Activation mechanism of sialidase by protease

蛋白酶激活唾液酸酶的机制

基本信息

项目摘要

An acid sialidase was activated by incubation at 37゚C.This activation showed both time and temperature dependencies, with the most effective activation observed at 37゚C in the pH range between 4.3 and 5.2. The influence of various protease inhibitors on its activation was investigated. Among the protease inhibitors tested, amastatin, an inhibitor of aminopeptidase A,significantly inhibited activation. Isolation of the aminopeptidase from human placenta was examined. Sephadex G-200 gel filtration of the glycoprotein fraction separated the two aminopeptidases. Both enzymes had pH optimum of 7, which was different from the optimum pH (5.0) for activation of sialidase. It was very difficult to purify human placental aminipeptidase, because of its lability and low activity. We do not succeed yet to establish the purification and characterization of the enzymes. To inquire the possibility that other factor is responsible for activation of the sialidase, the effects of buffer, cation, anion a … More nd pH were investigated Among the verious factors tested, halogen ions, especially chloride ion, significantly stimulated the activation of sialidase. The activation by chloride ion showed both time and concentration dependencies. Further work along this line is now in progress.Catepsine A,so called protective protein, occurs as an enzyme complex with lysosomal beta-galactosidase (beta-Gal) and is involved in the stable enzyme exprssion of lysosomal sialidase. We investigated the enzymatic properties of catepsin A in the bovine beta-Gal complex and how it is involved in the molecular multiplicities of the beta-Gal and sialidase complexes. Bovine protective protein homologus to the human protein had a molecular weight of 48kDa on SDS-PAGE and catepsin A activity optimum about pH 6.0. Immunoprecipitation using an anti beta-Gal antibody demonstrated that catepsin A is a component of both the sialidase and beta-Gal complexes. The over 700kDa sialidase complex depolymerized by a brief inicubation at pH 7.5 and the sialidase was inactivated irreversibly via formation of an enzyme active smaller species of sialidase. The 669kDa beta-Gal complex dissociated reversibly into a 120kDa beta-Gal and a 170 kDa catepsin A.Inactivation of catepsin A By heat and DIFP treatment sdid not affect its complex forming activity. Both beta-Gal and catepsin A activities were labile under the dissociated condition, indicating that it physiologically stabilizes not only beta-Gal but also itself by forming the complex. Chicken sialidase also occurs as a complex with beta-Gal and catepsin A.The complex reversibly dissociated into 120kDa beta-Gal dimer and 100kDa catepsin A dimer at pH 7.5, but the sialidase irreversibly inactivated during the depolymerization. Chicken sialidase seems to exist as a multienzyme complex, by which the sialidase activity appears to be stabilized. Less
37℃孵育活化酸性唾液酸酶。这种活化具有时间和温度依赖性,在pH值为4.3 ~ 5.2的37℃条件下活化效果最好。研究了各种蛋白酶抑制剂对其活化的影响。在所测试的蛋白酶抑制剂中,氨肽酶A的抑制剂阿马司他汀能显著抑制活性。研究了从人胎盘中分离氨基肽酶的方法。糖蛋白部分的Sephadex G-200凝胶过滤分离了两种氨基肽酶。两种酶的最适pH值均为7,不同于唾液酸酶激活的最适pH值(5.0)。人胎盘氨基肽酶由于稳定性差、活性低,纯化难度大。我们还没有成功地建立酶的纯化和表征。为了探讨其他因素对唾液酸酶活性的影响,我们考察了缓冲液、阳离子、阴离子和pH对唾液酸酶活性的影响。在所测试的各种因素中,卤素离子,尤其是氯离子对唾液酸酶的活性有显著的促进作用。氯离子活化具有时间依赖性和浓度依赖性。这方面的进一步工作目前正在进行中。catepsin A被称为保护性蛋白,与溶酶体-半乳糖苷酶(beta-Gal)形成酶复合体,参与溶酶体唾液酸酶的稳定酶表达。我们研究了牛β -半乳糖复合物中catepsin A的酶学性质,以及它是如何参与β -半乳糖和唾液酸酶复合物的分子多样性的。与人蛋白同源的牛保护蛋白在SDS-PAGE上分子量为48kDa,在pH为6.0时蛋白酶a活性最佳。使用抗β - gal抗体的免疫沉淀证明catepsin A是唾液酸酶和β - gal复合物的组成部分。超过700kDa的唾液酸酶复合物在pH 7.5下短暂孵育后解聚,唾液酸酶通过形成一种活性较小的唾液酸酶而不可逆地失活。669kDa β - gal复合物可逆地分解成120kDa β - gal和170kda catepsin a。加热和DIFP处理对catepsin a失活不影响其复合物的形成活性。在解离条件下,β - gal和catepsin A的活性都是不稳定的,这表明它通过形成复合物在生理上稳定了β - gal,也稳定了自身。鸡唾液酸酶也与β - gal和猫蛋白酶a形成复合物,在pH为7.5时,复合物可逆地解离成120kDa的β - gal二聚体和100kDa的猫蛋白酶a二聚体,但在解聚过程中唾液酸酶不可逆地失活。鸡唾液酸酶似乎以一种多酶复合物的形式存在,通过这种复合物,唾液酸酶的活性似乎是稳定的。少

项目成果

期刊论文数量(44)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
長岡めぐみ: "ヒト胎盤シアリダーゼの活性化に関与する因子について" 日本薬学会第117年会講演要旨集.
Megumi Nagaoka:“关于人胎盘唾液酸酶激活的因素”日本药学会第 117 届年会摘要。
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M.Hiraiwa, T.Yamauchi, S.Tsuji, M.Nishizawa, T.Miyatake, K.Oyanagi, F.Ikuta and Y.Uda: "Activation of human lysosomal sialidase.J.Biochem." 114. 901-905 (1993)
M.Hiraiwa、T.Yamauchi、S.Tsuji、M.Nishizawa、T.Miyatake、K.Oyanagi、F.Ikuta 和 Y.Uda:“人溶酶体唾液酸酶的激活。J.Biochem。”
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T.Takeuchi, M.Saitoh, T.Shiraishi, M.Hiraiwa, K.Izumi, M.T.Sawada, N.Takahashi and Y.Uda: "Purification and characterization of sialidase from starfish, Asterina pectinifera" Bull.Marine Biomed.Inst., Sapporo Med.Univ.3. 55-61 (1996)
T.Takeuchi、M.Saitoh、T.Shiraishi、M.Hiraiwa、K.Izumi、M.T.Sawada、N.Takahashi 和 Y.Uda:“海星海星唾液酸酶的纯化和表征”Bull.Marine Biomed.Inst。
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斎藤麻由: "ヒト胎盤β-ガラクトシダーゼ保護蛋白(カルボキシペプチダーゼについて" 第66回日本生化学会大会発表抄録集. 65. 739 (1993)
Mayu Saito:“关于人胎盘β-半乳糖苷酶保护蛋白(羧肽酶)”日本生化学会第66届年会论文集65. 739(1993)。
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Masao Hiraiwa: "Protective protein in the bovine lysosomal β-galactosidase complex" Biochim. Biophys. Acta. (in press). (1997)
Masao Hiraiwa:“牛溶酶体 β-半乳糖苷酶复合物中的保护蛋白”Biochim。(出版中)。
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UDA Yutaka其他文献

UDA Yutaka的其他文献

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{{ truncateString('UDA Yutaka', 18)}}的其他基金

Molecular interaction between the components in lysosomal sialidase complex
溶酶体唾液酸酶复合物成分之间的分子相互作用
  • 批准号:
    12672130
  • 财政年份:
    2000
  • 资助金额:
    $ 1.41万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Molecular structure and function of lysosomal sialidase
溶酶体唾液酸酶的分子结构和功能
  • 批准号:
    04671376
  • 财政年份:
    1992
  • 资助金额:
    $ 1.41万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Molecular and biological study on sialidase
唾液酸酶的分子生物学研究
  • 批准号:
    02671018
  • 财政年份:
    1990
  • 资助金额:
    $ 1.41万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Biomedical Studies on Sialidase and Beta-Galactosidase
唾液酸酶和β-半乳糖苷酶的生物医学研究
  • 批准号:
    63571064
  • 财政年份:
    1988
  • 资助金额:
    $ 1.41万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)

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