Characteristics of the chromaffin granule aspartic proteinase involved in proenkephalin processing.
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.
Azaryan
A V
AV
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
Schiller
M
M
Mende-Mueller
L
L
Hook
V Y
VY
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002837:Chromaffin Granules / Q000201:enzymology*; D004745:Enkephalins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins
D004745:Enkephalins; D015335:Molecular Probes; D011480:Protease Inhibitors; D011498:Protein Precursors; D011994:Recombinant Proteins; C029992:proenkephalin; D016282:Aspartic Acid Endopeptidases