amide,. Tyr-Pro-Ser-Phe-NH2, and the a-lactalbumin. (50~ 53) amide, Tyr-Gly-Leu-Phe-NH2, were also active in the guinea pig ileum assay. These were named ...
Agric.
BioL
Chem.,
50 (9),
2419-2421,
2419
1986
Rapid Paper
ceptors.5) Amongthese peptides, Tyr-Pro-PheVal-NH2 and Tyr-Pro-Phe-D-Val-NH2 had
Opioid Peptides from Milk Proteins
high affinities for the receptors. We named them valmuceptin and devalmuceptin, respec-
Masaaki Yoshdcawa, Fumito Tani, Takashi Yoshimura and Hideo Chiba
tively. Brantl reported opioid activity of synthetic
tetra- and penta-peptide
fragments of
human /^-casein and called them human /?-
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received June 5, 1986
casomorphins.6)
Koch et al. also
reported
opioid activity of human jS-casomorphins.7)
In this paper, opioid activities of synthetic fragment peptides of human jS-casein were
evaluated in various assay systems. Other fragment peptides of milk proteins containing posFragment peptides of milk proteins containing Tyr-XPhe or Tyr-X1-X2-Phe were synthesized and their opioid activities were evaluated by the guinea pig ileum and radioreceptor assays. Among the peptides tested, the
human /?-casein(51 ~ 54) amide, Tyr-Pro-Phe-Val-NH2 (valmuceptin) was the most active, more than the bovine counterpart, morphiceptin. The human /?-casein(41 ~ 44) amide,
Tyr-Pro-Ser-Phe-NH2,
and
the
a-lactalbumin
(50~ 53) amide, Tyr-Gly-Leu-Phe-NH2, were also active in the guinea pig ileum assay. These were named /?-casor-
phin and a-lactorphin, respectively. All of these peptides were not as active in the mouse and rabbit vas deferens assays.
Exogenous opioid peptides have been iso-
lated from casein hydrolysates. Brand et al. isolated opioid peptide from casein peptone.1} The peptide Tyr-Pro-Phe-Pro-Gly-Pro-Ile
cor-
responded to the 60~66th residues of bovine /?-casein and Morphiceptin,
was named /?-casomorphin.2) Tyr-Pro-Phe-Pro-NH2, is the
most active opioid
peptide
of the bovine /?-
casomorphin group.3) Another opioid peptide,
a-casein exorphin, Arg-Tyr-Leu-Gly-Tyr-LeuGlu, was isolated from a peptic digest of bovine
asl-casein.4)
Although
structures
of
these exogenous opioid peptides are different from those of endogenousones, which have a Tyr-Gly-Gly-Phe-Met/Leu sequence at their amino termini, they had similar biological effects.1~4) We have found the Tyr-Pro-Phe
sequence, which is commonto bovine /?-casomorphin,
in the primary
structure
of a hu-
man jS-casein fragment. Wehave synthesized various humanjS-casein fragments containing
Tyr-Pro-Phe sequences at their amino termini and evaluated their affinities for opioid re-
sible opioid-like sequences, Tyr-X-Phe and Tyr-X1-X2-Phe were also synthesized and their opioid activities were evaluated. MATERIALS AND METHODS
Peptides were synthesized by a solid-phase method on chloromethyl resin.8) Peptide amides were synthesized on benzhydrylamine resin.9) r-Butyloxycarbonyl amino acids were coupled successively in the presence of 1-hydroxybenzotriazole and dicyclohexyl carbodiimide.10) Peptides
were deprotected by the anisole/hydrogen fluoride method11* and purified
on reverse-phase
liquid
chromatog-
raphy. The radioreceptor assay was done as described previously.5} Opioid activities of peptides were evaluated by the inhibition of electrically evoked contraction of myenteric plexus of guinea pig ileum longitudinal mus-
cle.12) Opioid activities were also tested similarly in mouse and rabbit vas deferens preparations. RESULTS AND DISCUSSION
/. Opioid activity of milk protein fragments containing Tyr-X-Phe sequences Opioid activities of human /^-casein
frag-
ments were measuredin various assay systems (Table I). All of these fragments inhibited the contraction of the myenteric plexus of guinea pig ileum longitudinal muscle and the inhibitory effects were specifically reversed by naloxone, an opioid antagonist. Therefore, all of the human /^-casein fragments synthesized are opioid agonists. In the radioreceptor and
guinea pig ileum assays, human/?-casomor-
phins 4 and 5 were less active than the bovine counterparts. On the other hand, valmuceptin, the humanjS-casomorphin 4 amide, was more
2420
M. Yoshikawa et al.
Table I. Opioid Activity
of /^Casein Fragments lC so(jm )
IQ o
MV D
P ep tid es R A* H um an T y r-P ro T y r-P ro T y r-P ro T y r-P ro T y r-P ro T y r-P ro T y r-P ro T y r-P ro
-P -P -P -P -P -P -P -P
he h e-V h e-D h e-V h e-D h e -V h e-V h e -V
al -V a l a l-N H 2 (v a lm u ce p tin ) -V a l-N H 2 (d e v a lm u ce p tin ) al-G lu a l-G lu -P ro a l-G lu -P ro -Ile -P ro
200 170 27 2.0 0.5 8 600 540 1300
T y r-G ly -P h e-L e u -P ro T y r-P ro -S e r-P h e -N H 2 (tf -ca so rp h in ) B o v in e T y r-P T y r-P T yr -P T yr -P
ro ro ro ro
-P -P -P -P
h e -P ro h e -P ro -N H 2 (m o rp h ice p tin ) h e -P ro -G ly h e -P ro -G ly -P ro -Ile
300 35
GPI fc
M V D C
-4 2 19 7 .6 0 .0 66 0 .0 60 14 25 25
nd 750 600 l l. 5 28 nd 350 540
270 30
30 3. 0 8 .4 30
IC50 GPI
3 9.5 7 8.9 5 7.5 16 5 1 4.0 2 1. 6
> 1 0 00 > 1 0 00
7. 5 0. 1 3 0. 69 6.9
nd 1 8.5 4 .2 nd
1 42 6 .1
Radioreceptor assay in the presence of 1 nM[3H]naloxone. Guinea pig ileum assay. Mouse vas deferens assay, nd, not determined.
active than morphiceptin, the bovine /?-caso- //. Opioid activity of milk protein fragments morphin 4 amide. The opioid activities of hu- containing Tyr-X1-X2-Phe sequences Other possible opioid-like sequences, Tyrman jS-casomorphin 4 and valmuceptin were X1-X2-Phe, were searched for in the primary increased by the replacement of Val4 by D-Val. Like morphiceptin, valmuceptin and deval- structures of milk proteins. One such is Tyrmuceptin were not as active in mouse and rabbit vas deferens assays and are specific lig^nds for the //-receptor. Recently, the for-
mation of morphiceptin from bovine jS-casomorphin 5 in the digestive suggested.13) Valmuceptin,
tracts has been however, would
not be formed from human jS-casomorphin 5 because of the absence of the carboxy-terminal Gly, the amidation codon. A [Leu]enkephalin-like sequence, Tyr-Gly-
Phe-Leu, is found in human /?-casein next to the human /?-casomorphin 8 sequence.5'140 The human jS-casein (59-63), Tyr-Gly-PheLeu-Pro was synthesized and its opioid activity was evaluated (Table I). The peptide
had weak opioid activity.
Pro-Ser-Phe,
which
corresponds
to
the
41 ~44th residues of human j8-casein.14) A tetrapeptide amide containing this sequence was synthesized. The peptide had opioid activity (Table I). This peptide was named /?casorphin. The counterpart of this peptide is not found in the primary structure of either bovine
or rat /?-casein.15'16)
Another example of this group, Tyr-GlyLeu-Phe, is found in a-lactalbumins of various animal species, including human and cow.17~20) Another example, Tyr-Leu-LeuPhe, is found in bovine /Mactoglobulin.21)
Tetrapeptide amides corresponding to these sequences were synthesized and their opioid activities were evaluated. As shown in Table II, the activities of these peptides were rather small. In the guinea pig ileum assay, the activity of the a-lactalbumin fragment was
2421
Opioid Peptides from Milk Proteins Table II. Opioid Activity
3)
of
WheyProtein Fragments IC50G"M)
Peptides RAa Tyr-Gly-Leu-Phe-NH2 (a-lactorphm)
m
Tyr-Leu-Leu-Phe-NH2
160
a Radioreceptor [3H]naloxone.
assay
in
the
GPIb
MVDC
5Q >mQ 160
presence
of
1nM
Biol. 6) 7)
fragment.
The
a-lactalbumin
fragment was nameda-lactorphin. Thus many potential opioid sequences were found in milk analgesic
activity,
endo-
Chem.,
cal roles.22) It is an interesting whether these exogenous opioid
problem sequences
9)
G.
W. Konig (1970).
and
(1967).
12) J. 13) /.
Lett., R.
REFERENCES
Lottspeich, 2)
Hoppe-Seyler's
1211 (1979). A. Henschen,
Z. Physiol.
F. Lottspeich,
Teschemacher, Hoppe-Seyler's 360,
1217
(1979).
V.
and F.
Chem., 360, Brantl
Z. Physiol.
P.
and
H.
Chem.,
Geiger,
and J.
1969, Chem.
Ber.,
M. Peffee,
3505. 103,
788
Y. Shimonishi, Y. Kishida, M. Okada Bull Chem. Soc. Jpn., 40, 2164
H. W. Kosterlitz, R. J. Lydon and A. J. Watt., Brit. Pharmacol, 39, 398 (1970). K.-J. Chang, Y. F. Su, D. A. Brent andJ.-K. Chang, Biol Chem., 260, 9706 (1985).
14) R. Greenberg, Chem.,
259,
M. L. Groves and H. J. Dower, /. 5132 (1984).
15) B. Ribadeau-Dumas, G. Brignon, F. Grosclaude and J.-C.
Mercier,
Eur. J. Biochem.,
D. E. Blackburn,
Nucleic Acid Res., 17) J. B. C. Findlay
Acknowledgment.This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
A. Henschen
and
22, 4567 (1983). and H. Chiba, Agric.
G. S. Brooks
65 (1972).
H. Teschemacher,
Hazum
/. Am. Chem. Soc, 85, 2149 (1963).
L. Southard,
ll) S. Sakakibara, and H. Sugihara,
16)
would have physiological importance.
V. Brantl,
E.
4%, 3185 (1984).
8) R. B. Merrifield,
Biol
genous opioid peptides have various pysiologi-
1)
Killian,
V. Brand, Eur. J. Pharmacol, 106, 213 (1985). G. Koch, K. Wiedemann and H. Teschemacher, Naunyn-Schmiedeberg's Arch. Pharmacol, 331, 351 (1985).
10)
antagonized by naloxone. However, the antagonism by naloxone was only partial for the fi-
Besides
A.
Tetrahedron
c Mouse vas deferens assay.
proteins.
Chang,
and W. A. Klee, Biochemistry, 5) M. Yoshikawa, T. Yoshimura
>1000
b Guinea pig ileum assay.
lactoglobulin
K.-J.
Cuatrecasas, Science, 212, 75 (1981). 4) S. Loukas, D. Varoucha, C. Zioudrou, R.A. Streaty
/. Biol.
Chem.,
and
J. M. Rosen,
10, 2295 (1982). and K. Brew, Eur. J. Biochem.,
18) K. Brew, F. J. Castellino, Hill,
25, 505 (1972).
A. A. Hobbs
245,
27,
T. C. Vanaman and R. J. 4570
(1970).
19) S. Kaminogawa, H. A. Mckenzie and D. C. Shaw, Biochem. Int., 9, 539 (1984). 20) O. U. Beg, H. von Bahr-Lindstrom, Z. H. Zaidi and H. Jornvall, Eur. J. Biochem., 147, 233 (1985).
21) G. Braunitzer, R. Chen, B. Schrank and A. Stangl, Hoppe-Seyler's Z. Physiol Chem., 345, 867 (1973). 22) V. Clement-Jones and G. M. Besser, "The Peptides," Vol. 6, ed. by S. Udenfriend and J. Meienhofer, Academic Press Inc., Orlando, Florida, 1984 pp. 323-389.
