Chemical structure of angiotensin formed with kidney renin in the Japanese eel, Anguilla japonica

Angiotensin was produced by incubating kidney extract with homologous plasma in the Japanese eel. The pressor activity in the incubation product was separated into two peaks in an SP-Sephadex C-25 column chromatogram. The major peak was purified further, and examined by amino acid analysis and the fluorescent peptide mapping technique. It was identified as [Asp1, Vals, Gly°] angiotensin (ANG) I. The chemical structures of nonmammalian angiotensins produced by incubating the kidney tissues with homologous plasma are said to differ from mammalian angiotensins, [Asp1, Ile5, His°], angiotensin (ANG) I, or [Asp1, Iles] and [Asn1, Vals] ANG II (4). We have previously found [Asp1, Vals, Ser9] ANG I in the fowl Gallus gallus v. domesticus (5); X-[Asx1, Vals, Tyr°] ANG I in the snake Elaphe climacophora (6); [Asp1, Val5, His°] ANG I in the turtle Pseudemys scripta (1); and [Asp1, Vals, Asn°] ANG I in the frog Rana catesbeiana (2). In teleostean angiotensins, two peaks were always seen in the SE-Sephadex chromatogram (1). The major peak of the Japanese goosefish (Lophius litulon) angiotensin was identified as [Asn1, Vals, His°] ANG I (6). The major and minor peaks of salmon (Oncorhynchus keta) angiotensins were identified as [Asn1, Vals, Asn°] ANG I and [Asp1, Val5, Asn°] ANG I, respectively (7). In the present study, we isolated the Japanese eel (Anguilla japonica) angiotensin produced by incubating the kidney extract with homologous plasma and analyzed its amino acid sequence. Crude angiotensin was prepared by the modified Boucher’s procedures reported previously (2). The kidney tissue, 220 g, was homogenized with 220 ml of 5.9 mM EDTA and centrifuged at 0°C and 18,000g for 15 min. The kidney extract and the eel plasma were dialyzed against 10 volumes of 5.9 mM EDTA at 4°C for 24 h. The dialyzed materials were incubated with 80 ml of Dowex 50W-X2 resin at 20°C, pH 7.4 for 6 h. The incubation mixture was applied to a column with 40 ml of the Dowex resin. The column was washed successively with 800 ml of 0.2 M ammonium acetate-acetic acid (pH 6.0), 2,400 ml of 10% acetic acid, and 2,400 ml of distilled water. Angiotensin was eluted with 1.01 each of 0.1 M diethylamine and 0.5 M ammonium hydroxide. Pressor activity in the eluates was determined in anesthetized rats as described previously (2). The total activity was 238.6 pg equivalent to [Asp1, Iles] ANG II. The crude angiotensin of 75 Hg equivalent to [Asp1, Iles] ANG II was used for further purification. The starting material was suspended with 75 ml of 0.1% formic acid and applied to SEPPAK C13 cartridges (Waters Associates). Three-fourths of the activity was eluted with 50% methanol and the eluate was evaporated to dryness. The residue was dissolved with 3 ml of 0.05 M ammonium formate-formic acid (pH 3.0) and chromatographed on an SPSephadex C-25 column (9.5 >< 820 mm) by linear gradient elution (Fig. 1). Pressor activity was separated into major and minor peaks which contained 20 and 7 pg equivalent to [Asp1, Iles] ANG II, respectively. The major active fractions were further rechromatographed on an SP-Sephadex C-25 column (9><550 mm) using 0.2 M ammonium