• Title/Summary/Keyword: 인산효소

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Reaction Mechanism of Purine Nucleoside Phosphorylase and Effects of Reactive Agents for SH Group on the Enzyme in Saccharomyces cerevisiae (Saccharomyces cerevisiae에서 얻은 Purine Nucleoside Phosphorylase의 반응기작과 효소에 대한 Sulfhydryl Reagent의 영향)

  • Choi, Hye-Seon
    • Korean Journal of Microbiology
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    • v.32 no.3
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    • pp.222-231
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    • 1994
  • Kinetic analysis was done to elucidate the reaction mechanism of purine nucleoside phosphorylase (PNP) in Saccharomyces cerevisiae. The binary complexes of PNP${\cdot}$phosphate and PNP${\cdot}$ribose 1-phosphate were involved in the reaction mechanism. The initial velocity and product inhibition studies demonstrated were consistent with the predominant mechanism of the reaction being an ordered bi, bi reaction. The phosphate bound to the enzyme first, followed by nucleoside and base were the first product to leave, followed by ribose 1-phosphate. The kinetically suggested mechanism of PNP in S. cerevisiae was in agreement with the results of protection studies against the inactivation of the enzyme by sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and 5,5'-dithiobisnitrobenzoate (DTNB). PNP was protected by ribose 1-phosphate and phosphate, but not by nucleoside or base, supporting the reaction order of ordered bi, bi mechanism. PCMB or DTNB-inactivated PNP was totally reactivated by dithiothreitol (DTT) and the activity was returned to the level of 77% by 2-mercaptoethanol, indicating that inactivation was reversible. The kinetic behavior of the PCMB-inactivated enzyme had been changed with higher $K_m$ value of inosine and lower $V_m$, and was restored by DTT. Inactivation of enzyme by DTNB showed similar pattern of K sub(m) value with that by PCMB, but had not changed the $V_m$ value, significantly. Negative cooperativity was not found with PCMB or DTNB treated PNP at high concentration of phosphate.

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Purification of Recombinant CTP-Conjugated Human prostatic acid phosphatase for activation of Dendritic Cell (수지상세포 활성화를 위한 세포투과 펩타이드가 결합된 재조합 전립성 산성 인산분해효소의 정제)

  • Yi, Ki-Wan;Ryu, Kang
    • KSBB Journal
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    • v.24 no.1
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    • pp.80-88
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    • 2009
  • Human prostatic acid phosphatase (PAP), with comprehensive homology to glandular kallikrein, are representative serum biomarkers of prostate cancer. Dendritic cell (DC), which is the potent antigen-presenting cells(APC) in the immune system, can induce strong T cell responses against viruses, microbial pathogens, and tumors. Therefore, the immunization using DC loaded with tumor-associated antigens is a powerful method for inducing anti-tumor immunity. The CTP (Cytoplasmic Transduction Peptide) technology developed by Creagene which can transport attached bio-polymers like nucleic acids or proteins into the cell with high permeation efficiency. As the active forms of PAP can mediate apoptotic processing, we used multimer forms of PAP as an inactive form for antigen pulsing of DCs. In this study, multimeric forms of CTP-rhPAP was obtained according to the advanced purification process and subsequently confirmed by gel filtration chromatography, western blot and Dynamic Light Scattering. Therefore, CTP-conjugated PA multimers transduced into the cytoplasm were efficiently presented on the cell surface without any harm effect on cells via MHC class I molecules and result in induction of a large number of effector cell.

Effects of Low-Level Laser Irradiation on the ALP Activity and Calcified Module Formation of Rat Osteoblastic Cell (저수준레이저(GaAs 반도체)조사가 골모세포의 알칼리성 인산분해효소의 활성과 석회화결절의 형성에 미치는 영향)

  • Kyung-Hun Lee;Ki-Suk Kim
    • Journal of Oral Medicine and Pain
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    • v.21 no.2
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    • pp.279-291
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    • 1996
  • 저수준레에저요법에 대해서는 지난 10여년간 의학계 및 치과계에서 임상적으로 사용하여 좋은 결과가 있다고 많은 보고가 발표되고 있다. 특히 치근의 골결손에 관한 연구에서는 전기요법, 초음파요법, 전자장요법 등 뿐만아니라 저수준레이저를 사용하여 골절부내 Callus형성이 촉진되었음을 보고하고 새로운 치료법의 하나가 될 수 있음을 제안한 바도 있다. 본 연구에서는 칼륨비소를 다이오드로 사용한 저수준레이저조사가 골결손의 치유에 어떠한 영향을 미치는지 확인하고자 골모세포의 알칼리성 인산분해효소의 활성화와 석회화결절의 형성을 평가함으로 골모세포의 기능을 조사코저하였다. 실험은 첫째, 9개군으로 나누어 레이저 조사기간에 따른 알칼리성 인산분해효소의 활성화를 조사하였고, 둘째, 이를 근거로 9일간 계속 매일 1회 1.3 J/cm2의 레이저를 조사한 후 펄스의 종류별 차이를 비교하였으며, 세째,레이저펄스별 석회차 결절의 형성 정도를 광학현미경으로 관찰하여 비교분석하였다. 결과, 7일 계속 레이저를 조사한 경우 다른 군에 비해 서서히 ALP의 활성이 증가하였으나 유의한 차이는 없었으며. 따라서 9일동안 레이저를 계속 조사한 경우에는 전체 에너지량이 5.895 J/cm2 인 펄스13과 15가 뚜렷하게 유의한 증가를 보여주었다. 그러나 석회화결절의 형성은 전체 에너지량이 2.546 J/cm2 인 펄스11에서 가장 많았다. 결론적으로 골형성이나 알칼리성 인산분해효소의 활성을 촉진하는 데에는 적절한 레이저 조사조건이 필요하나, 알칼리성 인산분해효소의 활성을 촉진한 펄스와 석회화결절의 형성을 촉진하는 펄스가 서로 다르게 나타난 것은 골형성을 촉진하는 여러요인 들이 저수준레이저에 자극받았을 가능성이 높음을 보여준다 이러한 결과들로 보아 저수준레이저는 골모세포의 기능을 자극하여 골결손의 치유를 개선하는 데 도움될 것이라 사료된다.다. 각 백서의 양측 창상중 하나는 1,3,5,7일 마다 각 실험의 방법에 따라 레이저를 조사하고 실험동물의 다른 창상은 대조군으로서 사용하였다. 모든 창상의 면적은 실험 1,3,5,7 일째에 일정한 거리에서 사진촬영하여 면적계를 이용, 측정한 후 통계적인 의의를 조사하였다. 본 연구의 결과는 저수준레이저는 특정 조건하에서 S. aureus의 증식을 촉진하였다. 그러나 S. aureus에 감염된 창상을 저수준레이저로 조사시 치유가 촉진되었다. 중앙 조사법고 주변조사법에 의한 창상치유효과는 통계적인 의의가 보이지 않았다. 따라서 결론적으로 S. aureus 에 감염된 창상에 직접 또는 간접적이든 pulse의 종류에 관계없이 조사하는 경우 치유효과가 나타나는 것은 정사주위 조직의 LLLI 자극효과가 염증의 확산을 억제한다고 말할수 있다.4/1$0^{\circ}C$에서는 Shoa-Nan-Tsan과 Lenkwang이 가장 높았으며 백앙벼는 3 온도 조건 모두에서 활성이 낮았다. 발아소요일수와 amylase 활성과는 유의적인 정의 상관관계를 보였다., 다다조, 미국의 건답직파재배 품종 등이었으며 우리 나라 육성종들은 모두 지중에서 신장이 멈추어 제1본엽이 지중에서 추출하였으며, Scm파종심에서 불완전엽이 지면을 뚫고 나오는 품종은 Chinsura Boro뿐이었고 Nato, Labelle, Weld Pally, Italliconaverneco 등도 지면 가까이 까지 신장하였다. 6. 50% 출아일수는 제2절간장을 제외 한 모든 유아 형질의 신장도와 유의한 부의 상관을 보였는데 가장 높은 상관을 보인 것은 중배축장+제1절간장+불완전옆장이었으며, 다음이 불완전엽장이 었다. 7. 출아율은 중배축장+제1절간장+불완전엽장, 중배축장+초엽 장과 모든 파종심에서 높은 정의 상관을 보여 제1본엽의 추출 위치가 높을수록

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인슐린의 신호전달 기전 : Transcription Factor AP-1 의 역활

  • 김성진
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1995.10a
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    • pp.17-21
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    • 1995
  • 대부분의 인슐린의 작용들은 인슐린 수용체를 통하여 이루어진다. 인슐린이 수용체에 결합하면, 수용체 고유의 tyrosine kinase 효소활성의 증가를 유발시키며, 결과적으로 세포내에 존재하는 기질 단백질, IRS-1, 의 tyrosine 잔기의 인산화를 증가시키게 된다. 이후, 여러 형태의 serine / threonine protein kinase 의 연속적인 활성화가 일어난다. 이들에 부가해서, 인슐린의 효자는 세포핵 내에까지 전달되어 유전자 발현의 조절과 같은 세포핵 고유의 활동에도 관여한다. 현재, 세포막에서 시작된 인슐린의 신호들이 세포핵까지 전달되는 정확한 기전에 대해서는 알려진 바 없지만, 최근의 연구에 의하면 MAP Kinase 와 S6 Kinase 그리고 Transcription Factor AP-1의 중요성이 제시되고 있다. 특히 유전자 조절 기전에는 핵단백질인 transcription factor의 인산화 반응이 큰 역할을 한다고 보고되고 있는바, 본 연구에서 AP-1. transcription factor 의 인산화 반응이 인슐린의 신호전달계에 미치는 역할에 대하여 고찰하였다. 요약하면, AP-1 transcription factor의 구성원인 c-Jun, c-Fos 그리고 Fos 관련 단백질들의 인산화가 인슐린에 의해 증가되며, 동시에 그들의. DNA-binding activity 와 유전자 발현의 활성이 증가됨을 밝힘으로써, AP-1 transcription factor의 인산화 반응이 인슐린의 핵 내에서의 작용기전에 중요한 역할을 함이 제시되고 있다. 또한 AP-1 의 인산화 반응에 관여하는 세포핵 protein kinase로서 Casein Kinase II 의 중요성이 밝혀졌다.

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Anti-inflammatory and Anti-allergic Effects of Herbal Extracts on Atopic Dermatitis ( Part II ) (약용식물 추출물의 아토피성 피부염에 대한 항염증 및 항알레르기 효과 (제 2 보))

  • Rang, Moon-Jeong
    • Journal of the Korean Applied Science and Technology
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    • v.30 no.1
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    • pp.173-182
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    • 2013
  • Atopic dermatitis is a chronic, relapsing inflammatory skin disease associated with dysfunction of skin barrier and cutaneous hyper-reactivity to environmental triggers. In the previous study, cytotoxicity, antioxidant, anti-inflammatory and anti-allergic activities were investigated for various herbal extracts such as Aloe vera L. (AV), Viola mandshurica W. Becker (VM), Punica granatum L. (PG), and Dendrobium nobile L. (DN) in order to develop effective therapeutic herbal extracts for atopic dermatitis, In this study, anti-inflammatory activities of these herb extracts in lipopolysaccharide (LPS)-induced macrophage RAW264.7 cells were further examined to find the underlying molecular mechanisms. The RT-PCR (reverse transcription polymerase chain reaction) analysis showed that PG, DN and AV inhibited effectively the gene expression of pro-inflammatory cytokines IL-6 and IL-$1{\beta}$ in LPS-stimulated macrophages, while VM did not. The transfection and luciferase analysis exhibited that all herbal extracts hindered the activation of transcription nuclear factor kappa B (NF-${\kappa}B$). The western blot analysis indicated that AV blocked the activation of only JNK MAP (c-Jun N-terminal kinase mitogen-activated protein) kinase not p38 MAP kinase, while VM, PG and DN did not show the activation of both JNK and p38 MAP kinases. These results suggest that AV, VM, PG, and DN have anti-inflammatory activities and thus have the potential to reduce and alleviate the symptoms of atopic dermatitis.

Studies on the Differentiation of Skeletal Muscle Cells in vitro:Protein Kinase C in the Differentiation of Skeletal Muscle Cells (근세포 분화에 관한 연구 : 근세포 분화에 있어서 Protein Kinase C)

  • 최원철;김한도;김정락
    • The Korean Journal of Zoology
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    • v.34 no.2
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    • pp.131-141
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    • 1991
  • Treating 12-O-tetradecanoyIphorboI 13-acetate -TPA) or platelet~derived growth factor(PDGF), the signal transduction of protein Idnase C (PKC) is occurred by the phosphoryladon. However the targeting proteins phosphorylated by PKC were found to be different proteins in molecular weights when WA or PDGF wa~ treated to the myoblast. In the WA-treated myoblast cells, the protein of Mr. 20 I(d was phosphorylated. In the PDGF-treated cells, the protein of Mr. 40 Kd was phosphrylated, while the protein of Mr. 20 Kd which phosphorylated in the WA-treatment was dephosphorylated. These results indicate that not only WA and PDGF &e different in activating the signal transduction pathways, but also they may involve in the down reguladon of PI(C during the long-term treatment But PDGF gave rise more rapidly down reguladon than in the case of WA. Using immunocytochemical approach, two disdnct PKC isozymes, PKC II and PKC III, have been localized in cytoplasm and both cytoplasm and nuclsolus, respectively. Ther'efore, the expression of two types of PKC in the myoblast suggests that the isozymes of PKC may involve in each different pathway of signal transduction or down-reguladon.

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Dephosphorylation Study of Phosphorylated Myelin Basic Protein: A Model Substrate for Protein Phosphatase (인산화된 신경수초 염기성 단백질의 탈인산화 연구: 단백질 탈인산화 효소의 기질 모델)

  • Kim, Jin Hahn;Choi, Myung Un
    • Journal of the Korean Chemical Society
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    • v.41 no.4
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    • pp.205-209
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    • 1997
  • The site specificity of dephosphorylation of myelin basic protein(MBP) was studied in vitro. To assign amino acid site of dephosphorylation, MBP was phosphorylated by protein kinase C(PKC) and dephosphorylated by protein phosphatase PP2A. The phosphorylated MBP was digested by trypsine and the digested peptides were separated by a reverse phase HPLC chromatography. The radioactivity of each fraction was counted and partially sequenced. Seven radioactive peptides were observed and $Ser^{55}$ in the second peak($P_2$) shows the best susceptibility for the phosphorylation. However in the dephosphorylation, the fifth peak($P_5$) appeared to release it's phosphate group most rapidly. This result demonstrates that MBP is a suitable substrate for protein phosphatase.

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Evaluation of Bioavailability of Phosphorus Accumulated in Arable Soils (농경지 토양에 집적된 인산의 생물이용가능성 평가)

  • Lee, Seul-Bi;Lee, Chang-Hoon;Kim, Gun-Yeob;Lee, Jong-Sik;So, Kyu-Ho;Kim, Sang-Yoon;Kim, Pil-Joo
    • Korean Journal of Environmental Agriculture
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    • v.31 no.4
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    • pp.293-299
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    • 2012
  • BACKGROUND: Soil utilization pattern can be the main factor affecting soil physico-chemical properties, especially in soil phosphorus (P). Understanding the distribution and bioavailability of P is important for developing management to minimize P release from arable soils to environment. This study was conducted to evaluate the potential bioavailability of soil organic P by using phosphatase hydrolysis method. METHODS AND RESULTS: Twenty-four soils from onion-rice double cropping and 30 soils from plastic film house were selected from Changyeong and Daegok in Gyeongnam province, respectively. The P accumulation pattern (total P, inorganic P, organic P, residual P) and water soluble P were characterized. Commercial phosphatase enzymes were used to classify water-extractable molybdate unreactive P from arable soils into compounds that could be hydrolysed by (i) alkaline phosphomonoesterase (comprising labile orthophosphate monoesters), (ii) a combination of alkaline phosphomonoesterase and phosphodiesterase (comprising labile orthophosphate monoesters and diesters), and (iii) phytase (including inositol hexakisphosphate). Available P was highly accumulated with 616 and 1,208 mg/kg in double cropping system and plastic film house, respectively. Dissolved reactive P (DRP) and dissolved unreactive P (DUP) had similar trends with available P, showing 24 and 109 mg/kg in double cropping and 37 and 159 mg/kg in plastic film house, respectively, indicating that important role of dissolved organic P in the environments had been underestimated. From the result of phosphatase hydrolysis, about 39% and 66% of DUP was evaluated as bioavailable in double cropping and plastic film house, respectively. CONCLUSION(S): Orthophosphate monoester and orthophosphate diester accounted for high portion of dissolved organic P in arable soils, indicating that these organic P forms give important impacts on bioavailability of P released from P accumulated soils.