Title

Characteristics of the chromaffin granule aspartic proteinase involved in proenkephalin processing

Document Type

Article

Abstract

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.

Keywords

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

Disciplines

Biochemistry | Life Sciences | Medicine and Health Sciences

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