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.

Project Start
1989-05-01
Project End
1994-04-30
Budget Start
1989-05-01
Budget End
1990-04-30
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
Schools of Medicine
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
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