ENZYME DEFECT IN ALCOHOLIC PATIENTS

酒精患者的酶缺陷

• 批准号: 2044130
• 负责人: PAUL MANOWITZ
• 金额: $ 21.4万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2044130
• 关键词: acetaldehyde; alcoholism /alcohol abuse; arylsulfatases; chemical kinetics; electron microscopy; enzyme mechanism; enzyme structure; gel electrophoresis; genetic polymorphism; high performance liquid chromatography; histochemistry /cytochemistry; human tissue; immunofluorescence technique; phosphorylation

This work focuses on electrophoretic variants of arylsulfatase A (ASA) which have been proposed as a predisposing factor for dysmyelination due to alcohol intake. The studies in this grant application will examine structural, kinetic, and biological consequences of normal and variant ASA's. Extensive structural studies of the glycan and primary polypeptide structure will be performed. The effect of specific hydrolytic enzymes, lectin binding, and mannose-6-phosphate receptor binding on ASA will be studied to explore the structural elements of the glycan moiety. Since previous work has delineated the importance of the phosphorylated oligomannose glycan of the normal ASA on electrophoretic migration, particular attention will be paid to this parameter for the variant ASA's. The purified proteins will be fragmented and subjected to high performance liquid chromatography. Individual peaks which differ between the variant and normal enzymes will be analyzed in terms of both amino acid and carbohydrate content and sequence. The kinetic parameters of the variant and normal ASA's will be assessed in vitro using sulfatide as substrate in the presence of activator protein. A galactose biosensor will be used to determine the concentration of cerebroside, the product of the ASA reaction. The effect of ethanol and acetaldehyde on the reaction will also be measured. The biological consequences of ethanol on sulfatide metabolism and subcellular localization of normal and variant enzyme will be determined within fibroblast cells grown from skin biopsies. Subcellular localization of ASA will be performed by electron microscope cytohistochemistry as well as immunofluorescence analysis utilizing specific anti-ASA serum. In addition, high resolution subcellular fractionation will be performed to identify biochemically the location of ASA. In vitro cultured fibroblasts will be employed to metabolically label ASA with radioactive sulfate, phosphate, or glucosamine in order to assist in identifying sulfation and phosphorylation patterns of the normal and variant enzyme, as well as to allow detection of small amounts of glycan for complete structural analysis of ASA variants. It is anticipated that the integrated results of these various approaches will demonstrate structural and functional differences between the normal and variant ASA's.

这项工作的重点是芳基硫酸酯酶A（ASA）的电泳变体 已提出作为诱发障碍的诱发因素 饮酒。 该赠款申请中的研究将检查 正常和变体的结构，动力学和生物学后果 asa的。 聚糖和原发性多肽的广泛结构研究 结构将进行。 特定水解酶的影响， 凝集素结合，甘露糖-6-磷酸受体在ASA上的结合将是 研究以探索聚糖部分的结构元素。 自从 以前的工作描述了磷酸化的重要性 在电泳迁移时正常ASA的低聚糖聚糖， 将特别关注此参数的变体 asa的。 纯化的蛋白质将被碎裂并遭受高 性能液相色谱。 各个峰之间有所不同 将根据两种氨基分析变体和正常酶 酸和碳水化合物含量和序列。 将评估变体和正常ASA的动力学参数 在激活剂存在下，在体外使用亚磺胺作为底物 蛋白质。 半乳糖生物传感器将用于确定 大脑浓度是ASA反应的产物。 这 还将测量乙醇和乙醛对反应的影响。 乙醇对亚磺胺代谢和 将确定正常酶和变体酶的亚细胞定位 在皮肤活检中生长的成纤维细胞内。 亚细胞 ASA的定位将通过电子显微镜进行 利用细胞组织化学以及免疫荧光分析 特定的抗ASA血清。 另外，高分辨率亚细胞 将进行分馏以识别生化的位置 asa。 体外培养的成纤维细胞将用于代谢 用放射性硫酸盐，磷酸盐或葡萄糖胺标记ASA，以便为了 协助识别硫酸化和磷酸化模式 正常和变异酶，以及允许少量检测 聚糖的完整结构分析。 预计这些方法的综合结果 将证明正常之间的结构和功能差异 和变体ASA。