• The Korean Journal of Physiology
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• 제22권2호
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• pp.163-177
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• 1988

세포막을 구성하고 있는 지질의 물리학적 성상이 단백질의 세포막 속으로의 삽입과정 및 단백질의 기능에 미치는 영향을 관찰하기 위하여 골격근의 근세망(SR)으로부터 $Ca^{2+}-ATPase$ 단백질을 분리한 후 이를 세포막의 주 구성성분인 포스파티딜콜린(PC)과 포스파티딜에타노라민(PE)의 혼합지질과 재조합(reconstitution)시켰다. 이와같이 인공적으로 재조합된 구조물에서 $Ca^{2+}-ATPase$의 기능을 측정하기 위하여 칼슘지시색소인 아르세나죠III(AIII)를 이용한 분광방법과 방사선동위원소를 이용한 여과법으로 칼슘흡수율을 측정하였고 또한 ATP 가수분해 능력을 측정하였다. 실험결과 칼슘의 흡수율은 포스파티딜코린의 함량이 많은 혼합지질과 재조합시킬 때에 증가하였고, ATP 가수분해 능력은 포스파티딜함량이 25%까지는 포스타피딜코린의 양에 비례하여 증가하였으나 50%이상에서는 약간 감소하는 경향을 보였다. 한편 지질세포막속으로 단백질이 삽입되는 양은 포스파티딜 함량이 25%일 때 최고의 값을 보였으며 함량이 그 이하 또는 이상일 때는 감소하였다. 이상의 실험결과로보아 단백질의 기능은 세포막이 "bilayer" 구조를 갖출때에 증가하고 세포막속으로 단백질이 삽입되는 양은 세포막이 "non-bilayer" 구조를 형성할 때에 증가함을 알 수 있다.

• Mycobiology
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• 제43권1호
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• pp.49-56
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• 2015

The present study is the first report on the isolation of Penicillium menonorum from rhizosphere soil in Korea and its identification based on morphological characteristics and internal transcribed spacer gene sequence. The fungal isolate was named KNU-3 and was found to exhibit plant growth-promoting (PGP) activity through indole acetic acid (IAA) and siderophore production, as well as P solubilization. KNU-3 produced 9.7 mg/L IAA and solubilized 408 mg of $Ca_3PO_4/L$, and inoculation with the isolate significantly (p < 0.05) increased the dry biomass of cucumber roots (57%) and shoots (52%). Chlorophyll, starch, protein, and P contents were increased by 16%, 45%, 22%, and 14%, respectively, compared to plants grown in uninoculated soil. The fungus also increased soil dehydrogenase (30%) and acid phosphatase (19%) activities. These results demonstrate that the isolate KNU-3 has potential PGP attributes, and therefore it can be considered as a new fungus to enhance soil fertility and promote plant growth. Moreover, the discovery of PGP ability and traits of this fungus will open new aspects of research and investigations. In this study, plant growth promotion by P. menonorum KNU-3 is reported for the first time in Korea after its original description.

• 한국축산식품학회지
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• 제24권1호
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• pp.80-85
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• 2004

본 연구에서는 역미셀을 이용한 단백질 추출공정에서 적극적으로 활용되지 못했던 양이온 계면활성제에 의한 단백질 추출 가능성을 제시하였으며 초유로부터 IgG의 분리를 위한 반응조건을 조사하였다. IgG의 분리에 적합한 조건은 반응 수용액상의 경우 pH 8, 50 mM KCl었으며 유기용매상의 계면활성제(CDAB) 농도는 100 mM로 나타났다. 위의 조건에서는 초기시료에 존재하는 IgG의 90%이상이 회수되었으며 회수된 단백질의 93%가 IgG로 나타났다. 또한 본 연구는 기존의 역미셀을 이용한 일반적인 단백질 추출공정인 정추출 및 역추출 공정을 이용하지 않고 정추출 공정만을 이용함으로써 추출과정을 단순화하였다.

• 대한화장품학회지
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• 제25권4호
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• pp.145-154
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• 1999

A novel retinol derivative, polyethoxylated retinamide(Medimin A) was synthesized, as an anti-aging agent. Collagen synthesis, skin permeation, stability, and toxicity of Medimin A were evaluated and compared with those of retinol and retinyl palmitate. In vitro collagen synthesis was evaluated by quantitative assay of $[^3H]-proline$ incorporation into collagenase sensitive protein in fibroblast cultures. For in vitro skin permeation experiments, Franz diffusion cells(effective diffusion area: 1,766 $cm^2$) and the excised skin of female hairless mouse aged 8 weeks were used, The stabilities of retinoids were evaluated at two different temperature($25^{\circ}C\;and\;40^{\circ}C$) and under UV in solubilized state and in O/W emulsion. To estimate the safety, acute oral toxicity, acute dermal toxicity, primary skin irritation, acute eye irritation and human patch test were performed. The effect of Medimin A on collagen synthesis was similar to that of retinol. The skin permeability of Medimin A was higher than those of retinol and retinyl palmitate. The Medimin A was more stable than retinol and retinyl palmitate. Medimin A was nontoxic in various toxicological tests. These results suggest that Medimin A would be a good anti-aging agent for enhancing bioavailability and stability.

• BMB Reports
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• 제29권6호
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• pp.555-563
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• 1996

A membrane-bound phosphatidylinositol 4-kinase (PI 4-kinase) was separated in a sucrose gradient and solubilized with 1% Triton X-100 from mouse brain. The enzyme was purified 2,952-fold by various chromatographic techniques including DEAE-cellulose, PI-Sepharose and Sephacryl S-200 gel filtration. The molecular weight of PI 4-kinase was approximately 76 kDa by gel filtration and 70.8 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibited specific activity of 11.2 nmol/min/mg protein and pi value of 4.7. Kinetic analysis of the PI 4-kinase indicated apparent $K_m$, values of 190 ${\mu}M$ and 120 ${\mu}M$ for phosphatidylinositol and ATP, respectively. The maximal activity of this purified enzyme was observed at pH 7.4 at an incubation temperature of $37^{\circ}C$. The enzyme activity was significantly activated by $Mg^{2+}$, $Mn^{2+}$ and $Fe^{2+}$, and inhibited severely by $Ca^{2+}$. PI 4-kinase was proved to be pure in its immunoblot test by polyclonal antibody prepared from immunized rabbit sera. By this test, we were able to detect the existence of the same type of PI 4-kinase from other mouse organ tissues, such as liver, heart, kidney and spleen. Furthermore, similar immunoblot analysis with the same antisera recognized the different epitopes of PI 4-kinase proteins from various organs of rabbit, chinese hamster and rat.

• 미생물학회지
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• 제50권4호
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• pp.368-371
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• 2014

염 스트레스를 비롯한 다양한 비생물학적 스트레스는 식물의 생장저해와 작물 생산량을 감소시키는 요인으로 작용한다. 계화도 간척지 토양에서 식물생장 촉진 세균을 분리하여 Pseudomonas sp. G19로 명명하였다. G19 균주는 36시간 배양 후 $7.5{\mu}g/ml$의 indole acetic acid를 생산하고 불용성인산을 25% 가용화시켰으며, 식물의 에틸렌 감소와 관련된 1-aminocyclopropane-1-carboxylate deaminase를 발현하였다. 또한 150 mM NaCl에 침지된 염 조건의 토양에서 재배된 유묘기 배추를 이용한 실증실험에서 G19 균주는 배추의 생체중량을 증가시킴으로써 염 스트레스의 경감 및 생장 촉진에 관여함을 알 수 있었다.

• 한국식품과학회지
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• 제18권3호
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• pp.197-203
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• 1986

부분도정한 보리를 미생물효소(Asp. niger 및 oryzae)에 의한 분해반응 및 미세 고형분의 용출, 여과, 침강 또는 원심분리, 건조 등의 과정을 거쳐 제분할 때, 보리가루 획분$(73{\sim}76%)$, 맥강, 배아, 종구등의 잔유물 획분(3.6% 내외) 그리고 가용성물질 획분 등으로 분리하여 얻을 수 있었다. 이 때 가장 좋은 효소반응 조건은 온도 $45^{\circ}C$, 보리와 물의 비율 1 : 1.5(w/v)였고, ${\alpha}-a-mylase$역가 보다는 glucanase 와 Protease의 역가가 높은 효소를 사용할 때 더 좋은 효과를 보였다. 그리고 얻어진 세가지 종류 획분의 성분 조성은 단백질, 지방, 섬유소 등에서 각각 현저한 차이를 보여, 각 획분의 특성별 구분 이용이 가능하였다.

• Bulletin of the Korean Chemical Society
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• 제22권12호
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• pp.1361-1365
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• 2001

Recombinant immunotoxins are hybrid cytotoxic proteins designed to selectively kill cancer cells. A divalent immunotoxins, [B3(FabH1)-PE38]2, was constructed by recombining Fab domain of B3 antibody as a cell-targeting domain and Pseudo monas exotoxin A (PE) as a cytotoxic domain. Monoclonal antibody, B3, is the murine antibody (IgG1k) directed against Lewis Y-related carbohydrate antigens, which are abundant on the surface of many carcinomas. Fab fragment of this antibody was used in this study with the modified hinge sequence where last two cysteines out of three were mutated to serine. PE is a 66 kDa bacterial toxin that kills eukaryotic cells by inhibiting protein synthesis with ADP ribosylation of ribosomal elongation factor 2 (EF2). Fc region of B3 antibody was substituted with the truncated form of PE (38 kDa, PE38) on DNA level. [B3(FabH1)-PE38]2 was formed by disulfide bond between cysteines in the modified hinge region of B3(FabH1)-PE38. Each polypeptide for recombinant immunotoxins was overexpressed in Escherichia coli and collected as inclusion bodies. Each inclusion body was solubilized and refolded, and cytotoxic effects were measured. Divalent immunotoxins, [B3(FabH1)-PE38]2, had ID50 values of about 10 ng/mL on A431 cell lines and about 4 ng/mL on CRL1739 cell lines. Control immunotoxins, B3(scFv)-PE40, had ID50 values of about 28 ng/mL on A431 cell lines and about 41 ng/mL on CRL1739 cell lines. Divalent immunotoxins, [B3(FabH1)-PE38]2, had higher cytotoxic effects than B3(scFv)-PE40 control immunotoxins.

• Toxicological Research
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• 제8권2호
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• pp.343-357
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• 1992

Tumor necrosis factor-${\alpha}$(TNF), a polypeptide hormone secreted primarily by activated macrophages, was originally identified on the basis of its ability to cause hemorrhagic necrosis and tumor regression in vivo. Subsequently, TNF has been shown to be an important component of the host responses to infection and cancer and may mediate the wasting syndrome known as cachexia. These systemic actions of TNF are reflected in its diverse effects on target cells in vitro. TNF initiates its diverse cellular actions by binding to specific cell surface receptors. Although TNF receptors have been identified on most of animal cells, regulation of these receptors and the mechanisms which transduce TNF receptor binding into cellular responses are not well understood. Therefore, in the present study, the mechanisms how TNF receptors are being regulated and how TNF receptor binding is being transduced into cellular responses were investigated in rat liver plasma membranes (PM) and ME-180 human cervical carcinoma cell lines. $^{125}I$-TNF bound to high ($K_d=1.51{\pm}0.35nM$)affinity receptors in rat liver PM. Solubilization of PM with 1% Triton X-100 increased both high affinity (from $0.33{\pm}0.04\;to\;1.67{\pm}0.05$ pmoles/mg protein) and low affinity (from $1.92{\pm}0.16\;to\;7.57{\pm}0.50$ pmoles/mg protein) TNF binding without affecting the affinities for TNF, suggesting the presence of a large latent pool of TNF receptors. Affinity labeling of receptors whether from PM or solubilized PM resulted in cross-linking of $^{125}I$-TNF into $M_r$ 130 kDa, 90 kDa and 66kDa complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, $^{125}I$-TNF binding to control and TNF-pretreated membranes were assayed. Specific binding was increased by pretreatment with TNF (P<0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF. As a next step, the post-receptor events induced by TNF were examined. Although the signal transduction pathways for TNF have not been delineated clearly, the actions of many other hormones are mediated by the reversible phosphorylation of specific enzymes or target proteins. The present study demonstrated that TNF induces phosphorylation of 28 kDa protein (p28). Two dimensional soidum dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) resolved the 28kDa phosphoprotein into two isoforms having pIs of 6.2 and 6.1. The pIs and relative molecular weight of p28 were consistent with those of a previously characterized mRNA cap binding protein. mRNA cap binding proteins are a class of translation initiation factors that recognize the 7-methylguanosine cap structure found on the 5' end of eukaryotic mRNAs. In vitro, these proteins are defined by their specific elution from affinity columns composed of 7-methylguanosine 5'-triphosphate($m^7$GTP)-Sepharose. Affinity purification of mRNA cap binding proteins from control and TNF treated ME-180 cells proved that TNF rapidly stimulates phosphorylation of an mRNA cap binding protein. Phosphorylation occurred in several cell types that are important in vitro models of TNF action. The mRNA cap binding protein phosphorylated in response to TNF treatment was purifice, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor-4E(eIF-4E). These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the diverse cellular actions of TNF.

• 한국수산과학회지
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• 제15권2호
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• pp.123-136
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• 1982

방어의 배육 보통육과 측육 혈합육을 $2^{\circ}C$에 보장하여 두고 선도변화 총계별로 단백질의 조성과 각축의 구성 및 유리아미노산의 조성을 분석하였으며, 건향질단백질과 근원섬유단백질에 대하여는 구성단백질의 분포변화를 확인하기 위하여 $NaDodSO_4$변한 다음, polyacrylamide겔 전기영동상을 비교 해석함으로써 적색육어류의 선도변화와 보통육 및 혈합육의 단백질조성 변화와의 관련성을 검토하였다. K-값과 휘발성염기질소 및 pH값으로 판정했을 때, 혈합육은 보통육에 비하여 선도변화가 빨랐다. 사후 선도변화와 더불어 근원질단백질과 노원섬유단백질은 감소하는 반면, 세포내잔사단백질은 상대적으로 증가하였으며, 이러한 변화는 보통육에 비하여 혈합육이 훨씬 심한 편이었다. Sodium dodecyl sulfate 화한 단백질을 polyacrylamide겔 전기영동함으로 분석한 결과, 보통육의 근원질단백질은 16개 성분, 그리고 혈합육의 근원질단백질은 12개 성분으로 구성되어 있었으며, 보장 10일째부터 보통육의 근원질단백질은에는 41,000dalton과 18,000dalton의 양분이 조금씩 증가하였고, 26,000 dalton과 23,500 dalton 및 23,000 dalton되 각 성분은 점차 감소하였다. 혈합육의 근원질단백질에 있어서는 49,000 dalton의 성분은 감소하였고 47.000 dalton의 성분은 새로이 형성되었으며, 26,000 dalton의 성분은 소감하였다. 근원섬유단백질의 전기영동분석결과에 의하면, 보통육의 근원섬유단백질은 17개 성분, 그리고 혈합육의 근원조직단백질은 16개의 성분으로 구성되어 있었다. 보장 10일에부터 보통육의 근원섬유단백질중에는 40,000 dalton의 성분이 증가하였고, 37,500 dalton의 성분은 점차 감소하였으며, 32,000 dalton의 성분은 새로히 출현하였다. 혈합육의 근원섬유단백질은 보장 9일째부터 58,000 dalton과 64,000 dalton의 성분은 그 농도가 단가하였으며, 17,500의 light chain-2 단백질은 소감하였고, 32,000 dalton의 성분은 새로이 출현하였다. 이 같은 전기영동상의 변화는 근원질단백질이 근원섬유단백질에 비하여 빨랐으며, 근원섬유단백질의 경화에 있어서는 혈합육쪽이 보통육에 비하여 빨랐다. 양 육의 단백질을 구성하는 아미노산을 분석한 결과, 즉살한 방어의 보통육 단백질은 글루탐산, 아스팔트산, 류신. 알기닌, 알라닌 등은 그 양이 어느 정도 많았고, 프롤린과 트?토판은 그 양이 적었으며, 혈합육에 있어서도 비슷한 경형이었다. 그리고 10일이 경과한 보통육에 있어서는 글루탐산과 아스팔트산이 현저히 감소하였으며, 9일이 경과한 혈합육에 있어서는 알라닌, 글리신, 그리고 알기닌이 현저히 감소하였다. 유리아미노산의 조성을 분석 결과, 즉살한 방어의 보통육중에는 히스티딘이 총유리아미노산의 약 $63\%$를, 그리고 혈합육중에는 타우린이 총유리아미노산의 약 $67\%$를 차지하는 양을 보였다. 그리고 $2^{\circ}C$에서 10일이 경과했을 때, 보통육에서는 히스티딘, 발린, 타우린이 증가하였으며, 알라닌, 류신 및 글리신 등은 조금 감소하였다. 같은 온도에서 9일이 경과한 혈합육에서는 타우린, 페닐알라닌, 글리신은 증하였으며, 히스티딘, 알라닌, 세린 등은 감소하였다.