1990" Asiatic Research Vol. 3, I April Herpetological pp. 116-119|

Isolation and Amino Acid Sequence of a New Dodecapeptide from the Skin of Oreolalax pingii +

1 1 1 1 Jl 1 YIQUAN TANG , SHENGHAI TlAN , SHIXIANG WU , JlACHENG HUA , XlNQUAN , 2 2 1 GUANFU WU , ERMI ZHAO , AND GANG ZOU

^Shanghai Institute of Materia Medica, Academia Sinica, Shanghai, China 2 Chengdu Institute of Biology, P.O. Box 416, Academia Sinica, Chengdu, China

Abstract. -A novel dodecapeptide has been isolated by alumina column chromatography and HPLC from methanol extracts of the skin of the Chinese Oreolalax pingii. The sequence of the peptide is: Gly-

Leu-Val-Ser-Asp-Leu-Met-Tyr-Gly-Ile-Gly-Leu-NH 2 Th's peptide differs from all the other skin peptides and should be regarded as a member of a new peptide family.

Key Words: Amphibia, Anura, Pelobatidae, Oreolalax pingii, China, biochemistry, peptides.

Introduction columns. The column was eluted with ethanol-water mixtures of descending Many active peptides have been concentrations of ethanol (also see discovered from amphibian skin during the Montecucchi et al. 1981) past 20 years or more. Amphibian skin peptides have proved to be of considerable HPLC was performed on a Waters value not only in pharmacology, but also in HPLC system. Details of individual taxonomic and evolutionary domains chromatographic procedure are shown in (Erspamer 1984; Cei 1985; Lazarus et al. the figures. Amino acid analysis of 1985). In order to discover new active peptides after hydrolysis in HC1 were peptides, we have carried out research on carried out on a LKB 4400 amino acid amphibian skin peptides from Chinese analyzer. Sequence analysis of the peptides since 1983 (Hua et al. 1985; Tang et were performed by manual DABITC/PITC al. 1985). This paper concerns another procedure (Chang 1983). The complete novel dodecapeptide obtained from the skin sequence analysis of the dodecapeptide was of Oreolalax pingii. carried out on an Applied Biosystems Model 470A gas phase sequencer. Methods Enzymatic digestions of the peptide with The materials and experimental a-Chymotrypsin, carboxypeptidase A (CP- procedures were previously reported (Tang A) and Y (CP-Y) were also as before (Tang et al. 1985), with the following brief et al. 1985). Bioassays of each isolated mentions and additions. product were tested on the longitudinal muscle myenteric plexus preparation of the Six hundred specimens of Oreolalax guinea pig ileum (GPI), pingii were collected in May, 1983 from the Daliangshan of Province, China. Results The fresh skins (400 g) were removed and extracted with methanol. The methanol The water portion eluted from alumina extracts were evaporated until dry. The columns was lyophilized to give 214 mg of residue was dissolved in 95% ethanol and residue. The residue was separated by the solution distributed on the alumina HPLC semi-preparatively as in Fig. 1. Perks 32 and 33 each were single peak by * verifying on HPLC (analytical uBondapak This publication combines material previously Cj8 column, the elution conditions were the published in Chinese by Tang et al (1985) with additional information. same as in Fig. 1), respectively. The

ir»r»r\ i_. April 1990 Asiatic Herpetological Research Vol. 3, p. 117

100

o CM

40 60 time(min)

FIG. 1. RP-HPLC of the water eluate from the alumina columns. Column: uBondapak C\% 7.8X300 elution mm. Mobile phase: A=0.05% CF3COOH B=60% CH3CN in A. Concave gradient from 0-85%B (dotted line), 90 min, at 1.0 ml/min. Detected at UV 220 nm, 0.4 aufs.

Gly-Leu-Val-Ser-Asp-Leu-Met-Tyr-Gly-Ile-Gly-Leu-NH 2

GH-1- A h CH-2-

FlG. 2. Profile of sequence anlaysis of the dodecapeptide. (-*): DABITC/PITC method. (-»): gas

phase sequencing. («-): CP-Y digestion. CH: a-chymotryptic peptides.

difference of retention time of the two and did not release any amino acids by the peaks was 0.8 min. The peak 32 acted as former. This indicated a blocked C- the representative of the dodecapeptide for terminus; upon latter, however, Leu and further studies and the peak 33 was also Gly were obtained. Thus, we deduced that investigated simultaneously. the C-terminal structure of the peak 32 is Leu-NH2. Digestion of the peptide by a- Amino acid composition of the peak 32 chymotrypsin provided further structural was Asp (1), Ser (l),Gly (3), Val (1), Met confirmation. The fragment peptides, CH- (1), He (1), Leu (3), Tyr (1). Amino acid 1 and CH-2 were separated on HPLC as sequence of the peak 32 was determined by depicted in Fig. 3. Amino acid the DABITC/PITC method and gas phase compositions of the two fragments are in sequencing. The former method proceeded accordance with their sequences (see to the tenth but the latter to the step, Fig. 2), respectively. penultimate residue (Fig. 2). For C- terminal residue analysis, CP-A and CP-Y From the above results the complete digestions of the peak 32 were carried out, amino acid sequence of the peak 32 was Vol. 3, p. 118 Asiatic Herpetological Research April 1990

0.2-r 100

10 20 30 40 50 60 time(min)

FIG. 3. RP-HPLC of a-chymotryptic digests of the dodecapeptide. Column: uBondapak Cis 3.9X300 in mm. Mobile phase: A=0.1% CF3COOH B=60% CH3CN A. Linear gradient elution from 0-60%B , 40 min, at 0.6 ml/min. Detected at UV 220 nm, 0.2 aufs. Peak (*) is the unreacted dodecapeptide.

established as in Fig. 2. Discussion

The peak 33 had the same properties The dodecapeptide described above is a with the peak 32 in the amino acid completely novel peptide. The peptide may compositions and sequence analysis, be a member of a new peptide family, and it respectively. We do not know the should be regarded as the biochemical structural differences between the two characteristic of Oreolalax pingii in peaks. . Furthermore, the C-terminal portion of the dodecapeptide has some Based on the amino acid analysis, the homologies to the mammalian Leu- yield of the pure dodecapeptide (including enkephalin (Hughes et al. 1975) as shown peak 32 and 33 in Fig. 1) was estimated to below: be at least 1.0 nmol starting from 1.0 g of fresh skin, according to that the rate of The dodecapeptide: Gly-Leu-Val-Ser-Asp- recovery of all above isolation steps was Leu-Met-Try-Gly-Ile-Gly-Leu-NH2 10%. The dodecapeptide was inactive in GPI test, and its activity awaits to be Leu-enkephalin: Try-Gly-Gly-Phe-Leu established by assay methods other than those used in the present screening. In amphibian skin peptide research, it Vol. 119 April 1990 Asiatic Herpetological Research 3, p.

was observed that one peptide can be active peptides in amphibian skin and molluscan tissues. and separated by HPLC into two components— Comparative Biochemistry Physiology 79C: 1-7. the peak splitting, such as that discovered in HPLC separation of PGL-a (Andreu et HUA, J., S. WU, Y. TANG, W. ZHANG, AND G. al. 1985) and ranamargatin (Tang et al. ZOU 1985 [Isolation and characterization of 1988). The peak 32 and 33 (Fig. 1) both bradykinin and its two fragments from the skin have the same amino acid composition and of the Chinese frog Rana nigromaculata ]. Acta Therefore, be sequence. they may Biochemica Biophysica Sinica 17:171-173. (In one in HPLC produced by peptide Chinese). separation. The reason of the peak splitting is not mentioned above, however, presently HUGHES, J., T. W. SMITH, H. W. KOSTERLITZ, known. To our knowledge, there are no L. A. FOTHERGILL, B. A. MORGAN, AND H. similar findings besides the amphibian skin R. MORRIS. 1975. Identification of two peptides. Hence, it is necessary to related pentapeptides from the brain with potent Nature (London) understand if the peak splitting is unique to opiate agonist activity. 258:577-579. the amphibian skin peptides.

W. E. G. B. Acknowledgments LAZARUS, L.H., WILSON, GAUDINO, J. IRONS, AND A. GUGLIETTA 1985. between non- We thank Mr. C. Chen for amino acid Evolutionary relationships mammalian and mammalian peptides. Peptides analysis. 6(Suppl. 3):295-307. Literature Cited MONTECUCCHI, P.C., R. DE CASTIGLIONE, S. PIANI, L. GOZZIN, AND V. ERSPAMER 1981. ANDREU, D., H. ASCHAUER, G. KREIL, AND R. Amino acid composition and sequence of B. MERRIFIELD 1985. Solid-phase synthesis dermorphin, a novel opiate-like peptide from the of PYLa and isolation of its natural counterpart, skin of Phyllomedusa sauvagei. International PGLa[PYLa-(4-24) peptide amide] from skin Journal of Peptide Protein Research 17:275-284. secretion of Xenopus laevis. European Journal of 149:531-535. Biochemistry TANG, Y., S. TIAN, S. WU, J. HUA, G. HU, X. JI, G. ZOU, G. WU, AND E. ZHAO. 1985. CEI, J.M. 1985. Taxonomic and evolutionary [Separation and structure of a novel hexapeptide significance of peptides in amphibian skin. obtained from the skin of Oreolalax pingii]. Peptides 6(Suppl. 3): 13-16. Acta Herpetologica Sinica 1985, 4(2):99-102. (In Chinese). CHANG, J. 1983. Manual micro-sequence analysis of polypeptides using dimethylaminoazobenzene TANG, Y., S. TIAN, S. WU, J. HUA, G. WU, E. isothiocyanate. Methods in Enzymology ZHAO, Y. LU, Y. ZHU, AND G. ZOU. 1988. 91:455-466. [Isolation and structure of ranamargarin, a new tachykinin from the skin of Chinese frog Rana a of ERSPAMER, V. 1984. Half century margaratae ]. Scienua Sinica (B) 9:967-974. (In comparative researchs on biogenic amines and Chinese).