Probing epitopes on human prorenin during its proteolytic and non-proteolytic activation.
The conformational changes of prorenin (PR) that are associated with its reversible non-proteolytic activation and irreversible proteolytic activation were monitored with immunoradiometric assays, using antibodies against epitopes belonging to the propeptide or the renin part of PR. Binding of PR to the renin inhibitor remikiren or protonation of PR resulted in the slowly progressive and simultaneous expression (t1/2 congruent with3.5-5.0 h at 4 degreesC) of epitopes of the N-terminal and C-terminal halves of the propeptide and an epitope that is manifest on renin but not on native non-activated PR. During reversible PR activation-inactivation, expression and disappearance of these epitopes coincided with the appearance and disappearance of enzyme activity. Cleavage of the propeptide from the renin part of PR by plasmin, as demonstrated by the failure of remikiren to unmask the N-terminal and C-terminal propeptide epitopes, was, with some time lag, followed by the simultaneous expression (t1/2 congruent with60 min at 4 degreesC) of the renin-specific epitope and enzymatic activity. Based on these findings we propose a model for the non-proteolytic activation of PR that involves the formation of an intermediary form of activated PR with the following properties: (1) the covalently bound propeptide has moved out of the active-site cleft, so that binding sites are exposed to active site ligands, (2) the propeptide is still not in the 'relaxed' conformation that is characteristic for fully, non-proteolytically, activated PR, and (3) the N-terminal part of the renin polypeptide chain has not yet attained the proper location that is required for enzymatic activity.
Deinum
J
J
Department of Internal Medicine I, University Hospital Dijkzigt, Postbus 2040, 3000 CA Rotterdam, The Netherlands. deinum@inwl.azr.nl
Derkx
F H
FH
Schalekamp
M A
MA
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D004789:Enzyme Activation / Q000276:immunology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry* / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D008968:Molecular Conformation; D011487:Protein Conformation; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics; D012083:Renin / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D000939:Epitopes; D007093:Imidazoles; D011994:Recombinant Proteins; D005341:Fibrinolysin; D012083:Renin; C065581:remikiren