CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

用常规的体细胞杂交技术和 鼠半乳糖神经酰胺酶缺乏病，抽搐，人 编码半乳糖神经酰胺酶的基因将被定位到特定的染色体上。 将尝试对人半乳糖神经酰胺酶进行详细的表征 通过丙烯酰胺凝胶电泳和电聚焦。 目的是（1） 将人类酶与小鼠酶区分开来， 可作为上述染色体定位的补充手段 研究，和（2）获得少量相对纯的酶， 用作单克隆抗体生产的抗原，也可能用作 用于至少几个N-末端氨基酸的测序的来源。 更 将尝试从胎盘和/或尿中进行常规纯化 有着相似的最终目标。 使用新的“自杀”抑制剂， β-半乳糖苷酶--半乳糖基甲基-对硝基苯基三氮烯（gal-MNT）-- 将探索差异抑制GM 1-神经节苷脂 β-半乳糖苷酶。 如果成功的话，传统的荧光底物， 4-甲基伞形酮基β-半乳糖苷可用于测定 半乳糖基神经酰胺酶及其在电泳上的定位 凝胶 这将促进半乳糖神经酰胺酶研究的所有方面， 包括对患者的诊断。 将尝试诱导实验小鼠模型， GM 1-神经节苷脂沉积症与gal-MNT抑制剂， GM 1-神经节苷脂β-半乳糖苷酶，但不是半乳糖神经酰胺酶。 模型 将在病理学和生化学上详细描述。 然后， 该模型将被操纵来回答有关 发育中的大脑对酶的需求和那些关于 在这组遗传性神经系统疾病中， 发育中大脑的可塑性障碍。 平行实验 计划使用器官型CNS培养物。 一种可能的新因子，赋予葡糖神经酰胺酶 将表征水解葡糖神经酰胺。 这并不等同 这种因子长期以来被称为“戈谢激活蛋白”。 就其本身而言， 将是脂质酶学的一个重要发展。 再者是 可以想象，类似戈谢病的遗传状态可能存在， 这个因素的异常。 这种可能性将在 该因素具有充分的特征。

With the conventional somatic cell hybridization technique and with the murine galactosylceramidase-deficiency disease, the twitcher, the human gene coded for galactosylceramidase will be mapped to a specific chromosome. Detailed characterization of human galactosylceramidase will be attempted by acrylamide gel electrophoresis and electrofocusing. The aim is (1) to distinguish the human enzyme from the mouse enzyme so that the procedure can be used as a supplementary means for the above chromosomal localization study, and (2) to obtain a small amount of relatively pure enzyme to be used as the antigen for monoclonal antibody production and also possibly as the source for sequencing of at least several N-terminal amino acids. More conventional purification will be attempted from the placenta and/or urine with similar eventual aims. The use of the new "suicide" inhibitor of beta-galactosidase -- galactosylmethyl-p-nitrophenyltriazene (gal-MNT) -- will be explored to differentially inhibit GM1-ganglioside beta-galactosidase. If successful, the conventional fluorogenic substrate, 4-methylumbelliferyl beta-galactoside could be used for assays of galactosylceramidase and also for its localization on the electrophoretic gel. This will facilitate all aspects of the galactosylceramidase studies, including diagnosis of affected patients. Attempts will be made to induce an experimental mouse model of GM1-gangliosidosis with the gal-MNT inhibitor which specifically inhibits GM1-ganglioside beta-galactosidase but not galactosylceramidase. The model will be characterized in detail pathologically and biochemically. Then, the model will be manipulated to answer questions concerning the requirement for the enzyme by developing brain and those concerning the timing of therapeutic intervention in this group of genetic neurological disorders vis-a-vis plasticity of developing brain. Parallel experiments are planned with the use of the organotypic CNS cultures. A possible new factor that confers to glucosylceramidase the capacity to hydrolyze glucosylceramide will be characterized. This is NOT the same factor long known as the "Gaucher activator protein". By itself, this would be an important development in lipid enzymology. Furthermore, it is conceivable that genetic status resembling Gaucher disease might exist due to abnormality of this factor. Such a possibility will be tested when the factor is adequately characterized.