Study of Mucolipidosis by analysis of phosphorylation enzyme on carbohydrate moiety of lysosomal enzyme

UDP-GlcNAc：溶酶GlcNAc 1-磷酸转移酶，分别在II型粘脂病(I-细胞病)和III型粘脂病中缺失或部分表达活性，由于该酶在许多哺乳动物组织中表达较少且不稳定，因此尚未对其生化性质进行分析。此外，该酶需要[β-^&^&lt；32&gt；P]UDP-GlcNAc作为供体来测定其活性，而UDP-GlcNAc尚未商业化，需要合成。因此，这些缺点延误了对上述疾病的详细调查。本课题旨在对该酶蛋白进行免疫学分析，进一步阐明人酶纯化后粘脂病的发病机制。(1991)1.人白血病酶、血清和人肝细胞癌组织中该酶的活性明显高于正常对照。此外，在这所大学建立的大鼠纤维肉瘤细胞KMT-17和腹水细胞显示出最高的活性，提示有更好的酶来源进行纯化。2.本实验室从大鼠肝脏高尔基膜中分离纯化了该酶，并制备了兔抗该酶的多克隆抗体。然而，该抗体对人酶没有免疫反应，在大鼠和人(准备提交)中显示不同的蛋白质。(1992)1.大鼠酶的纯化方法已用于人酶，但由于没有来自人的新鲜或未冷冻的组织，导致纯化倍数较低(10^4倍)。2.由于该酶催化UDP-GlcNAc磷酸化为甘露聚糖，因此这些底物的类似物可能被用作光亲和探针。事实上，我们已经合成了[β-^&lt；32&gt；P]UDP-GlcNH_2和甘露聚糖的叠氮水杨酸衍生物，并将其应用于几个细胞中的酶的检测。

UDP-GlcNAc : lysosmal enzyme GlcNAc 1-phosphotransferase, which defects or partially expresses the activity in Mucolipidosis type II (I-cell disease) or type III, respectively, has not yet been analyzed for the biochemical property, since the enzyme is much less expressed and unstable in many mammalian tissues such as liver, kidney, etc. Furthermore the enzyme requires[beta -^<32>P]UDP-GlcNAc for assay of the activity as a donor, which is not available commercially and needs to be synthesized. These disadvantages, therefore, delay to investigate above diseases in detail. This project aims to analyze immunologically the enzyme protein and further clarify a mechanism of the defect in the Mucolipidosis after purification of the human enzyme.(1991) 1. The activity of the enzyme was much higher in human leukemic' enzyme and the serum, and human hepatocellular carcinoma tissue as compared to their normal control. Furthermore, rat fibrosarcoma cell KMT-17, established in this university, and the ascites cells demonstrated the highest activity, suggesting better source of the enzyme to purify. 2. The enzyme has been purified from rat liver Golgi membrane by us, and poly clonal antibody against the enzyme has been prepared by rabbit. However, the antibody has not reacted immunologically to human enzyme, showing different protein between rat and human (in preparation to be submitted).(1992) 1. The purification methods of the rat enzyme has been applied for the human enzyme, however, resulting low purification fold (10^4-fold), since fresh or unfrozen tissue from human has not been available. 2. Since the enzyme catalyzes the phosphorylation to mannan from UDP-GlcNAc, the analogues of these substrates might be employed as a photoaffinity probe. In fact, azidosalycilate derivatives of[beta -^<32>P]UDP-GlcNH_2 and mannan has been synthesized)and the derivatives are appling for detection of the enzyme in several cells.