BIOCHEMICAL BASIS OF SULFATASE ACTIVITY

硫酸酯酶活性的生物化学基础

• 批准号: 3324527
• 负责人: ALLEN L HORWITZ
• 金额: $ 18.23万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3324527
• 关键词: antibody; arylsulfatases; complementary DNA; electrofocusing; enzyme biosynthesis; enzyme structure; fibroblasts; genetic transcription; genetic translation; human tissue; immunoprecipitation; inborn metabolism disorder; laboratory mouse; laboratory rabbit; laboratory rat; messenger RNA; mucopolysaccharidosis type II; mucopolysaccharidosis type VI; oligosaccharides; protein sequence; regulatory gene; sulfatases; tissue /cell culture; transfection

Mammalian sulfatases are a diverse group of enzymes which hydrolize steroid, mucopolysaccharide and glycolipid sulfates. The sulfatase deficiency disorders include a number of lysosomal storage diseases, and are a significant cause of mental retardation and/or physical deformities. Multiple sulfatase deficiency (MSD) is a unique genetic disorder resulting from deficiencies of at least seven distinct sulfatases and appears to result from increased degradation of cellular sulfatases. These investigations are aimed at the definition of various controls of sulfatase activity and causes of defects at various levels, including transcription, translation and post-translational processes. The elucidation of the defect in MSD is likely to demonstrate new levels of control of cellular hydrolases. Antibody and cDNA probes for steroid sulfatase, iduronate sulfatase (Id-S) and arylsulfatase B (ASB) will be used to study the various levels of control of study the molecular basis of the genetic variation of ASB activity in mouse strains with high or low ASB activity. Synthesis and processing of ASB in these mice using immunoprecipitation and pulse-chase labeling will also be studied to detect the effect of regulatory genes on mouse ASB. cDNA clones and antibodies for human ASB and human Id-S will be isolated and used to characterize the mRNA and newly synthesized polypeptides in ASB deficient Maroteaux-Lamy syndrome and Id-S deficient Hunter disease. These probes plus those for steroid sulfatase will be used to characterize the synthesis and processing of sulfatase in MSD. cDNA for sulfatase will be transfected into MSD and normal cells, and the translation and processing of the transfected STS will be followed. The subcellular compartmentalization and degradation of microinjected STS from normal and MSD cells will be evaluated. Immunoprecipitated labeled sulfatases from normal and MSD cells will be compared by isoelectric focussing, peptide mapping, oligosaccharide content and subcellular location. The role of cellular stabilization factors for sulfatases will be investigated. The route and location of post-translational processing and the role of the Golgi complex in sulfatase processing, including degradation, will be determined. The understanding of all these processes will be important in devising strategies for the treatment of enzyme deficiency diseases.

哺乳动物的硫酸盐酶是一组不同的酶，它们 使类固醇、粘多糖和糖脂硫酸盐水解物。 硫酸酯酶缺乏症包括一些溶酶体 储藏疾病，是智力低下的一个重要原因 和/或身体畸形。多发性硫酸酶缺乏症(MSD) 是一种独特的遗传性疾病，至少是由于缺乏 七种不同的硫酸盐酶，似乎是由于 细胞硫酸盐酶的降解。这些调查的目的是 关于硫酸盐酶活性的各种控制的定义和 不同层次缺陷的原因，包括转录， 翻译和翻译后流程。阐明了 MSD的缺陷可能会显示出新的控制水平 细胞水解酶。 艾杜酸类固醇硫酸酯的抗体和cDNAs探针 将使用硫酸酯酶(ID-S)和芳基硫酸酯酶B(ASB)来研究 不同水平的控制研究的分子基础 高OR小鼠品系ASB活性的遗传变异 ASB活性低。ASB在这些小鼠体内的合成和加工 使用免疫沉淀和脉冲追逐标记也将 研究检测调控基因对小鼠ASB的影响。 抗人ASB和人ID-S基因的克隆和抗体 被分离出来并用于鉴定mRNA和新的 ASB缺陷型Maroteaux-Lamy合成多肽 证候与ID-S虚型亨特病。这些探头加上 类固醇硫酸酯酶的那些将被用来表征 MSD中硫酸酯酶的合成与加工。硫酸酯酶基因的cDNA 将其导入MSD和正常细胞，并翻译成 并且随后将对转染型STS进行处理。这个 细胞的亚细胞区划和降解 来自正常细胞和MSD细胞的显微注射STS将被评估。 免疫沉淀标记的正常和MSD细胞中的硫酸酯酶 将通过等电聚焦，肽图谱， 低聚糖含量和亚细胞定位。的作用 硫酸盐酶的细胞稳定因素将被研究。 翻译后处理的路线和位置以及 高尔基复合体在硫酸盐酶加工中的作用，包括 退化，将被确定。对这一切的理解 在制定战略方面，进程将是重要的 酵素缺乏症的治疗。