GENETIC AND BIOCHEMICAL STUDIES ON KRABBE'S DISEASE

克拉伯病的遗传学和生物化学研究

• 批准号: 3231553
• 负责人: DAVID A WENGER
• 金额: $ 4.51万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1986-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3231553
• 关键词: Krabbe's disease; antibody formation; beta galactosidase; brain metabolism; fibroblasts; gangliosidosis GM1; human tissue; leukodystrophy; lysosomes; molecular pathology; orphan disease /drug; tissue /cell culture

The aim of this research is to investigate the enzyme and activator protein which control the lysosomal degradation of galactosylceramide (gal-cer), psychosine monogalactosyldiglyceride and lactosylceramide (lac-cer). This enzyme, gal-cer Beta-galactosidase, is found in very low levels in all tissues from patients with Krabbe disease, an autosomal recessive disease of children, and in leukocyte and fibroblast cultures from some healthy people. We plan to purify this enzyme from control human brain using techniques routinely in use in this laboratory including hydrophobic affinity chromatography, chromatofocusing and specific affinity chromatography using psychosine (galactosylsphingosine) and the activator protein coupled to Sepharose. The purity of our enzyme will be checked by measuring other possible contaminating enzymes, by running polyacrylamide gel electrophoresis on different pH and concentration gels and by reverse phase liquid chromatography. The pure enzyme will be injected into rabbits for the production of antibodies which will then be used for measuring the level of crossreacting material (CRM) in tissues and cells from controls and patients with gal-cer Beta-galactosidase deficiency. These antibodies will then used for studies of processing of the protein chains after cells are given (3H)leucine in the medium. The nature of the mutation will also be examined in tissues and cells after extraction, SDS-PAGE, electroblotting to nitrocellulose filter paper, reaction with specific antibody and then with 125I-Protein A or horse-radish peroxidase conjugated goat antirabbit lgG. A study of this enzyme would not be complete without careful examination of the heat stable protein required for reaction with the lipid substrates. This activator protein will be purified from human brain, liver and spleen using methods currently in use in this laboratory. It's amino acid composition will be determined and its specificity to activate other enzymes will be evaluated. Antibodies to this activator protein will be made in rabbits and the levels of this activator will be determined in tissues and cells using rocket immunoelectrophoresis. The possibility that a defect in the activator protein is responsible for certain genetic diseases or the cause of some clinical heterogeneity will be investigated. Processing and determination of the molecular forms will be studied using the specific antibodies. This research will provide information related to the defect in Krabbe disease and other genetic diseases.

这项研究的目的是研究酶和激活蛋白 控制半乳糖酰胺（GAL-CER）的溶酶体降解， 心理单乳糖基甘油三酸酯和乳糖基酰胺（LAC-CER）。 这 酶，gal-cerβ-半乳糖苷酶，在所有水平中都发现很低 来自Krabbe病患者的组织，一种常染色体隐性疾病 儿童，以及一些健康的白细胞和成纤维细胞培养物 人们。 我们计划使用控制人的大脑净化这种酶 该实验室通常使用的技术（包括疏水） 亲和色谱，色谱和特定亲和力 使用Psychosine（半乳糖基肾上腺素）和激活剂色谱法 蛋白质耦合到sepharose。 我们的酶的纯度将由 通过运行聚丙烯酰胺来测量其他可能的污染酶 在不同的pH和浓度凝胶上的凝胶电泳以及反向 相液相色谱。 纯酶将被注入兔子 用于生产抗体，然后将其用于测量 来自对照组的组织和细胞中的交叉反应材料（CRM）水平 gal-cerβ-半乳糖苷酶缺乏症患者。 这些抗体 然后将用于研究细胞后蛋白质链的加工 在培养基中给出（3H）亮氨酸。 突变的性质也将 提取后，在组织和细胞中检查SDS-PAGE， 电印迹到硝酸纤维素滤纸，与特定的反应 抗体，然后使用125-蛋白A或马羽流过氧化物酶结合 山羊抗trabbit lgg。 没有该酶的研究将是不完整的 仔细检查与反应所需的热稳定蛋白 脂质底物。 该激活蛋白将从人中纯化 大脑，肝脏和脾脏使用当前在该实验室中使用的方法。 它的氨基酸成分将被确定，并具有 将评估其他酶。 该激活剂的抗体 蛋白质将在兔子中制成，该激活剂的水平将是 使用火箭免疫电泳在组织和细胞中确定。 这 激活蛋白中缺陷的可能性是 某些遗传疾病或某些临床异质性的原因将 被调查。 分子形式的处理和确定将 使用特定抗体研究。 这项研究将提供 与Krabbe病和其他遗传缺陷有关的信息 疾病。