Characteristics of the chromaffin granule aspartic proteinase involved in proenkephalin processing

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Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a chromaffin granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([35S]Met) preproenkephalin. The 70-kDa CG aspartic proteinase was purified by concanavalin A-Sepharose, Sephacryl S-200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val-D-Leu-Pro-Phe-Val-D-Leu--an inhibitor of pepsin, cathepsin D, and cathepsin E--distinguishes the CG aspartic proteinases from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys172-Arg173 pair located at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8, as assessed by peptide microsequencing. The importance of full-length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze 35S-labeled proenkephalin and proopiomelanocortin and its inability to cleave tri- and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.


Amino acid sequence; Animals; Aspartic acid; Aspartic Acid Endopeptidases/isolation & purification; Aspartic Acid Endopeptidases/metabolism; Aspartic proteinases; Aspartic proteinases—Inhibitors; Base Sequence; Chromaffin; Chromaffin Granules/enzymology; Endopeptidases; Enkephalins; Enkephalins/metabolism; Enzymology; Escherichia coli; Escherichia coli/metabolism; Genetics; Molecular probes; Molecular Probes/genetics; Molecular Sequence Data; Neurotransmitters; Nucleotide sequence; Pharmacology; Protease inhibitors; Protease Inhibitors/pharmacology; Protein precursors; Protein Precursors/metabolism; Protein Processing; Post-Translational; Rats; Recombinant Proteins


Biochemistry | Life Sciences | Medicine and Health Sciences

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