Molecular structure and function of lysosomal sialidase

兔抗血清抗唾液酸酯酶制剂，抗血清有效地沉淀唾液酸酯酶活性。用抗唾液酸酶抗体对牛脑cDNA文库进行筛选，得到6个与该抗体反应的克隆。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出一种酸性唾液酸酶，用孵育剂在37℃下活化。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。在测试的蛋白酶抑制剂中，氨基肽酶A的抑制剂阿马伐他汀显著抑制活性。部分纯化的酶制剂含有氨基肽酶活性，被阿马伐他汀抑制。锌离子抑制了酶制剂中唾液酸酶和氨基肽酶的活性。这些结果进一步表明，氨基肽酶的功能可能参与唾液酸酶的激活过程。已知β -半乳糖不仅与羧肽酶（CP），而且与唾液酸酶以酶复合物的形式存在。CP蛋白可能是稳定β -半乳糖以及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子。为了阐明CP的功能，我们对纯化的牛肝脏β -gal/CP复合物中的CP进行了表征。CP活性在pH为6时最佳，可被二价阳离子激活，但被丝氨酸蛋白酶抑制剂DIFP强烈抑制。通过^3[H]-DIFP亲和标记，确定了30K蛋白上CP活性的催化位点。通过凝胶过滤分离两种形式的β -gal/CP复合物（700K,100K）。700K配合物在pH值为7时解聚为110K配合物，而在pH值为4.5时解聚为700K配合物。即使在CP活性失活时也观察到这种转化。30K和/或20K蛋白是CP的组成部分，它们可能是形成高分子量β -gal复合物以稳定溶酶体中β -gal的必要条件，然而，形成β -gal/CP复合物似乎不需要CP活性。为了弄清溶酶体唾液酸酶复合物的活性成分，尝试合成抑制唾液酸酶活性的唾液酸衍生物。在所测试的新合成化合物中，氨基苯基或硝基苯基硫代唾液苷显著抑制唾液酸酶。目前，含光反应官能团的唾液酸衍生物的合成正在进行中。少

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