서 peroxidase의 불활성이 유발됨을 알았다. 이때 팽이 및 만가닥버섯 peroxidase의 활성에너지$(E_a)$는 59.58 kcal/mol, 43.05 kcal/mol이며 z값은 $9.0^{\circ}C,\;12.4^{circ}C$였다. 반면 Blanching에 의한 두 버섯 peroxidase의 활성화에너지($E_a$)와 z값은 각각 7.97 kcal/mol, 6.55 kcal/mol와 $59.8^{\circ}C,\;74.1^{circ}C$로 나타났다. 이는 peroxidase 자체의 열적 특성과 blanching에 의해 유발된 peroxidase의 열적 특성이 다르다는 것을 보여주는 것으로 blanching시에는 버섯자체와 버섯내의 성분들에 의해 열에 대한 저항성이 증대되어 z값이 높아지는 결과를 유발, 활성화에너지($E_a$)가 감소하는 결과로 나타났다.

• 한국진공학회:학술대회논문집
• /
• 한국진공학회 2012년도 제43회 하계 정기 학술대회 초록집
• /
• pp.270-270
• /
• 2012

Low temperature atmospheric pressure plasmas (APPs) have been known to be effective for living cell inactivation in the water [1]. Many earlier research found that pH level of the solution was changed from neutral to acidic after plasma treatment. The importance of the effect of acidity of the solution for cell treatments has already been reported by many experiments. In addition, several studies have demonstrated that the addition of a small amount of oxygen to pure helium results in higher sterilization efficiency of APPs [2]. However, it is not clear yet which species are key factors for the cell treatment. To find key factors, we used GMoo simulation. We elucidate the processes through which pH level in the solution is changed from neutral to acidic after plasma exposure and key components with pH and air variation with using GMoo simulation. First, pH level in a liquid solution is changed by He+ and He(21S) radicals. Second, O3 density decreases as pH level in the solution decreases and air concentration decreases. It can be a method of removing O3 that cause chest pain and damage lung tissue when the density is very high. H2O2, HO2 and NO radicals are found to be key factors for cell inactivation in the solution with pH and air variation.

• Bulletin of the Korean Chemical Society
• /
• 제27권5호
• /
• pp.657-662
• /
• 2006

Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of $Mg^{2+}$. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.

• 조명전기설비학회논문지
• /
• 제23권9호
• /
• pp.17-23
• /
• 2009

자외선 살균기술을 바탕으로 한 수처리 분야에 대한 관심이 최근 증가하고 있다. 수처리분야에서 살균처리방식의 효율은 유체 살균챔버나 살균강도 그리고 미생물의 불활성역학에 따라 수처리 성능이 결정된다. 광산화법에 이용되는 빛은 주로 자외선이 이용되며 매우 깨끗하고 높은 에너지를 가지고 있어 화학살균방법에 비해 잔류물이 없고 높은 안정성으로 최근 관심이 높아지고 있다. 그러나 자외선살균방식은 조사시간과 조사량에 직접적인 영향을 받아 이를 위한 최적의 설계가 이루어져야 한다. 본 연구에서는 유수살균장치의 챔버 내의 자외선 램프와 유수에 대한 3D-CFD discrete ordinates model 모델을 제시하고 이를 시뮬레이션을 통해 설계방식의 최적화 여부를 검증하고 향후 유수형 자외선살균방식의 시뮬레이션방법을 제안하고자 한다.

• BMB Reports
• /
• 제33권2호
• /
• pp.172-178
• /
• 2000

The lipoxygenase was purified 35 fold to homogeneity from the Korean red potato by an ammonium sulfate precipitation and DEAE-cellulose column chromatography. The simple purification method is useful for the preparation of pure lipoxygenase. The molecular weight of the enzyme was estimated to be 38,000 by SDS-polyacrylamide gel electrophoreses and Sepharose 6B column chromatography. The purified enzyme with 2 M $(NH_4)_2SO_4$ in a potassium phosphate buffer, pH 7.0, was very stable for 5 months at $-20^{\circ}C$. Because the purified lipoxygenase is very stable, it could be useful for the screening of a lipoxygenase inhibitor. The optimal pH and temperature for lipoxygenase purified from the red potato were found to be pH 9.0. and $30^{\circ}C$, respectively. The Km and Vmax values for linoleic acid of the lipoxygenase purified from the red potato were $48\;{\mu}M$ and $0.03\;{\mu}M$ per minute per milligram of protein, respectively. The enzyme was insensitive to the metal chelating agents tested (2 mM KCN, 1 and 10mM EDTA, and 1 mM $NaN_3$), but was inhibited by several divalent cations, such as $Cu^{++}$, $Co^{++}$ and $Ni^{++}$. The essential amino acids that were involved in the catalytic mechanism of the 5-lipoxygenase from the Korean red potato were determined by chemical modification studies. The catalytic activity of lipoxygenase from the red potato was seriously reduced after treatment with a diethylpyrocarbonate (DEPC) modifying histidine residue and Woodward's reagent (WRK) modifying aspartic/glutamic acid. The inactivation reaction of DEPC (WRK) processed in the form of pseudo-first-order kinetics. The double-logarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order 2, indicating that two histidine residues (carboxylic acids) were essential for the lipoxygenase activity from the red potato. The linoleic acid protected the enzyme against inactivation by DEPC(WRK), revealing that histidine and carboxylic amino acids residues were present at the substrate binding site of the enzyme molecules.

• Applied Biological Chemistry
• /
• 제30권3호
• /
• pp.278-284
• /
• 1987

사과의 건조, 가공 중의 갈변을 방지하기 위한 기초 조사로서 후지 사과로부터 추출한 crude polyphenol oxidase의 특성과 열에 대한 저항성, 갈변 저해제의 저해 효과 등을 조사하였다. Catechol을 기질로 사용하였을 때 polyphenol oxidase의 최적 pH는 5.5, 최적온도는 $20^{\circ}C$, $K_m$ 값은 0.14M이었고, 열불활성화는 유사일차반응을 보였으며 이때 활성화에너지$(E_a)$와 Z값은 각각 23.0cal/kmol, $19.7^{\circ}C$였다. 기질에 따른 친화력은 o-diphenol, 특히 chlorogenic acid에 대하여 높았고, monophenol과 m-diphenol, p-diphenol에 대해서는 나타나지 않았다. Polyphenol oxidase에 의한 갈변은 thiourea와 potassium metabisulfite는 10mM에서, L-cysteine과 ascorbic acid, sodium diethyldithiocarbamate는 1mM에서 현저하게 저해되었다.

• BMB Reports
• /
• 제36권4호
• /
• pp.409-416
• /
• 2003

The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-$\beta$-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants ($k_{inactivation}$) of $29.5\;M^{-1}s^{-1}$. The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of $3,200\;M^{-1}cm^{-1}$. It was discovered that methyl-$\beta$-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with $R_t$ 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.

• Animal cells and systems
• /
• 제12권3호
• /
• pp.137-141
• /
• 2008

Molecular cloning revealed the three isoforms($Ca_v3.1,\;Ca_v3.2,\;and\;Ca_v3.3$) of the T-type calcium channel subfamily. Expression studies exhibited their distinctive electrophysiological and pharmacological properties, accounting for diverse properties of T-type calcium channel currents previously characterized from isolated cells. However, electrophysiological properties of ion channels have shown to be more diversified by their splice variants. We here searched splice variants of rat $Ca_v3.1$ T-type channel by reverse-transcription-polymerase chain reaction(RT-PCR) to further explore diversity of $Ca_v3.1$. Interestingly, analyses of cloned RT-PCR products displayed that there were at least four splicing variants of rat $Ca_v3.1$ in the loop connecting repeats III and IV. Southern blot analyses indicated that the predominantly detected variant in brain was $Ca_v3.1a$(492 bp), which were rarely detected in most of peripheral tissues. Other two variants($Ca_v3.1c$, 546 bp; $Ca_v3.1d$, 525 bp) were detected in most of the tissues examined. The smallest isoform($Ca_v3.1b$, 471 bp) was rarely detected all the tissues. Electrophysiological characterization of the splicing variants indicated that the splice variants differ in inactivation kinetics and the voltage dependence of activation and inactivation as well.