Prorenin is the inactive precursor of renin (EC 3.4.23.15), which is a key enzyme in the regulation of blood pressure and electrolyte balance. Prorenin has a prosegment with 43 residues attached to the N terminus of mature renin with 339 -341 residues (1-4). The prosegment has been considered to associate with mature renin to prevent interaction with angiotensinogen, its macromolecular renin substrate (5-7). Prorenin does not proteolytically self-activate like pepsinogen, and its blood circulating level is 10 times higher than that of mature renin (8, 9). Some investigators have recently proposed that prorenin is a useful marker of diabetic microvascular complications and Wilms' tumor (11-13). However, much information regarding prorenin is unclear or lacking. The intrinsic activation enzyme, the activation mechanism in vivo, and its physiological role and source in the circulation remain unknown.Prorenin has reportedly been activated in vitro by endopeptidases such as trypsin and cathepsin B (3, 14, 15) and has also been non-proteolytically activated under acidic pH and/or low temperature (17)(18)(19)(20)(21)(22). We recently showed that specific antibodies to the prosegment (L 1P PTDTTTFKRIFLKR 15P ) activated human prorenin non-proteolytically (23). More recently, a renin/prorenin receptor was found in several tissues with nonproteolytically activated renin as well as prorenin (24). These non-proteolytic activations have generally been thought to arise from a conformational change of the prorenin molecule in vivo.The inactivation mechanism for prorenin has been reported using recombinant prorenins mutated at single to triple residues in the prosegment that formed ionic bonds (25-27) or a hydrophobic bond (27) between the prosegment and mature renin. Advanced research on the role of the prosegment in the non-proteolytic activation of prorenin may provide clues to solving those problems. Recently, we found that the acid activation rate of rat prorenin was less than one-fifth of that of human prorenin (4), and the speed of this process was only dependent on the amino acid sequence in the prosegment with 43 amino acid residues (28). These results led to our working hypothesis that there was an essential region in the N-terminal side of the prosegment for non-proteolytic activation of prorenin. In this study, we propose a hypothesis that there are two key regions, "gate" and "handle," in prorenin non-proteolytic activation using several kinds of prorenin-specific antibodies. EXPERIMENTAL PROCEDURESDesignation of Antigen Peptides-Antigen peptides in several regions of the prorenin prosegment were designed on the basis of the primary structure of the prosegment and the stereo structure of human prorenin predicted by the homology modeling method, as shown in Fig.

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