We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 degrees C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 degrees C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 degrees C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 degrees C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 degrees C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 degrees C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 degrees C. These results indicate that prorenin has "gate" (T7PFKR10P) and "handle" (I11PFLKR15P) regions critical for its non-proteolytic activation.