STRUCTURE AND FUNCTION OF CARBOXYPEPTIDASE M

具有C-末端碱性氨基酸的多肽和蛋白质(例如， 缓激肽、过敏性毒素)是5月参与的强有力的介体 病理性的炎症过程。去掉C-末端 碱性氨基酸是一种羧基肽酶，可以灭活或改变 这类调解人的活动。这项提案的重点是 新发现的羧基肽酶(CP)-M，是一种血浆- 结合在许多细胞和组织中的膜，裂解C-末端 碱性氨基酸。这种酶显然不同于血浆中的CP- 胞内分泌颗粒中的N或CP-H。这项研究 Cp-M的研究将填补我们对碱性多肽和 蛋白质可以被加工或分解当它们获得 CP-N(即血管外)或CP-H无法访问的站点 (即细胞外)。我们的长期目标是了解 CP-M如何在生理和生理状态下控制肽活性 病理情况。具体目标是：1.纯化CP-M和 提高抗血清水平。2.比较酶法、体力法和 CP-M与其他B型的免疫学特征 羧基肽酶。3.测定CP-M的N-端序列和 一些内部多肽的序列。4.分离和测序 一个与CP-M相对应的cDNA克隆，以推导该蛋白 序列。5.根据CP-M的一级序列确定： 信号或激活肽，如果存在，潜在的膜 结合区域(S)、糖基化部位和可能的活性部位 残留物。6.确定CP-M与细胞的附着方式 薄膜。7.确定CP-M在肾脏中的定位。 免疫电子显微镜观察胎盘和培养细胞。8. CP-M在细胞膜上的酶活性研究 通过比较生物活性多肽的水解动力学 通过可溶性CP-M与膜结合CP-M的比较。9.调查 CP-M与一系列合成肽的底物专一性。 10.合成、膜附着和膜的研究 极性、翻译后加工及可能的机制 在培养的肾脏细胞中释放。实现这些目标 将提供有关本地化和生化的新信息 CP-M的结构和酶学性质。希望是这样的 这一结果将适用于防治的研究。 在正常或病理情况下的多肽活性。

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.