Enzymatic characterization of immunopurified prohormone convertase 2: potent inhibition by a 7B2 peptide fragment.
Prohormone convertase (PCs) are thought to mediate the controlled proteolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possible to produce recombinant PC2. We have used conditioned medium obtained from the mouse insulinoma cell line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Immunopurified PC2 cleaved the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC in a time- and calcium-dependent manner. It was half-maximally stimulated at 75 microM Ca2+, had an optimum pH of 5, and exhibited PCMS and EDTA sensitivity similar to that reported for furin and PC1. The tight-binding inhibitor 27 kDa 7B2 was used to calculate the Kd for this inhibitor and the active enzyme concentration. The Kd was 7.3 +/- 1.7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per enzyme molecule per minute. The specific activity was 4.9 nmol/micrograms/h (assuming a molecular mass for PC2 of 64 kDa). The enzyme preparation was able to cleave recombinant proenkephalin at at least four of the expected paired basic sites in the absence, but not in the presence, of 27 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase inhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used to demonstrate that the 7B2 CT-peptide (a) represents a potent inhibitor of PC2 (Ki = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Lindberg
I
I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112, USA.
van den Hurk
W H
WH
Bui
C
C
Batie
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D004745:Enkephalins / Q000378:metabolism; D006801:Humans; D007340:Insulinoma; D007700:Kinetics; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D045706:Proprotein Convertase 2; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D014407:Tumor Cells, Cultured; D014462:Ultrafiltration
D004745:Enkephalins; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; C029992:proenkephalin; D013381:Subtilisins; D045706:Proprotein Convertase 2