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Characteristics of the Chromaffin Granule Aspartic Proteinase Involved in Proenkephalin Processing
Author(s) -
Azaryan Anahit V.,
Schiller Martin,
MendeMueller Liane,
Hook Vivian Y. H.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.65041771.x
Subject(s) - pepstatin , proenkephalin , biochemistry , cathepsin d , biology , chemistry , enkephalin , microbiology and biotechnology , enzyme , protease , receptor , opioid
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 ([ 35 S]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 Lys 172 ‐Arg 173 pair located at the COOH‐terminus of (Met)enkephalin‐Arg 6 ‐Gly 7 ‐Leu 8 , as assessed by peptide microsequencing. The importance of full‐length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze 35 S‐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.