BIOCHEMISTRY OF HUMAN ALPHA-GALACTOSIDASE A

α-半乳糖苷酶A是一种催化转化的溶酶体酶 将神经酰胺三己糖苷转化为乳糖基神经酰胺和半乳糖。这种酶 在正常人的许多组织中都发现了活性，但缺乏活性 导致溶酶体降解的代谢受阻 糖皮糖脂。α-半乳糖苷酶A的生理作用 某些蛋白质的溶酶体降解由以下事实进一步定义 法布里病患者缺乏α-半乳糖苷酶A活性 在它们的内脏组织中。由于α-半乳糖苷酶A是 在正常的人体组织中含量很低，而且相当 它不稳定，其生化和分子特性还没有很好的定义。为 例如，α-半乳糖苷酶A蛋白的相对分子质量范围为 49,000-101,000个，没有准确的氨基酸序列数据可用。 α-半乳糖苷酶A在体内的许多组织中都有合成，但它是 约占肝脏总蛋白的0.02%。vbl.使用 亲和纯化的兔抗人多克隆抗体 α-半乳糖苷酶A，我们证明了它的单特异性和 筛选利用表达载体构建的人肝细胞c DNA文库 GT-11。一个可能的人α-半乳糖苷酶A克隆被鉴定为 在文库中筛选200万个重组子。这份报告的真实性 通过展示相应的人 染色体基因映射到x染色体，这是已知的 人类的法布里氏病。这是通过使用假定的 α-半乳糖苷酶A基因克隆用于基因剂量和体细胞检测 细胞杂交DNA面板。分离出人α-半乳糖苷酶A基因 克隆，我们建议进行详细的生化和分子检测 人α-半乳糖苷酶A全长cDNA克隆的鉴定 将被分离并确定其核苷酸序列，从中 将推导出该蛋白质的氨基酸序列。染色体 α-半乳糖苷酶A基因的分离及其分子结构 将会被定义。突变的α-半乳糖苷酶A基因也将被分离出来 从法布里的病人身上提取并进行了测序。这些研究将定义 Fabry病的分子基础，并应为 产前诊断和携带者分析方法的发展 通过基因作图检测遗传性疾病。

Alpha-galactosidase A is a lysosomal enzyme that catalyzes the conversion of ceramide trihexoside to lactosylceramide and galactose. This enzyme activity is found in many tissues of normal individuals but its absence leads to a metabolic block in the lysosomal degradation of glycoshpingolipids. The physiological role of alpha-galactosidase A in lysosomal degradation of certain proteins is further defined by the fact that individuals with Fabry's disease lack alpha-galactosidase A activity in their visceral tissues. Due to the fact that alpha-galactosidase A is found in very low quantities in normal human tissues and it is fairly labile, its biochemical and molecular properties are not well defined. For example, the alpha-galactosidase A protein has a molecular weight range ot 49,000 - 101,000 and no accurate amino acid sequence data is available. Alpha-galactosidase A is synthesized in many tissues in vivo, but it is found to represent approximately 0.02% of the total liver proteins. Using an affinity purified rabbit polyclonal antibody to human alpha-galactosidase A, we have demonstrated its monospecificity and screened a human liver cDNA library constructed using the expression vector gt-11. A putative human alpha-galactosidase A clone was identified after screening 2 million recombinants in the library. The authenticity of the cDNA clone was verified by demonstrating the corresponding human chromosomal gene maps to the x chromosome, which is the known locus for Fabry's disease in man. This was accomplished by using the putative alpha--galactosidase A cDNA clone to probe both gene dosage and somatic cell hybrid DNA panels. Having isolated a human alpha-galactosidase A cDNA clone, we propose to perform detailed biochemical and molecular characterization of human alpha-galactosidase A. A full-length cDNA clone will be isolated and its nucleotide sequence determined, from which the amino acid sequence of the protein will be deduced. The chromosomal alpha-galactosidase A gene will be isolated and its molecular structure will be defined. Mutant alpha-galactosidase A genes will also be isolated from Fabry's patients and sequenced. These studies will define the molecular basis of Fabry's disease, and should provide the foundation for development of analytical methodologies for prenatal diagnosis and carrier detection of the herediatry disorder by gene mapping.