I

Asiatic Herpetological Research

April 1990"

Vol. 3, pp. 116-119|and Amino Acid Sequence of a New Dodecapeptide from the Skin

Isolationof Oreolalax pingii +YIQUAN TANG SHENGHAI TlAN SHIXIANG WU JlACHENG HUA XlNQUAN Jl
GUANFU WU 2 ERMI ZHAO2 AND GANG ZOU
11,

11,,,1,1,2,^Shanghai Institute of Materia Medica, Academia Sinica, Shanghai, China
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 frommethanol extracts of the skin of the Chinese frog Oreolalax pingii. The sequence of the peptide is: GlyLeu-Val-Ser-Asp-Leu-Met-Tyr-Gly-Ile-Gly-Leu-NH 2 Th' s peptide differs from all the other amphibian skinpeptides and should be regarded as a member of a new peptide family.

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

Introduction

Many active peptides have been
discovered from amphibian skin during thepast 20 years or more. Amphibian skinpeptides have proved to be of considerable
value not only in pharmacology, but also intaxonomic and evolutionary domains(Erspamer 1984; Cei 1985; Lazarus et al.
1985). In order to discover new active
peptides, we have carried out research on
amphibian skin peptides from Chinese
frogs since 1983 (Hua et al. 1985; Tang etal. 1985).
This paper concerns anothernovel dodecapeptide obtained from the skinof Oreolalax pingii.

columns. The column was eluted withethanol-water mixtures of descending
concentrations of ethanol (also see
Montecucchi et al. 1981)

HPLC was performed on a Waters
HPLC system. Details of individual
chromatographic procedure are shown inthe figures.
Amino acid analysis ofpeptides after hydrolysis in HC1 were4400 amino acidcarried out on aanalyzer. Sequence analysis of the peptides

LKB

were performed by manual DABITC/PITC
procedure (Chang 1983). The completesequence analysis of the dodecapeptide wascarried out on an Applied Biosystems

Model 470A gas phase sequencer.

Methods
The

materials and experimental
procedures were previously reported (Tanget al. 1985), with the following brief
mentions and additions.
Six hundred specimens of Oreolalax
pingii were collected in May, 1983 from theDaliangshan of Sichuan Province, China.

The fresh skins (400 g) were removed andextracted with methanol. The methanol
extracts were evaporated until dry. The
residue was dissolved in 95% ethanol andthe solution distributed on the alumina

Enzymatic digestions of the peptide witha-Chymotrypsin, carboxypeptidase A (CPA) and Y (CP-Y) were also as before (Tanget al. 1985).
Bioassays of each isolated
weretested on the longitudinal
product
muscle myenteric plexus preparation of theguinea pig ileum (GPI),Results

The water portion eluted from alumina
columns was lyophilized to give 214 mg ofresidue.
The residue was separated by

HPLC semi-preparatively as in Fig. 1.
Perks 32 and 33 each were single peak by*This publication combines material previously
published in Chinese by Tang et al (1985) withadditional information.

ir»r»r\ i_.

verifying on HPLC (analytical uBondapak
Cj8 column, the elution conditions were thesame as in Fig. 1), respectively. The

Asiatic Herpetological ResearchApril 1990

Vol. 3, p. 117

100oCMC\J<60

40time(min)1. RP-HPLC of the water eluate from the alumina columns. Column: uBondapak C\% 7.8X300
mm. Mobile phase: A=0.05% CF3 COOH B=60% CH3CN in A. Concave gradient elution from 0-85%B

FIG.

(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 hCH-2-(-*): DABITC/PITC method.
CH:
CP-Y
digestion.
a-chymotryptic peptides.
phase sequencing. («-):

FlG. 2.

Profile of sequence anlaysis of the dodecapeptide.

difference of retention time of the twopeaks was 0.8 min. The peak 32 acted as
the representative of the dodecapeptide forfurther studies and the peak 33

was also

investigated simultaneously.

Amino acid composition of the peak 32(-»):

gas

and did not release any amino acids by theThis indicated a blocked Cformer.
terminus; upon latter, however, Leu and
Gly were obtained. Thus, we deduced that
the C-terminal structure of the peak 32 is

Leu-NH2. Digestion of the peptide by a-was Asp (1), Ser (l),Gly (3), Val (1), Met(1), He (1), Leu (3), Tyr (1). Amino acidsequence of the peak 32 was determined by
the DABITC/PITC method and gas phasesequencing. The former method proceeded

chymotrypsin provided further structuralconfirmation. The fragment peptides, CH1 and CH-2 were separated on HPLC as
Amino aciddepicted in Fig. 3.
are inof
thetwofragments
compositions
accordance with their sequences (seeto the tenth step, but the latter to theFig. 2), respectively.

For Cterminal residue analysis, CP-A and CP-Y
digestions of the peak 32 were carried out,

From the above results the completeamino acid sequence of the peak 32 waspenultimate residue (Fig.

2).

Asiatic Herpetological ResearchVol. 3, p. 118

April 1990

1000.2-r

102040305060time(min)FIG. 3. RP-HPLC of a-chymotryptic digests of the dodecapeptide. Column: uBondapak Cis 3.9X300mm. Mobile phase: A=0.1% CF3COOH B=60% CH3CN in 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.

,Discussion

established as in Fig. 2.

The peak 33 had the same properties
the peak 32 in the amino acidwithcompositions and sequence analysis,
do not know therespectively.
structural differences between the twoWepeaks.

Based on the amino acid analysis, theyield of the pure dodecapeptide (including

peak 32 and 33 in Fig. 1) was estimated to
be at least 1.0 nmol starting from 1.0 g of
fresh skin, according to that the rate ofrecovery of all above isolation steps was10%. The dodecapeptide was inactive in
GPI test, and its activity awaits to beestablished by assay methods other thanthose used in the present screening.

The dodecapeptide described above is acompletely novel peptide. The peptide may
be a member of a new peptide family, and it
should be regarded as the biochemical
characteristic of Oreolalax pingii intaxonomy. Furthermore, the C-terminal
portion of the dodecapeptide has somehomologies to the mammalian Leuenkephalin (Hughes et al. 1975) as shown
below:The dodecapeptide:

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

Leu-enkephalin: Try-Gly-Gly-Phe-Leu
Inamphibian skin peptide research, itAsiatic Herpetological Research

April 1990

one peptide can beseparated by HPLC into two components—

was observed

thatthe peak splitting, such as that discoveredin HPLC separation of PGL-a (Andreu et1985) and ranamargatin (Tang et al.
1988). The peak 32 and 33 (Fig. 1) bothhave the same amino acid composition andTherefore, they may besequence.
produced by one peptide in HPLC
separation. The reason of the peak splitting
mentioned above, however, is presently notknown. To our knowledge, there are no
similar findings besides the amphibian skinHence, it is necessary topeptides.
understand if the peak splitting is unique tothe amphibian skin peptides.
al.

Acknowledgments

We thank Mr. C. Chen for amino acidanalysis.

Vol. 3, p. 119

active peptides in amphibian skin and molluscan

tissues.

Comparative Biochemistry andPhysiology 79C:1-7.

HUA, J., S. WU, Y. TANG, W. ZHANG, AND G.

ZOU

1985

[Isolationand characterization ofbradykinin and its two fragments from the skinof the Chinese frog Rana nigromaculata ]. Acta
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HUGHES, J., T. W. SMITH, H. W. KOSTERLITZ,L. A. FOTHERGILL, B. A. MORGAN, AND H.

R.

MORRIS.

1975.

Identification oftwo

related pentapeptides from the brain with potent

opiate agonist activity.
258:577-579.

Nature (London)

LAZARUS, L.H., W. E. WILSON, G. GAUDINO, B.
1985.
IRONS, AND A. GUGLIETTA
Evolutionary relationships between nonmammalian and mammalian peptides. PeptidesJ.

6(Suppl. 3):295-307.

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