Chemical structure of angiotensin in the turtle, Pseudemys scripta

GENERAL

AND

Chemical YUKIO

Lbpartment

COMPARATIVE

ENDOCRINOLOGY

Structure

53, 1.59-162 (1984)

of Angiotensin

in the Turtle, Pseudemys scrip&

HASEGAWA, MARK

CIPOLLE,* TAKUSHI X. WATANABE, HIROFUMI SOKABE, AND JOHN E. ZEHR*

TERUMI

NAKAJD.%A,~

of Pharmacology, Jichi Medical School, ~~na~z~kawach~-muchj, Tochigi-ken, 329-04, Japan; *Department of Physiology and Biophysics, University of Iiiinois at Urbana-Champaign, Urbana, Illinois 61801; and iInstitute for Medical and Dental University, Tokyo, 101 Japan Accepted February 25, 1983 The chemical structure of angiotensin generated by incubating kidney extract with homologous plasma from the turtle, Pseudemys scripta, has been analyzed. The turtle angiotensin was proposed to be [Asp’, Va15, His91 ANG I by its amino acid composition and by its fluorescent peptide mapping. It was the same structure as angiotensin I in the ox and the sheep. The N-terminal amino acid of the turtle angiotensin was not blocked, unlike that of another reptilian angiotensin found in the snake, Elaphe climacophora.

MATERIALS AND METHODS Since Nakajima et al. (1971) suggested that the chemical structure of nonmamPlasma and kidney tissue. Ibrtles (red-eared termalian angiotensin produced by incubating rapin), Pseudemys scripta, weighing 0.8-1.5 kg, were anesthetized with pentobarbital sodium (23 mgikg, kidney extract with homologous plasma im). A catheter (PE 50) was inserted into a carotid might differ from mammalian angiotensins, artery, and 1600 units of sodium heparin (Sigma) was [Asp’, Ile5, His91 ANG I or [Asp’, Va15, administered. Turtles were then decapitated, hung His91 ANG I, sequential analyses of angiohead down, and allowed to bleed into a chilled polytensins have been carrie.d out in some rep- ethylene beaker containing 3 ml of 0.5 M ammonium resentative nonmammalian species. We EDTA. Generally, 60 ml of blood could be obtained a l-kg turtle, The plasma was immediately sephave found [Asp’, Va15, Ser9] ANG I in the from arated by centrifugation (14OOg) for 1.5 min at 1” and domestic fowl, Gullus gallus v. domesticus stored at -20” until dialyzed. The kidney was then (Nakayama et al., 1973); [Aspr, VaF, Asn9] removed and weighed. Preparation of crude angiotensin. Crude angioANG I in the bullfrog, Rana catesbeiana tensin prepared by the modified Boucher proce(Hasegawa et al., 1982); [Asn’, ValS, His91 dure aswas reported previously (Hasegawa et al., 1982; ANG I in the Japanese goosefish, Lophius Takemdto et al., 1982). Kidney tissue, 50 g, was litulon (Hayashi et al., 1978); and [Asnr, minced, frozen, and thawed three times and then homogenized with distilled water (1 ml/g tissue) using a Vals, Asn9] ANG I in the chum salmon, Onmortar and pestle. The suspension was centrifuged at corhynchus keta (Takemoto et al., 1982). 35OOgfor 20 min. Both kidney extract and plasma were In a reptile, the snake (Elaphe climoco- dialyzed (cutoff = 12,000-14,000 &&.) against a soluphora) angiotensin was identified as X- tion of 0.005 M sodium EDTA, 0.10 M NaCl, and 0.05 [Asxl, Va15, Tyr9] ANG I (Nakayama et al., M ammonium acetate at pH 5.5, l”, for 20-24 hr. 1977). The N-terminal amino acid was Kidney extract was acidified to pH 3.0 (I&l) for &O min at 1”. After t’he pH was brought back to 5.5 blocked with unidentified substituent(s). It (NaOH), the extract was recentrifuged at 3SOOgfor 15 was not determined whether the N-terminal min just prior to the incubation procedure, is asparatic acid or asparagine. In the In vitro incubation and angiotensin elutipn from the Dowex 50W’rX2 resin was performed in two batches. present study, we have isolated angiotensin Each batch contained 250 ml dialyzed plasma @!H 5.5), produced by incubating kidney extract with 125 ml Dow& SOW:X2 resin suspended ,tiith 0.2 M plasma in the turtle, Pseudemys scripta, CH&ddNI&-CH,COOH (pH 5.5)., 25 ml dialyzed, and ‘proposed its amino acid sequence as acid-treated! kidney extract (pH 5.5), and 5 ml,ammonium EDNA (0.5 A4). Incubation with rapid shaking [Aspl, VaIs, His91 ANG I. 159 0016~6480184 $1.50 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form I-eserved.

160

HASEGAWA

was carried out at pH 5.5,25”, for 4 hr. For each batch, 125 ml of Dowex SOW-X2 resin (pH 6.0) was first added to a glass column (4.5 x 45 cm). The incubation mixture was placed in the column. The Dowex resin was successively washed with 750 ml of 0.2 M CHsCOONH.+-CHsCOOH buffer (pH 6.0), 750 ml of 10% CH$OOH, and 1500 ml of distilled water. Angiotensins adsorbed on the Dowex resin were then eluted off with 750 ml of 0.02 M diethylamine followed by 750 ml of 0.02 Mammonium hydroxide. The eluates from the two batches were concentrated by rotary evaporation at 45” and combined. This residue was washed with 50 ml of 80% ethanol and reevaporated four times. The dry residue was used as the starting material. The pressor activity in the materials was determined in the rat anesthetized with pentobarbital sodium (50 mg/kg, ip) and treated with pentolinium tartrate (5 mg/ kg, iv) as described previously (Hasegawa et al., 1982). [Asp’, Ile5] ANG II (Protein Research Foundation) was used as the standard. The starting material contained 115 Fg equivalent to [Asp’, Ile5] ANG II. Purification of angiotensin. The starting material was dissolved with 60 ml of 0.1% formic acid and centrifuged at 70,OOOg for 30 min. The supematant containing 68 p,g equivalent to [Asp’, Ile5] ANG II was vortexed in 200 ml of Dowex 5OW-X2 resin suspended with 0.2 M CHsCOONH4-CHsCOOH buffer (pH 6.5). The mixture was applied to a column and the resin was washed successively with 2 liters of 0.2 M CHsCOONH4-CHsCOOH buffer (pH 6.5), 4 liters of 10% CHsCOOH, and 6 liters of distilled water. Angiotensin adsorbed on the resin was eluted with 1.95 liters of 0.1 M diethylamine and 1.8 liters of 0.5 M ammonium hydroxide. The active fraction of 0.1 M diethylamine was concentrated to 200 ml by a rotatory evaporator. Further purification was followed in a manner similar to that described previously (Hasegawa et al., 1982). The concentrated fraction was applied to SEPPAK Cis cartridges (Waters Associates). The active principle, 51 yg equivalent to [Asp’, Ile5] ANG II, was eluted with 50% CHsOH and dried with the evaporators. It was then dissolved in 1 ml of 0.05 M HCOONH4-HCOOH buffer (pH 3.0) and applied to an SP-Sephadex C-25 column (120 x 800 mm). The active principle, 38 p,g equivalent to [Asp’, Iles] ANG II, was eluted with a linear concentration gradient from 0.05 M HCOONHd-HCOOH buffer (pH 3.0) to 0.5 M HCOONHd--HCOOH buffer (pH 6.5). The active fraction was applied to the SEP-PAK Cis cartridge for desalting. The active principle was eluted with 100% CH30H and dried. It was further purified with high-performance liquid chromatography (HPLC) with a FBondapak Cis column (Waters Associates). The active principle, 11.6 pg equivalent to [Asp’, Be51 ANG II, was eluted with 50 mM KHzPO~-H~PO~ buffer (pH 3.0) containing 50 mM NapSO and 18% CHsCN at a

ET AL. flow rate of 1 mlimin. Then, the active principle was rechromatographed with an SP-Sephadex C-25 column two times using 0.25 and 0.3 M HCOONH,-HCOOH buffers (pH 5.0) as eluants. The purity was checked as a single spot on high-performance thin-layer chromatography (HPTLC). We finally obtained the purified turtle angiotensin of 6.7 ug equivalent to [Aspl, Be51 ANG II. Amino acid analysis. Amino acid composition of purified angiotensin was determined by an amino acid analyzer (Toy0 Soda HLC-805) using the o-phthaldialdehyde method as described previously (Hasegawa et al., 1982). Fluorescent peptide mapping technique. Purified turtle angiotensin was cochromatographed on a HPTLC plate with synthetic [Asp’, Ile5, His91 ANG I, [Aspl, Va15, His91 ANG I, [AspI, Va15, Ser9] ANG I, [Asp’, Va15, Asng] ANG I, [Am’, Va15, His91 ANG I, and [Asni, Va15, Asn9] ANG I. Two fluorescent peptide maps were made by labeling with fluorescamine (Roche) before or after the development as reported previously (Hasegawa et al., 1982). Purified turtle angiotensin (200 ng equivalent to [Asp’, IleT] ANG II) was digested with 99 x low3 units of TPCK-trypsin (Worthington Biochemicals) or 18 x 10T3 units of ochymotrypsin (Worthington Biochemicals). Synthetic [Asp’, Va15, His7 and [Asn’, Va15, Hiss] ANG I were also digested under the same conditions to make reference standards. Chymotryptic or tryptic digests of these two angiotensins and the purified turtle angiotensin were cochromatographed and identified on the HPTLC plate by labeling before or after the development.

RESULTS AND DISCUSSION

Crude angiotensin of 115 p,g equivalent to [Aspl, Ile5] ANG II was obtained from 500 ml of the plasma. Crude angiotensin, 68 pg equivalent to [Asp’, Be51 ANG II, was purified by ion-exchange chromatography with Dowex 5OW-X2 and SP-Sephadex C25 columns, adsorbtion with SEP-PAK C,s cartridges, and HPLC with a p,Bondapak Crs column. When pressor activity was determined, only a single peak was seen in each step of purification. The active principle was eluted as a single peak in the HPLC pattern detected by the optical density at 214 nm. Its retention time was 48 min, and agreed with that of synthetic [Asp’, Va15, His91 ANG I. Purity of purified turtle angiotensin was detected as a single spot on HPTLC.

TURTLE

ANGIOTENSIN

a

3

4

5

6

7

2

3

45

6

7

FIG. 1. Identification of turtle angiotensin labeled with fluorescamine before (a) and after (b) the development. (1) [Asn’, Vals, His? ANG I; (2) [Asp’, Ile5, His91 ANG I; (3) [Asp’, Vals, Ser9] ANG I; (4) [Asp’, Va15, His91 ANG I; (5) turtle angiotensin; (6) [Asn’, Va15, Asng] ANG I; (7) [Asp’, Va15, Asn9] ANG I.

Amino acid composition of the purified angiotensin after acid hydrolysis was Aspi, Va&, Leq, Tyrt, Phet, His2, and Argt. This composition was the same as that of [Asp’, VaP, His91 ANG I. Purified turtle angiotensin and six synthetic angiotensins were cochromatographed on an HPTLC plate and identified by labeling with fluorescamine before or after the development (Fig. 1). Turtle angiotensin coincided with [Asp], Va15, His91 ANG I on HPTLC. After tryptic digestion of [Asp’, Va15, His91 ANG I, two fragments, Asp-Arg and Val-Tyr-Val-His-ProPhe- His - Leu, were produced. Tryptic digests of [Asnl, Va15, His91 ANG I were Asn-Arg and Val-Tyr-Val-His-ProPhe-His-Leu. Tryptic digests of these two synthetic angiotensins and turtle angiotensin were cochromatographed on an

HPTLC plate (Fig. 2). Two fluorescent peptide maps were also made by labeling before or after the development (the latter map was not shown). Two fragments of [Asp*, Va15, His91 ANG I and turtle angiotensin accorded with each other. Chymotryptic digests of [Asp’, Va15, PIis? ANG I, [Asn’, Va15, His91 ANG I, and turtle angiotensin were also cochromatographed on HPTLC (Fig. 2). Three fragments of [Asp I, Va15, His91 ANG I and turtle angiotensin accorded with each other. From the above results, we proposed that turtle angiotensin was [Asp], Va15, Nisg] ANG I. It was the same structure as angiotensin I in the ox and the sheep (Sokabe and Nakajima, 1980). The snake (E. climacophoua) angiotensin I, X-[Asx’, Va15, Tyrg] ANG I, was blocked at the N-terminal amino acid, which was

A fr.

--'

or.

12 i_

J

2. Identification of fluorescamine-labeled fragments of turtle angiqtensin digested with trypsin and chymotrypsinP(B). The peptides were labeled with fluorescamine before the developmeat. (1) Enzyme blank;:(2) [Asn’, Vals, His91 ANG I -t trypsin; (3) [Asp’, Val*, His7 ANG I + irypsin; turtle angiotensin I + trypsin. (B) (1) Enzyme blank; (2) [Asn’, Vals, His:] ANG I + chymotrypsin; [Asp*, Va15, His91 ANG I + chymotrypsin; (4) turtle angiotensin I + cbymotrypsin.

FIG.

(A) (A) (4) (3)

34

162

HASEGAWA

not detected by the dansyl method and remained to be identified as asparatic acid or asparagine (Nakayama et al., 1977). The Nterminal amino acid of turtle angiotensin was not blocked, because it could be detected by labeling with fluorescamine which bound with the primary amine of the N-terminal amino acid. It was asparatic acid, which is common to angiotensins elucidated in tetrapods. The N-terminal blocking may not be common in reptilian species. The active component of the renin-angiotensin system in the turtle may be an octapeptide, [Asp’, Va15] ANG II, because Stephens (1981) reported that teprotide, a converting enzyme inhibitor, reduced the vasopressor response to [Asp’, Ile5, His91 ANG I in the turtle, Pseudemys scripta eleguns, suggesting that an angiotensin-converting enzyme-like mechanism may exist. ACKNOWLEDGMENTS We thank Dr. S. Sakakibara, Peptide Institute, Protein Research Foundation, for supplying synthetic angiotensins. Part of this work was supported by a grant from the National Institutes of Health (NIH-HL 15307) to J.E.Z.

REFERENCES Hasegawa, Y., Watanabe, T. X., Sokabe, H., and Nakajima, T. (1983). Chemical structure of angio-

ET AL. tensin in the bullfrog Comp.

Rana catesbeiana. 50, 75-80.

Endocrinol.

Gen.

Hayashi, T., Nakayama, T., Nakajima, T., and Sokabe, H. (1978). Comparative studies on angiotensins. V. Structure of angiotensin formed by the kidney of Japanese goosefish and its identification by Dansyl method. Chem. Pharm. Bull. 26, 21% 219. Nakajima, T., Nakayama, T., and Sokabe, H. (1971). Examination of angiotensin-like substances from renal and extrarenal sources in mammalian and nonmammalian species. Gen. Comp. Endocrinol. 17, 4.58-466. Nakayama, T., Nakajima, T., and Sokabe, H. (1973). Comparative studies on angiotensins. III. Structure of fowl angiotensin and its identification by DNS-method. Chem. Pharm. Bull. 21, 20852087. Nakayama, T., Nakajima, T., and Sokabe, H. (1977). Comparative studies on angiotensins. IV. Structure of snake (Elaphe climocophora) angiotensin. Chem.

Pharm.

Bull.

25, 3255-3260.

Sokabe, H., and Nakajima, T. (1980). Chemical evolution of angiotensins. Zrz “Hormones, Adaptation and Evolution” (S. Ishii et al., eds.), pp. 281-286. Japan Sci. Sot. Press, Tokyo/Springer-Verlag, Berlin/New York. Stephens, G. A. (1981). Blockade of angiotensin pressor activity in the freshwater turtle. Gen. Comp.

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45, 364-371.

Takemoto, Y., Nakajima, T., Hasegawa, Y., Watanabe, T. X., Sokabe, H., Kumagae, S., and Sakakibara, S. (1983). Chemical structures of angiotensins formed by incubating plasma with the kidney and the corpuscles of Stannius in the chum salmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 51, 219-227.