STRUCTURE AND FUNCTION OF CARBOXYPEPTIDASE M

羧肽酶 M 的结构和功能

• 批准号: 3463832
• 负责人: Randal A Skidgel
• 金额: $ 10.67万
• 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/3463832
• 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.

含有c端碱性氨基酸的肽和蛋白质(例如，