Abstract
Incubation of human plasma prorenin (PR), the enzymatically inactive precursor of renin (EC 3.4.23.15), with a number of nonpeptide high-affinity active site-directed renin inhibitors induces a conformational change in PR, which was detected by a monoclonal antibody that reacts with active renin but not with native inactive PR. This conformational change also occurred when inactive PR was activated during exposure to low pH. Nonproteolytically acid-activated PR, and inhibitor-"activated" PR, as well as native PR, were retained on a blue Sepharose column, in contrast to proteolytically activated PR. Kinetic analysis of the activation of plasma prorenin by renin inhibitor (INH) indicated that native plasma contains an open intermediary form of prorenin, PRoi, in which the active site is exposed and which is in rapid equilibrium with the inactive closed form, PRc. PRoi reacts with inhibitor to form a reversible complex, PRoi.INH, which undergoes a conformational change resulting in a tight complex of a modified open form of prorenin, PRo, and the inhibitor, PRoi.INH-->PRo.INH. The PRoi-to-PRo conversion leads to the expression of an epitope on the renin part of the molecule that is recognized by a renin-specific monoclonal antibody. Presumably, PRo corresponds to the enzymatically active form of PR that is formed during exposure to low pH. Thus, it seems that the propeptide of PR interacts with the renin part of the molecule not only at or near the enzyme's active site but also at some distance from the active site. Interference with the first interaction by renin inhibitor leads to destabilization of the propeptide, by which the second interaction is disrupted and the enzyme assumes its active conformation. The results of this study may provide a model for substrate-mediated prorenin activation and increase the likelihood that enzymatically active prorenin is formed in vivo.
Highlights
Incubation of human plasma prorenin (PR), the en- which causes the prosegment to be cleaved from the renin zymatically inactive precursoorf renin (EC 3.4.23.15), polypeptide chain [1,2,3,4,5]
Proteolytic Activation of Gel-bound Plasma Proreninby Plasmin and Nonproteolytic Activation by Renin InhibitorsIncubation of gel-bound kidney renin with plasmin or with the renin inhibitors Ro 42-5892, A 64662, or CGP 38-560A for 24 h at 4 "C did not alter the binding of renin to the radiolabeled monoclonal antibody mAb4G1, an antibody that reacts with reninandnot with native inactive prorenin
Complex formation with the renin inhibitors is associated with a conformational change in proreninb,y which it becomes recognizable by the antibody
Summary
The second interaction is disrupted and the enzyme Chemicals-Two monoclonal antirenin antibodies were used. Proteolytic Activation of Gel-bound Prorenin by Incubation with Plasmin-Magnogel-Ab3E8 beads with bound prorenin from 250 pl of plasma were suspended in 250 p1 of PBS containing 0.2 p~ plasmin, and incubated for 24 h a t 4 "C, while the tubes were continuously shaken. Complete activation of prorenin by plasmin and the absence of any effect of the plasmin treatment on the MRC human kidney renin standard was demonstrated with the immunoradiometric assay, in which the washed plasmin-treated beads were incubated in a buffer containing 1251-labeledmAb4G1 and the quantityof antibody bound to the beads was measured after incubation, by y counting (immunoradiometric assay, see below). Nonproteolytic "Activation" of Gel-bound Prorenin by Incubation with Renin Inhibitor-Magnogel-Ab3E8 beads with bound prorenin from 250 p1 of plasma were suspended in 250 p1 of PBS containing renin inhibitor in concentrations ranging from lo-@to 10 -4 M. Kinetic Analysis of Renin Inhibitor-induced Activation of Prorenin-We assumed the renin inhibitor to interact with prorenin in themanner of active site-directed irreversible inhibitors [24] as follows
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