Molecular structure and function of lysosomal sialidase

制备的唾液酸酶兔抗血清能有效地沉淀唾液酸酶活性。用抗唾液酸酶抗体筛选牛脑cDNA文库，获得了6个与该抗体反应的克隆。目前，该基因的核苷酸序列分析正在进行中。从人胎盘中部分纯化的一个酸性唾液酸酶在37ﾟC下被孵化棒激活。该激活具有时间和温度依赖性，在37ﾟC时激活效果最好，在pH为4.3到5.2之间。考察了不同的蛋白水解酶抑制剂对其活性的影响。在所测试的酶抑制剂中，氨基肽酶A的抑制剂阿司他丁对激活有明显的抑制作用，部分纯化的酶制剂含有氨基肽酶活性，而阿司他丁对其活性有抑制作用。锌离子对酶制剂中唾液酸酶的激活或氨基肽酶的活性均有抑制作用。这些结果可用于…进一步推测氨基肽酶功能参与唾液酸酶激活过程的可能性。已知，β-半乳糖不仅与羧基肽酶(CP)存在，而且与唾液酸酶存在。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体来表达唾液酸酶活性的重要因素。为了阐明CP的功能，我们对纯化的牛肝β-半乳糖/CP复合体中的CP进行了鉴定。该酶的最适pH为6，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。用^3[H]-DIFP亲和标记，证明了CP活性的催化部位在30K蛋白上。通过凝胶过滤分离了两种形式的β-半乳糖/CP复合体(700K和100K)。虽然700K络合物在pH=7时解聚为110K络合物，但在pH 4.5时它与700K络合物结合在一起。即使在CP活性失活的情况下，也能观察到这种转化。30K和/或20K蛋白是CP的组成部分，可能是形成高分子量β-半乳糖复合体以稳定溶酶体内β-半乳糖所必需的，然而，似乎不需要CP活性来形成β-半乳糖/CP cpmlex.为了阐明溶酶体唾液酸酶复合体的活性成分，尝试合成抑制唾液酸酶活性的唾液酸衍生物.在测试的新合成的化合物中，氨基苯基或硝基苯硫代唾液酸苷对唾液酸酶有显著的抑制作用。目前，含光活性官能团的唾液酸衍生物的合成正在进行中。较少

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