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Molecular structure and function of lysosomal sialidase

溶酶体唾液酸酶的分子结构和功能

基本信息

批准号：
04671376
负责人：
UDA Yutaka
金额：
$ 0.38万
依托单位：
Niigata College of Pharmacy
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

Rabbit antiserum was raised against sialidase preparation and the antiserum precipitated sialidase activity effectively. By the screening of bovine brain cDNA library with the anti-sialidase antibody, six clones which react with the antibody was obtained. Now, nucleotide sequence analysis of the cDNA is in progress.An acid sialidase, partially purified from human placenta, was activated by incubaton 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 The partially purified enzyme preparation contained aminopeptidase activity, which was inhibited by amastatin. Zinc ions inhibited either the activation of sialidase or the aminopeptidase activity in the enzyme preparatin. These results su … More ggest the possibility of participation of aminopeptidase function in the activation process of sialidase.It has been known that beta-gal exists as an enzyme complex with not only carboxypeptidase(CP) but also sialidase. CP protein is likely to be an essential factor to stabilize beta-gal as well as to express sialidase activity by forming the complex in lysosome. In order to clarify the function of CP,we have characterized CP in the purified beta-gal/CP complex from bovine liver. CP activity was optimum at pH 6 and activated by divalent cations, but strongly inhibited by DIFP,a serine protease inhibitor. By affinity labelling with ^3[H]-DIFP,the catalytic site of CP activity was demonstrated on the 30K protein. Two forms of beta-gal/CP complex(700K,100K)were separated by gelfiltration. Although 700K complex were de-polymerized to 110K one at pH 7, it was associated to 700K complex at pH 4.5. This conversion was observed even when CP activity was inactivated. 30K and/or 20K proteins, which are components of CP,may be necessary for forming high molecular weight beta-gal complex to stabilize beta-gal in lysosome, however, it seems that CP activity is not needed to form beta-gal/CP cpmlex.In order to be clarified the active component of lysosomal sialidase complex, synthesis of the sialic acid derivatives which inhibits sialidase activity was tried. Among the newly synthesized compounds tested, aminophenyl or nitrophenyl thio sialosides significantly inhibited sialidase. Now, synthesis of the derivatives of sialic acid containing photoreactive functional groups is in progress. Less

制备了兔抗唾液酸酶的抗血清，并能有效地沉淀唾液酸酶的活性。用抗唾液酸酶抗体筛选牛脑cDNA文库，得到6个与抗体反应的克隆。目前，cDNA的核苷酸序列分析正在进行中。从人胎盘中部分纯化的酸性唾液酸酶在37 ℃孵育下被激活。这种激活表现出时间和温度依赖性，在37 ℃和pH 4.3至5.2之间观察到最有效的激活。研究了不同蛋白酶抑制剂对其活性的影响。在测试的蛋白酶抑制剂中，氨肽酶A的抑制剂amastatin显著抑制活化。部分纯化的酶制剂含有氨肽酶活性，其被amastatin抑制。锌离子抑制唾液酸酶的活化或氨肽酶的活性，在酶Escheratin。这些结果表明， ...更多信息 β-半乳糖不仅与羧肽酶（CP）形成复合物，而且与唾液酸酶形成复合物。CP蛋白可能是稳定β-半乳糖的重要因素，也可能是通过在溶酶体中形成复合物来表达唾液酸酶活性的重要因素。为了阐明CP的功能，我们对牛肝中纯化的β-gal/CP复合物中的CP进行了表征。CP活性在pH 6时是最佳的，并且被二价阳离子激活，但被丝氨酸蛋白酶抑制剂DIFP强烈抑制。通过用^3[H]-DIFP进行亲和标记，在30 K蛋白上证实了CP活性的催化位点。用凝胶过滤法分离了两种形式的β-gal/CP复合物（700 K、100 K）。700 K络合物在pH 7时解聚为110 K络合物，而在pH4.5时与700 K络合物缔合。甚至当CP活性失活时也观察到这种转化。作为CP组分的30 K和/或20 K蛋白可能是形成高分子量β-gal复合物以稳定溶酶体中β-gal所必需的，然而，似乎CP活性不是形成β-gal/CP cpmlex所必需的。在新合成的化合物测试，氨基苯基或硝基苯基硫代唾液酸苷显着抑制唾液酸酶。目前，含光反应性官能团的唾液酸衍生物的合成正在进行中。少