Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing

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 proteinase 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.