Isolation and molecular cloning of mast cell carboxypeptidase A

摘要: Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and a Kirsten sarcoma virus-immortalized line (KiSV-MC), cDNA that encodes this exopeptidase cloned KiSV-MC-derived library. was purified with potato-derived carboxypeptidase-inhibitor affinity column found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be Mr 36,000 protein. Secretory granule proteins KiSV-MC CTMC were then resolved preparative transblotted polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences obtained for prominent protein present in both types. Based on amino-terminal sequence, an oligonucleotide probe synthesized used isolate 1,470-base pair exopeptidase. The deduced sequence revealed that, after cleavage 15-amino hydrophobic signal peptide 94-amino activation 417-amino preproenzyme, mature core predicted 35,780 high positive charge [Lys + Arg) - (Asp Glu) = 17) at neutral pH. Although critical zinc-binding acids (His67, Glu70, His195), substrate-binding (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), cysteine residues participate intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) pancreatic carboxypeptidases also A, overall identities relative rat A1, A2, B only 43, 41, 53%, respectively. RNA DNA blot analyses CTMC, KiSV-MC, bone marrow-derived all express 1.5-kilobase mRNA which is transcribed single gene. We conclude enzyme subclass represents novel addition gene family.