STRUCTURE AND FUNCTION OF CARBOXYPEPTIDASE M

羧肽酶 M 的结构和功能

• 批准号: 3463834
• 负责人: Randal A Skidgel
• 金额: $ 10.47万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-05-01 至 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3463834
• 关键词: carboxypeptidase; enzyme mechanism; enzyme structure; enzyme substrate; glycosylation; hormone binding protein; human tissue; immunocytochemistry; laboratory rabbit; membrane proteins; molecular cloning; peptide hormone; protein signal sequence; protein structure function; tissue /cell culture

Peptides and proteins with a C-terminal basic amino acid (e.g., bradykinin, anaphylatoxins) are potent mediators involved in may pathological, inflammatory processes. Removal of the C-terminal basic amino acid byu a carboxypeptidase can inactivated or alter the activity of this type of mediator. This proposal focuses on the newly described carboxypeptidase (CP)-M, which is plasma- membrane bound in many cells and tissues and cleaves C-terminal basic amino acids. The enzyme definitely differs from plasma CP- N or CP-H which is in intracellular secretory granules. The study of CP-M will fill a gap in our knowledge of how basic peptides and proteins can be processed or catabolized when they gain access to sites that are inaccessible to CP-N (i.e, extravascular) or CP-H (i.e., extrascellular). Our long-term objective is to understand how CP-M controls peptide activity under physiological and pathological conditions. The specific aims are: 1. Purify CP-M and raise antiserum. 2. Compare the enzymatic, physical and immunological characteristic of CP-M to those of other B-type carboxypeptidase. 3. Determine the N-terminal sequence of CP-M and the sequences of some internal peptides. 4. Isolate and sequence a cDNA clone corresponding to CP-M in order to deduce the protein sequence. 5. From the primary sequence of CP-M determine: the signal or activation peptide, if present, potential membrane binding region(s), glycosylation sites and possible active site residues. 6. Determine the mode of attachment of CP-M to the cell membrane. 7. Determine the localization of CP-M in kidney, placenta and cultured cells by immuno-electron microscopy. 8. Investigate the enzymatic activity of CP-M on the cell membrane by comparing kinetics of hydrolysis of biologically active peptides by soluble CP-M vs.membrane-bound CP-M. 9. Investigate the substrate specificity of CP-M with a series of synthetic peptides. 10. Study the synthesis, membrane attachment and membrane polarity, postranslational processing and possible mechanisms of release in cultured kidney cells. Accomplishing these objectives will provide new information on the localization and biochemical structural, and enzymatic characteristics of CP-M. It is hoped that the results will be applicable to the study of the control of peptide activity in normal or pathological situations.

具有C-末端碱性氨基酸的肽和蛋白质（例如， 缓激肽、过敏毒素）是参与可能的 病理性炎症过程 移除c末端 碱性氨基酸被羧肽酶灭活或改变 这类中介的活动。 该提案的重点是 新描述的羧肽酶（CP）-M，其是血浆- 膜结合在许多细胞和组织中，并切割C末端 碱性氨基酸 这种酶肯定不同于血浆CP- N或CP-H，存在于细胞内分泌颗粒中。 研究 CP-M的研究将填补我们对基本肽和 蛋白质可以被加工或分解代谢，当它们获得 CP-N（即血管外）或CP-H无法触及的部位 (i.e.,细胞外的）。 我们的长期目标是了解 CP-M如何在生理条件下控制肽活性， 病理条件。 具体目标是：1.纯化CP-M和 产生抗血清。 2.比较酶，物理和 CP-M与其他B型免疫学特性 羧肽酶 3.测定CP-M的N-末端序列， 一些内部肽的序列。 4.分离和测序 对应于CP-M的cDNA克隆，以推导蛋白质 顺序 5.从CP-M的一级序列确定： 信号肽或激活肽，如果存在，潜在膜 结合区、糖基化位点和可能的活性位点 残基 6.确定CP-M与细胞的连接方式 膜的 7.确定CP-M在肾脏中的定位， 胎盘和培养细胞的免疫电镜观察。 8. 研究CP-M在细胞膜上的酶活性 通过比较生物活性肽的水解动力学， 通过可溶性CP-M与膜结合CP-M。 9.探讨 CP-M与一系列合成肽的底物特异性。 10. 研究了合成、膜附着和膜 极性，翻译后加工和可能的机制， 在培养的肾细胞中释放。 实现这些目标 将提供新的信息定位和生化 CP-M的结构和酶特性。 希望 该结果将适用于控制的研究 在正常或病理情况下的肽活性。