No. 6] Proc. Japan Acad., 62, Ser. B (1986) 197

52. : Purification, Structure Determination and

By Tadashi INACIAMI and Kunio MISONO Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.

(Communicated by Hisateru MITSUDA, M. J. A., June 10, 1986)

Renin is a peptidase whose function is dedicated exclusively to the forma- tion of angiotensin I from its prohormone angiotensinogen by a highly specific proteolysis of a unique leucyl peptide bond in the angiotensinogen molecule. The decapeptide angiotensin us converted in turn to the octapeptide angiotensin II by angiotensin I converting . Due to the potent vasoconstrictor and mineralocorticoid secretagogue activities of this octapeptide, renin is considered to play a pivotal role in the regulation of blood pressure. However, the molecular properties and regulatory mechanism of this pathophysiologically important enzyme had remained unclear for a long time, because it was not possible to obtain it in a pure form. It was because of exceedingly small quantities of renin in the kidney and instability of partially purified renin which was susceptible to proteolysic degradation by contaminating . To cope with these problems in the purification of renin, we have set up an affinity chromatographic method for efficient purification and employed in- hibitors for chemical elimination o:f contaminating proteases. Pepstatinl) was found to be a useful affinity ligand for the purification. It was attached to aminohexyl-Sepharose.2> The affinity chromatographic step was followed by ion exchange chromatography on DEAE- and CM-cellulose and gel filtration chroma- tography to complete the purification. We isolated renal renin in pure and stable states from the pig,3) rat4> and manse for the first time since its original dis-

Fig. 1. Inhibition of mouse submandibular gland renin by diazoacetyl-D,L-norleucine methyl ester (DAN) . In- corporation of a stoichiometric amount of DAN com- pletely inactivated mouse renin. 198 T. INAGAMI and K. MISONO [Vol. 62(B), No. 6] Renin : Structure and Active Site 199 covery in 1898 by Tiegerstedt and Bergman. Renin was also purified from mouse submandibular gland by conventional chromatographic techniques.7> This renin was shown to cross-react with mouse kidney renin immunologically. The unique substrate specificity and its exclusive role in the prohormone- to-hormone conversion in angiotensin formation aroused questions concerning the mechanism of its enzymatic action. Specific active site-directed inhibitors such as diisopropylfluorophosphate for se:rine proteases, EDTA for metaloproteases or iodoacetic acid for cysteine proteases did not inhibit the enzyme activity of renin. However, we found that renin was inhibited completely by acid -specific reagents diazoacetyl-D,L-norleucine methyl ester (DAN) (Fig. 1) and 1,2-epoxy- 3-nitrophenoxypropane (EPNP).g) These two reagents inhibited renin in a mutu- ally independent manner by esterifying-carboxyl groups of two aspartyl residues independently in much the same way as and D. These findings led to the conclusion that renin belongs to the family of acid (aspartyl) proteases which include the digestive pepsin, , the lysosomal enzyme , and the fungal protease penicillopepsin. This conceptual recognition of renin as a biochemically definable enzyme protein stimulated vigorous studies on renin. Very small quantities of renin available in pure forms had not permitted the determination of renin structure. We developed a large scale purification method for mouse submandibular gland renin for the sequence analysis of renin.g) By reductive carboxymethylation of renin it became clear that a mature renin molecule consists of two polypeptide chains, one heavy (H) and one light (L) chain, linked by a disulfide bridge. The H- and L-chains were fragmented by tryptic digestion and cyanogen bromide treatment. The amino acid sequences of these fragments were determined by automated Edman degradation. The entire amino acid sequence was determined10) as shown in Fig. 2. The structure consists of 328 amino acid residues. At about the same time the mouse renin amino acid sequence was also deduced from the base sequence of its cDNA by Pantier et al.11) However, their amino acid sequence contained considerable errors which were corrected later.12) The corrected sequence was in agreement with that determined by the direct method by us.10~ Human renin sequence was also determined later by using its cDNA by Imai et at.13.)

Fig. 3. Amino acid sequence identity in the vicinity of catalytically essential Asp-32 and Asp-215. Mouse renin structure is compared with rat cathepsin D13' and pig pepsinl4'. 200 T. INAGAMI and K. MIsoNO [Vol. 62(B),

The amino acid sequence of renin showed 49% sequence identity with rat spleen cathepsin D13 and 42% identity with porcine pepsin.14) The mouse renin also showed sequence homology with bovine chymosin (34%) and even with the fungal protease penicillopepsin (22%). Of particular importance are amino acid sequences in the vicinity of catalytically essential Asp-32 and Asp-215 as shown in Fig. 3. Asp-32 of pepsin was shown to be esterified by EPNP. Asp-215 was specifically esterified by DAN. Amino acid residues in the vicinity of these two residues such as Ser-35, Thr-Gly (216-217) have been implicated in penicillopepsin catalysis. Thus, renin seems to share a catalytic mechanism similar or identical to these aspartyl proteases. In spite of the extensive sequence identity, antibodies to renin and cathepsin D did not cross-react each other. In contrast to these general proteases, renin lacks the general proteolytic activity and hydrolyzes only one unique leucyl peptide bond in the angiotensinogen molecule. Structural basis for such a high degree of specificity is not yet known. The successful purification of renin and determination of the primary structure provide a great impetus to the further development of studies on three dimensional structure of renin. The clarification of the molecular properties of renin has greatly facilitated the investigation of regulatory mechanisms of physiological function of renin. Acknowledgements. Authors express sincere gratitude to Professor Hisateru Mitsuda, M. J. A., for his valuable discussion. Collaboration by Drs. K. Murakami, T. Matoba, H. Yokosawa, J.-J. Chang, S. Cohen, A. M. Michelakis, E. Haas and L. A. Holladay is gratefully acknowledged.

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