Proocytocin/neurophysin convertase from bovine neurohypophysis and corpus luteum secretory granules: complete purification, structure-function relationships, and competitive inhibitor.
Structure-function relationship studies were conducted on the proocytocin/neurophysin endoprotease previously characterized in both bovine neurohypophyseal and corpus luteum granules, using as a reference substrate a synthetic peptide reproducing the entire (1-20) NH2-terminal domain of the precursor. The [D-Arg12] derivative of proocytocin/neurophysin (1-20) was found to be a good competitive inhibitor of the enzyme (Ki = 30 microM), while the [D-Lys11] derivative was not. This allowed the complete purification of two isoforms of the endoprotease (Mr 58,000 and 52,000, respectively) by affinity chromatography using covalently immobilized [D-Arg12] proocytocin/neurophysin (1-20) as the affinity adsorbent. The use of selectively modified or truncated forms of the reference substrate or of the [D-Arg12] competitive inhibitor of the endoprotease established clearly that this basic pair specific convertase is sensitive to modification of the substrate structure either at the basic residues of the cleavage locus or at amino acids around this site (i.e., Pro7 and Gly9). It is concluded that longer distance interactions between amino acids situated on both the NH2 and COOH sides of the basic doublet Lys11Arg12 may contribute to the stabilization of a preferred substrate conformation allowing recognition by the enzyme subsites.
Plevrakis
I
I
Unité de Recherche Associée 003 au Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris, France.
Clamagirand
C
C
Créminon
C
C
Brakch
N
N
Rholam
M
M
Cohen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003338:Corpus Luteum / Q000201:enzymology*; D003594:Cytoplasmic Granules / Q000201:enzymology*; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis; D010455:Peptides / Q000138:chemical synthesis; D010904:Pituitary Gland, Posterior / Q000201:enzymology*; D011480:Protease Inhibitors; D013379:Substrate Specificity
D009842:Oligopeptides; D010455:Peptides; D011480:Protease Inhibitors; D010450:Endopeptidases; C056535:pro-ocytocin-neurophysin convertase