Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor.
The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.
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Unité de Neurobiologie et Pharmacologie (U. 109) de l'Institut National de la Santé et de la Recherche Médicale, Centre Paul Broca, Paris, France.
Martres
M P
MP
Schwartz
J C
JC
Gros
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000494:pharmacology; D007700:Kinetics; D008322:Mammals; D008407:Mast Cells / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism*; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D013552:Swine; D014158:Transcription, Genetic
D000941:Antigens; D018076:DNA, Complementary; D006023:Glycoproteins; C086812:Lxn protein, rat; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D002268:Carboxypeptidases