Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOLATION OF PEPTIDES WITH OPIATE ACTIVITY FROM SHEEP AND HUMAN PITUITARIES: RELATIONSHIP TO BETA-LIPOTROPIN
M. C h r # t i e n ,
S. B e n j a n n e t ,
N. Dragon,
N.G. Seidah and M. L i s Clinical
Research
Institute
of Montreal.
Received July 22,1976 SUMMARY. Human and o v i n e p i t u i t a r i e s were found to c o n t a i n p e p t i d e s of i d e n t i c a l s i z e , c o m p o s i t i o n and N - t e r m i n a l r e s i d u e to the c a r b o x y - t e r m i n a l p o r t i o n 61-91 of b e t a - l i p o t r o p i n (I-91). These p e p t i d e s e x h i b i t m o r p h i n e - l i k e a c t i v i t y in the mouse vas deferens bioassay. Beta-lipotropins from both species have much lower potency than the new p e p t i d e s in the same b i o a s s a y . The f i n d i n g s f a v o r the h y p o t h e s i s t h a t b e t a - l i p o t r o p i n i s the p r e c u r s o r of endogenous m o r p h i n e - l i k e p e p t i d e s of p i t u i t a r y origin. Hughes et a l . structure like
(I)
recently
isolated
of a pentapeptide
from pig
brain
activity.
enkephalin al.
(I)
This
pentapeptide
(met-enkephalin)
to be s t r u c t u r a l y
beta-lipotropin discovered
and d e s c r i b e d
(gamma-LPH). to the f i r s t
It
was found
and Li
58 amino a c i d s
being
structure
(3),
that
that
of
of beta-MSH. beta-LPH
an i n t e r m e d i a t e .
peptide
beta-LPH.
is
It
Chr~tien
and Li
is
Moreover,
(3)
identical
both beta-LPH
41-58 sequence the
was t h e r e f o r e
the prohormone
472
by Hughes et
named g a m m a - l i p o t r o p i n
in t h e i r
The d i s c o v e r y
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
morphine-
the name m e t h i o n i n e -
gamma-LPH sequence
to c o n t a i n
the
to the 61-65 sequence of
In 1967,
another
and gamma-LPH were found complete
(2).
potent
was a l s o observed
identical
(beta-LPH)
with
was g i v e n
and i t
and d e s c r i b e d
proposed
of beta-MSH,
of m e t - e n k e p h a l i n
by C h r ~ t i e
gamma-LPH raised
the
Vol. 72, No. 2, 1976
possibility Chung
identical peptides (6,
that
(4)
to 61-91
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
beta-LPH
isolated
a peptide
peptide
while
tification
(l-91)
report
of p e p t i d e s
with
human and sheep p i t u i t a r i e s to
residues
activity.
61-91
found
an All
Seidah
fragments
the
morphine-like with
(5)
Finally,
i s much l e s s
and
corresponding
have c o n s i d e r a b l e
describes
Li
pituitaries.
the t r y p t i c
of sheep beta-LPH
The p r e s e n t
et al.
from p o r c i n e
shown t h a t
as w e l l .
pituitaries
Bradbury
morphine-like
beta-LPH
precursor
from camel
in e x t r a c t s
exhibit
and 61-91
activity
be i t s
sequence of beta-LPH.
7) have r e c e n t l y
61-81
could
these
et a l .
61-80,
morphine-like
active. isolation
activity
amino a c i d
and i d e n - from both
composition
corresponding
of beta-LPH.
MATERIAL AND METHODS Frozen human and sheep p i t u i t a r i e s were e x t r a c t e d to the method of Li (8) and the e x t r a c t chromatographed C M - c e l l u l o s e or CM-sephadex. Some of the human C M - c e l l u l o s e f r a c t i o n s were f u r t h e r purified by Sephadex G-75 and e l u t e d w i t h O.Ol M ammonium a c e t a t e pH 4 . 6 . The sheep p i t u i t a r y e x t r a c t was chromatographed on CM-Sephadex as d e s c r i b e d by Bradbury et a l . (5) p r i o r t o , t h e CM-cellulose step. The m o r p h i n e - l i k e a c t i v i t y o f i s o l a t e d f r a c t i o n s was assayed on mouse vas d e f e r e n s as d e s c r i b e d by Henderson e t a l . (9). The a c t i v i t y i s e x p r e s s e d as p e r c e n t a g e o f i n h i b i t i o n of electrically s t i m u l a t e d c o n t r a c t i o n s of mouse vas d e f e r e n s in vitro. The maximal response i s u s u a l l y in the range o f 70-80% inhibition. Amino a c i d a n a l y s i s was c a r r i e d out a c c o r d i n g to Spackman e t a l . ( l O ) using a Beckman 121C a u t o m a t i c a n a l y z e r . The N - t e r m i n a l r e s i d u e was i d e n t i f i e d by the dansyl p r o c e d u r e (ll). P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s was done a c c o r d i n g to Davis (12) at pH 4 . 5 . according on e i t h e r
RESULTS The C M - c e l l u l o s e according hormone,
to
Li
(8),
is
routinely
ACTH and beta-LPH
a new peak a d j a c e n t
to t h a t
possessed m o r p h i n e - l i k e
chromatography
(13).
of
human p i t u i t a r y
used f o r
purification
A slightly
slower
o f growth
activity.
hormone;
Gel f i l t r a t i o n
473
extract
o f growth
gradient
revealed
the c o n s t i t u e n t of
this
fraction
VoI. 72, No. 2, 1976
2,0
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
-
1.8 16
~,
1.4
~, LO
0.4
%0 i;o ,~o ~;o
2)0
~;o 2;o 2~o 3oo 32o 34o 36o 38o 4oo 42o TUBE NUMBER
Figure I: Purification o f human f r a c t i o n e l u t e d from CMcellulose ( 3 7 . 5 mg) p o s s e s s i n g m o r p h i n e - l i k e a c t i v i t y on a column ( 2 . 5 x 120 cm) of sephadex G-75 s u p e r f i n e , using 0.01 M NH4Ac pH 4.6 as e l u a n t . Peak D gave the p e p t i d e 61-91 of human beta-LPH (see t a b l e I ) . 2.0 LB ~VH4,~I NH, Ac oJ~ I 0.4M 1,6 pH4.6,I, pH ~.;'
NH4Ac
1,4
1.2 LO
~
O.B
~, o,6 0.4 0.2
.A 0
i
i
I
i
40
60
80
I00
120
140
160
IBO
200
220
240
i
I
260
280
I
300
TUBE NUMBER
F i g u r e 2: Purification of sheep p i t u i t a r y extract eluted from CH-sephadex w i t h 1 M NH4Ac on a column (I x 25 cm) o f C H - c e l l u l o s e , u s i n g a g r a d i e n t of NH4Ac from 0 . I to 1 M. Peak I I I , upon f u r t h e r p u r i f i c a t i o n on sephadex G-25, gave the p e p t i d e 61-91 of sheep beta-LPN (see t a b l e I ) .
474
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I AMINO ACID ANALYSIS OF HUMAN AND SHEEP 61-91 Hydrolysis
Time
Human* Amino Acid Lys His Asp Thr Ser Glu Pro GIy Ala Val Met lle Leu Tyr Phe
PEPTIDES
Sheep*
24 h
48 h
72 h
Nearest Integer
4.53
5.28
4.80
5
2.08 2.71 1.84 2.99 1.20 3.27 2.20 1.08 0.67 1.28 2.03 1.86 1.87
2.22 2.61 1.58 2.76 1.09 3.24 2.06 0.86 0.40 1.34 1.80 1.94 1.84
2.06 2.28 1.32 3.21 1.21 3.01 1.98 1.06 0.79 1.73 2.11 1.65 1.85
2 3 2 3 1 3 2 1 1 2 2 2 2
24 h
48 h
72 h
Nearest Integer
4.96 0.91 2.28 2.98 1.94 3.01 1.32 3.00 2.32 1.18 0.91 1.32 2.25 0.94 2.16
5.34 0.91 1.93 2.49 1.53 2.67 1.23 2.66 2.08 1.00 0.83 1.69 2.13 n.87 1.93
5.00 0.90 2.09 2.59 1.51 2.78 I.II 2.75 2.06 1.04 0.73 1.82 2.15 0.94 1.96
5 1 2 3 2 3 1 3 2 1 1 2 2 1 2
* D a n s y l a t i o n r e v e a l e d t h a t t h e N - t e r m i n a l r e s i d u e o f both p e p t i d e s is exclusively Tyr. Preliminary results ( S e i d a h , N.G. e t a l . , unpublished results) showed t h a t t h e c o m p l e t e sequence o f both p e p t i d e s i s i d e n t i c a l to t h e p o r t i o n 61-91 o f beta-LPH s t r u c t u r e (8, 14).
on Sephadex G-75 i s most o f
shown in
the morphine-like
corresponds N-terminal
to
that
residue
of is
Fig.
activity.
residues
by 1 M NH4Ac, was f u r t h e r 2.
Repurification
a pure m a t e r i a l 61-91
fragment
residue
(Table
with of I).
extract
the
amino-acid
amino-acid
of
(Table
eluted
biologically
active
composition
sheep beta-LPH and w i t h
475
from
on C M - c e l l u l o s e
The p o l y a c r y l a m i d e
to c o n t a i n
composition
human beta-LPH
tyrosine
purified of
Peak D was found Its
61-91
exclusively
The sheep p i t u i t a r y
Fig.
I.
gel
(14)
I). CM-Sephadex 25 as shown i n fraction
corresponding
tyrosine
and t h e
yielded to
the
as N - t e r m i n a l
electrophoresis
at
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
% OF
INHIBITION
.
PH (HUMAN) B-LPH ( O V I N E ) / / ~ 6 1 - 9 1
60
6,-9, / T / , ~
/ ~
/
'~0 30
I0 -8
i
i
tO-7
IO-6
M
F i g u r e 3: Morphine-like activity assayed on e l e c t r i c a l l y s t i m u l a t e d mouse vas d e f e r e n s . The a c t i v i t y is e x p r e s s e d as a % o f i n h i b i t i o n o f vas d e f e r e n s c o n t r a c t i o n s . A l l the p o i n t s are t h e means o f 4 measurements ± SE. Beta-LPH ( o v i n e ) 61-91 and beta-LPH (human) 61-91 mean t h a t such p e p t i d e s have t h e same a m i n o - a c i d c o m p o s i t i o n and t h e same N - t e r m i n a l residLae as t h e i d e n t i f i e d portion of both LPH's.
pH 4 . 5
showed an Rf 0 . 6 4
for
human p e p t i d e
and Rf 0 . 7 5 f o r
sheep p e p t i d e . The b i o l o g i c a l morphine-like to
that
of
activity
activities
their
of
is
both
respective
shown in
new p e p t i d e s
beta-LPH
(I-91)
Fig.
3 where t h e
are compared
peptides.
DISCUSSION Beta-LPH the
structures
to
be a u n i q u e m o l e c u l e
two b i o l o g i c a l l y
active
and m e t - e n k e p h a l i n .
Although,
is
that
prohormone
there
beta-LPH
of
now a p p e a r s
is
the
are many i n d i c a t i o n s
proposed supporting
by C h r ~ t i e n this
could
in
no d i r e c t
or p r e c u r s o r
which
and Li
theory
there
peptides:
favor
1967
beta-MSH evidence
both
t h e prohormone
(3).
as f o l l o w s :
showing
compounds, theory
The,experimental
be summarized
476
of
comprising
findings
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Beta-LPH i s a r e l a t i v e l y lated
beta-LPH f r a g m e n t s
due to the i s o l a t i o n Gilardeau
s t a b l e m o l e c u l e and the i s o -
are not l i k e l y
procedure,
to be d e g r a d a t i o n p r o d u c t s
as shown by C h r ~ t i e n and
(15). Chr#tien et al.
(16),
using in vitro
pulse-labelling
t e c h n i q u e s have a l r e a d y d e m o n s t r a t e d the t r a n s f o r m a t i o n beta-LPH i n t o (61-91)
gamma-LPH.
Beta-LPH,
o f beta-LPH are c o n s i s t e n t l y
of d i f f e r e n t recently
species
isolated
(2-5)
new p e p t i d e s , proinsulin
isolated Also,
from p i t u i t a r i e s
Guillemin
et a l .
(20)
Beta-LPH has much lower MSH a c t i v i t y
and much l o w e r m o r p h i n e - l i k e
activity
which would be an analogous s i t u a t i o n
- insulin
model
The d i f f e r e n t peptides
gamma-LPH and the p e p t i d e
a f r a g m e n t 61-76 from p o r c i n e n e u r o h y p o p h y s i s
and h y p o t h a l a m i c t i s s u e . than beta-MSH (8)
(17-19).
of
be e x p l a i n e d by the f a c t
to the
(21).
Rf v a l u e s found f o r
on p o l y a c r y l a m i d e
than the
the human and sheep
gel e l e c t r o p h o r e s i s
that
tyrosine
at pH 4.5 c o u l d
replaces
histidine
in
the human p e p t i d e . The b i o l o g i c a l rable.
activity
This is not surprising
of both p e p t i d e s
s i n c e t h e y both c o n t a i n
m e t - e n k e p h a l i n p e n t a p e p t i d e w h i c h , we b e l i e v e active
core
i s compa-
to be the
(6-7).
ACKNOWLEDGMENTS. Work s u p p o r t e d by the Medical Research Council o f Canada and "Le M i n i s t ~ r e de l ' E d u c a t i o n du Quebec" Authors thank Mrs F. Rousseau, Mrs O. Th~berge and Hr R. R o u t h i e r f o r t e c h n i c a l a s s i s t a n c e and Mrs D. r4arcil f o r s e c r e t a r i a l help.
REFERENCES 1.
Hughes, J . , S m i t h , Morgan, B.A. 577-579.
T.W., K o s t e r l i t z , and M o r r i s , H.R.
477
H.W., (1975)
Fothergill, L.A., Nature 258,
Vol. 72, No. 2, 1976
2. 3.
4. 5.
6.
7. . 9,
I0. II. 12. 13. 14. 15. 16 17 18 19 2O 21
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C h r # t i e n , M. (1973) In: Methods in I n v e s t i g a t i v e and D i a g n o s t i c E n d o c r i n o l o g y , Ed. by S.A. Berson and R.S. Yalow, pp. 617-632. C h r ~ t i e n , !I. and L i , C.H. (1967) Can. J. Biochem. 45, 1163-1174. L i , C.H. and Chung, D. (1976) Proc. Nat. Acad. S c i . 73, 1145-1148. Bradbury, A . F . , Smyth, D.G. and S n e l l , C.R. (1975) I n : Peptides C h e m i s t r y , S t r u c t u r e and B i o l o g y . Ed. R. ~Jalter and J. M e i n h o f e r , Ann Arbour Science P u b l i s h e r , pp. 609-615. Seidah, N.G., L i s , M., G i a n o u l a k i s , C., S c h i l l e r , P. and C h r ~ t i e n , M. (1976) Lancet (In P r e s s ) . Seidah, N.G., L i s , M., G i a n o u l a k i s , C., Benjannet, S., R o u t h i e r , R., S c h i l l e r , P. and C h r ~ t i e n , M. (1976) M a n u s c r i p t sent f o r p u b l i c a t i o n . L i , C.H., B a r n a f i , L . , C h r # t i e n , M. and Chung, D. (1966) I n : Excerta Medica I n t e r n a t i o n a l Congress 112, 249-255. Henderson, G., Hughes, J. and K o s t e r l i t z , H.H. (1972) B r i t . J. Pharmacol. 4_6_6, 764-766. Spackman, D.H., S t e i n , W.H. and Moore, B. (1958) Anal. Chem. 30, 1190-1206. I,Joods,--K.R. and Wang, K.T. (1967) Biochim. Biophys. Acta 133, 369-370. D a v i s , D.J. (1965) Ann. N.Y. Acad. S c i . 121, 404. C h r ~ t i e n , M., G i l a r d e a u , C., Seidah, N.G. and L i s , M (1976) Can. J. Biochem. (In P r e s s ) . L i , C.H. and Chung, D. (1976) Nature 260, 622-624. C h r # t i e n , M. and G i l a r d e a u , C. (1970) Can. J. Biochem. 4__~8, 511-515. C h r ~ t i e n , M., L i s , M., G i l a r d e a u , C. and Benjannet, S. (1976) Can. J. Biochem. ( I n P r e s s ) . Lohmar, P. and L i , C.H. (1971) Biochim. Biophys. Acta 147, 381-383. G i l a r d e a u , C. and C h r # t i e n , M. (1970) Can. J. Biochem. 48, 1017-1021. Graf, L . , B a r a t , E., Cseh, G. and Sajgo, Mo (1971) Biochim. Biophys. Acta 229, 276-278. G u i l l e m i n , R., L i n g , N. and Burgus, R. (1976) C.R. Acad. Sc. Paris 282, 783-785. S t e i n e r , D . F . , Kemmler, W., Tager, H.S. and P e t e r s , n , J.D. (1974) Fed. Proc. 3__3_3,2105-2115.
478
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISOLATION OF PEPTIDES WITH OPIATE ACTIVITY FROM SHEEP AND HUMAN PITUITARIES: RELATIONSHIP TO BETA-LIPOTROPIN
M. C h r # t i e n ,
S. B e n j a n n e t ,
N. Dragon,
N.G. Seidah and M. L i s Clinical
Research
Institute
of Montreal.
Received July 22,1976 SUMMARY. Human and o v i n e p i t u i t a r i e s were found to c o n t a i n p e p t i d e s of i d e n t i c a l s i z e , c o m p o s i t i o n and N - t e r m i n a l r e s i d u e to the c a r b o x y - t e r m i n a l p o r t i o n 61-91 of b e t a - l i p o t r o p i n (I-91). These p e p t i d e s e x h i b i t m o r p h i n e - l i k e a c t i v i t y in the mouse vas deferens bioassay. Beta-lipotropins from both species have much lower potency than the new p e p t i d e s in the same b i o a s s a y . The f i n d i n g s f a v o r the h y p o t h e s i s t h a t b e t a - l i p o t r o p i n i s the p r e c u r s o r of endogenous m o r p h i n e - l i k e p e p t i d e s of p i t u i t a r y origin. Hughes et a l . structure like
(I)
recently
isolated
of a pentapeptide
from pig
brain
activity.
enkephalin al.
(I)
This
pentapeptide
(met-enkephalin)
to be s t r u c t u r a l y
beta-lipotropin discovered
and d e s c r i b e d
(gamma-LPH). to the f i r s t
It
was found
and Li
58 amino a c i d s
being
structure
(3),
that
that
of
of beta-MSH. beta-LPH
an i n t e r m e d i a t e .
peptide
beta-LPH.
is
It
Chr~tien
and Li
is
Moreover,
(3)
identical
both beta-LPH
41-58 sequence the
was t h e r e f o r e
the prohormone
472
by Hughes et
named g a m m a - l i p o t r o p i n
in t h e i r
The d i s c o v e r y
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
morphine-
the name m e t h i o n i n e -
gamma-LPH sequence
to c o n t a i n
the
to the 61-65 sequence of
In 1967,
another
and gamma-LPH were found complete
(2).
potent
was a l s o observed
identical
(beta-LPH)
with
was g i v e n
and i t
and d e s c r i b e d
proposed
of beta-MSH,
of m e t - e n k e p h a l i n
by C h r ~ t i e
gamma-LPH raised
the
Vol. 72, No. 2, 1976
possibility Chung
identical peptides (6,
that
(4)
to 61-91
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
beta-LPH
isolated
a peptide
peptide
while
tification
(l-91)
report
of p e p t i d e s
with
human and sheep p i t u i t a r i e s to
residues
activity.
61-91
found
an All
Seidah
fragments
the
morphine-like with
(5)
Finally,
i s much l e s s
and
corresponding
have c o n s i d e r a b l e
describes
Li
pituitaries.
the t r y p t i c
of sheep beta-LPH
The p r e s e n t
et al.
from p o r c i n e
shown t h a t
as w e l l .
pituitaries
Bradbury
morphine-like
beta-LPH
precursor
from camel
in e x t r a c t s
exhibit
and 61-91
activity
be i t s
sequence of beta-LPH.
7) have r e c e n t l y
61-81
could
these
et a l .
61-80,
morphine-like
active. isolation
activity
amino a c i d
and i d e n - from both
composition
corresponding
of beta-LPH.
MATERIAL AND METHODS Frozen human and sheep p i t u i t a r i e s were e x t r a c t e d to the method of Li (8) and the e x t r a c t chromatographed C M - c e l l u l o s e or CM-sephadex. Some of the human C M - c e l l u l o s e f r a c t i o n s were f u r t h e r purified by Sephadex G-75 and e l u t e d w i t h O.Ol M ammonium a c e t a t e pH 4 . 6 . The sheep p i t u i t a r y e x t r a c t was chromatographed on CM-Sephadex as d e s c r i b e d by Bradbury et a l . (5) p r i o r t o , t h e CM-cellulose step. The m o r p h i n e - l i k e a c t i v i t y o f i s o l a t e d f r a c t i o n s was assayed on mouse vas d e f e r e n s as d e s c r i b e d by Henderson e t a l . (9). The a c t i v i t y i s e x p r e s s e d as p e r c e n t a g e o f i n h i b i t i o n of electrically s t i m u l a t e d c o n t r a c t i o n s of mouse vas d e f e r e n s in vitro. The maximal response i s u s u a l l y in the range o f 70-80% inhibition. Amino a c i d a n a l y s i s was c a r r i e d out a c c o r d i n g to Spackman e t a l . ( l O ) using a Beckman 121C a u t o m a t i c a n a l y z e r . The N - t e r m i n a l r e s i d u e was i d e n t i f i e d by the dansyl p r o c e d u r e (ll). P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s was done a c c o r d i n g to Davis (12) at pH 4 . 5 . according on e i t h e r
RESULTS The C M - c e l l u l o s e according hormone,
to
Li
(8),
is
routinely
ACTH and beta-LPH
a new peak a d j a c e n t
to t h a t
possessed m o r p h i n e - l i k e
chromatography
(13).
of
human p i t u i t a r y
used f o r
purification
A slightly
slower
o f growth
activity.
hormone;
Gel f i l t r a t i o n
473
extract
o f growth
gradient
revealed
the c o n s t i t u e n t of
this
fraction
VoI. 72, No. 2, 1976
2,0
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
-
1.8 16
~,
1.4
~, LO
0.4
%0 i;o ,~o ~;o
2)0
~;o 2;o 2~o 3oo 32o 34o 36o 38o 4oo 42o TUBE NUMBER
Figure I: Purification o f human f r a c t i o n e l u t e d from CMcellulose ( 3 7 . 5 mg) p o s s e s s i n g m o r p h i n e - l i k e a c t i v i t y on a column ( 2 . 5 x 120 cm) of sephadex G-75 s u p e r f i n e , using 0.01 M NH4Ac pH 4.6 as e l u a n t . Peak D gave the p e p t i d e 61-91 of human beta-LPH (see t a b l e I ) . 2.0 LB ~VH4,~I NH, Ac oJ~ I 0.4M 1,6 pH4.6,I, pH ~.;'
NH4Ac
1,4
1.2 LO
~
O.B
~, o,6 0.4 0.2
.A 0
i
i
I
i
40
60
80
I00
120
140
160
IBO
200
220
240
i
I
260
280
I
300
TUBE NUMBER
F i g u r e 2: Purification of sheep p i t u i t a r y extract eluted from CH-sephadex w i t h 1 M NH4Ac on a column (I x 25 cm) o f C H - c e l l u l o s e , u s i n g a g r a d i e n t of NH4Ac from 0 . I to 1 M. Peak I I I , upon f u r t h e r p u r i f i c a t i o n on sephadex G-25, gave the p e p t i d e 61-91 of sheep beta-LPN (see t a b l e I ) .
474
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I AMINO ACID ANALYSIS OF HUMAN AND SHEEP 61-91 Hydrolysis
Time
Human* Amino Acid Lys His Asp Thr Ser Glu Pro GIy Ala Val Met lle Leu Tyr Phe
PEPTIDES
Sheep*
24 h
48 h
72 h
Nearest Integer
4.53
5.28
4.80
5
2.08 2.71 1.84 2.99 1.20 3.27 2.20 1.08 0.67 1.28 2.03 1.86 1.87
2.22 2.61 1.58 2.76 1.09 3.24 2.06 0.86 0.40 1.34 1.80 1.94 1.84
2.06 2.28 1.32 3.21 1.21 3.01 1.98 1.06 0.79 1.73 2.11 1.65 1.85
2 3 2 3 1 3 2 1 1 2 2 2 2
24 h
48 h
72 h
Nearest Integer
4.96 0.91 2.28 2.98 1.94 3.01 1.32 3.00 2.32 1.18 0.91 1.32 2.25 0.94 2.16
5.34 0.91 1.93 2.49 1.53 2.67 1.23 2.66 2.08 1.00 0.83 1.69 2.13 n.87 1.93
5.00 0.90 2.09 2.59 1.51 2.78 I.II 2.75 2.06 1.04 0.73 1.82 2.15 0.94 1.96
5 1 2 3 2 3 1 3 2 1 1 2 2 1 2
* D a n s y l a t i o n r e v e a l e d t h a t t h e N - t e r m i n a l r e s i d u e o f both p e p t i d e s is exclusively Tyr. Preliminary results ( S e i d a h , N.G. e t a l . , unpublished results) showed t h a t t h e c o m p l e t e sequence o f both p e p t i d e s i s i d e n t i c a l to t h e p o r t i o n 61-91 o f beta-LPH s t r u c t u r e (8, 14).
on Sephadex G-75 i s most o f
shown in
the morphine-like
corresponds N-terminal
to
that
residue
of is
Fig.
activity.
residues
by 1 M NH4Ac, was f u r t h e r 2.
Repurification
a pure m a t e r i a l 61-91
fragment
residue
(Table
with of I).
extract
the
amino-acid
amino-acid
of
(Table
eluted
biologically
active
composition
sheep beta-LPH and w i t h
475
from
on C M - c e l l u l o s e
The p o l y a c r y l a m i d e
to c o n t a i n
composition
human beta-LPH
tyrosine
purified of
Peak D was found Its
61-91
exclusively
The sheep p i t u i t a r y
Fig.
I.
gel
(14)
I). CM-Sephadex 25 as shown i n fraction
corresponding
tyrosine
and t h e
yielded to
the
as N - t e r m i n a l
electrophoresis
at
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
% OF
INHIBITION
.
PH (HUMAN) B-LPH ( O V I N E ) / / ~ 6 1 - 9 1
60
6,-9, / T / , ~
/ ~
/
'~0 30
I0 -8
i
i
tO-7
IO-6
M
F i g u r e 3: Morphine-like activity assayed on e l e c t r i c a l l y s t i m u l a t e d mouse vas d e f e r e n s . The a c t i v i t y is e x p r e s s e d as a % o f i n h i b i t i o n o f vas d e f e r e n s c o n t r a c t i o n s . A l l the p o i n t s are t h e means o f 4 measurements ± SE. Beta-LPH ( o v i n e ) 61-91 and beta-LPH (human) 61-91 mean t h a t such p e p t i d e s have t h e same a m i n o - a c i d c o m p o s i t i o n and t h e same N - t e r m i n a l residLae as t h e i d e n t i f i e d portion of both LPH's.
pH 4 . 5
showed an Rf 0 . 6 4
for
human p e p t i d e
and Rf 0 . 7 5 f o r
sheep p e p t i d e . The b i o l o g i c a l morphine-like to
that
of
activity
activities
their
of
is
both
respective
shown in
new p e p t i d e s
beta-LPH
(I-91)
Fig.
3 where t h e
are compared
peptides.
DISCUSSION Beta-LPH the
structures
to
be a u n i q u e m o l e c u l e
two b i o l o g i c a l l y
active
and m e t - e n k e p h a l i n .
Although,
is
that
prohormone
there
beta-LPH
of
now a p p e a r s
is
the
are many i n d i c a t i o n s
proposed supporting
by C h r ~ t i e n this
could
in
no d i r e c t
or p r e c u r s o r
which
and Li
theory
there
peptides:
favor
1967
beta-MSH evidence
both
t h e prohormone
(3).
as f o l l o w s :
showing
compounds, theory
The,experimental
be summarized
476
of
comprising
findings
Vol. 72, No. 2, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Beta-LPH i s a r e l a t i v e l y lated
beta-LPH f r a g m e n t s
due to the i s o l a t i o n Gilardeau
s t a b l e m o l e c u l e and the i s o -
are not l i k e l y
procedure,
to be d e g r a d a t i o n p r o d u c t s
as shown by C h r ~ t i e n and
(15). Chr#tien et al.
(16),
using in vitro
pulse-labelling
t e c h n i q u e s have a l r e a d y d e m o n s t r a t e d the t r a n s f o r m a t i o n beta-LPH i n t o (61-91)
gamma-LPH.
Beta-LPH,
o f beta-LPH are c o n s i s t e n t l y
of d i f f e r e n t recently
species
isolated
(2-5)
new p e p t i d e s , proinsulin
isolated Also,
from p i t u i t a r i e s
Guillemin
et a l .
(20)
Beta-LPH has much lower MSH a c t i v i t y
and much l o w e r m o r p h i n e - l i k e
activity
which would be an analogous s i t u a t i o n
- insulin
model
The d i f f e r e n t peptides
gamma-LPH and the p e p t i d e
a f r a g m e n t 61-76 from p o r c i n e n e u r o h y p o p h y s i s
and h y p o t h a l a m i c t i s s u e . than beta-MSH (8)
(17-19).
of
be e x p l a i n e d by the f a c t
to the
(21).
Rf v a l u e s found f o r
on p o l y a c r y l a m i d e
than the
the human and sheep
gel e l e c t r o p h o r e s i s
that
tyrosine
at pH 4.5 c o u l d
replaces
histidine
in
the human p e p t i d e . The b i o l o g i c a l rable.
activity
This is not surprising
of both p e p t i d e s
s i n c e t h e y both c o n t a i n
m e t - e n k e p h a l i n p e n t a p e p t i d e w h i c h , we b e l i e v e active
core
i s compa-
to be the
(6-7).
ACKNOWLEDGMENTS. Work s u p p o r t e d by the Medical Research Council o f Canada and "Le M i n i s t ~ r e de l ' E d u c a t i o n du Quebec" Authors thank Mrs F. Rousseau, Mrs O. Th~berge and Hr R. R o u t h i e r f o r t e c h n i c a l a s s i s t a n c e and Mrs D. r4arcil f o r s e c r e t a r i a l help.
REFERENCES 1.
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T.W., K o s t e r l i t z , and M o r r i s , H.R.
477
H.W., (1975)
Fothergill, L.A., Nature 258,
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2. 3.
4. 5.
6.
7. . 9,
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C h r # t i e n , M. (1973) In: Methods in I n v e s t i g a t i v e and D i a g n o s t i c E n d o c r i n o l o g y , Ed. by S.A. Berson and R.S. Yalow, pp. 617-632. C h r ~ t i e n , !I. and L i , C.H. (1967) Can. J. Biochem. 45, 1163-1174. L i , C.H. and Chung, D. (1976) Proc. Nat. Acad. S c i . 73, 1145-1148. Bradbury, A . F . , Smyth, D.G. and S n e l l , C.R. (1975) I n : Peptides C h e m i s t r y , S t r u c t u r e and B i o l o g y . Ed. R. ~Jalter and J. M e i n h o f e r , Ann Arbour Science P u b l i s h e r , pp. 609-615. Seidah, N.G., L i s , M., G i a n o u l a k i s , C., S c h i l l e r , P. and C h r ~ t i e n , M. (1976) Lancet (In P r e s s ) . Seidah, N.G., L i s , M., G i a n o u l a k i s , C., Benjannet, S., R o u t h i e r , R., S c h i l l e r , P. and C h r ~ t i e n , M. (1976) M a n u s c r i p t sent f o r p u b l i c a t i o n . L i , C.H., B a r n a f i , L . , C h r # t i e n , M. and Chung, D. (1966) I n : Excerta Medica I n t e r n a t i o n a l Congress 112, 249-255. Henderson, G., Hughes, J. and K o s t e r l i t z , H.H. (1972) B r i t . J. Pharmacol. 4_6_6, 764-766. Spackman, D.H., S t e i n , W.H. and Moore, B. (1958) Anal. Chem. 30, 1190-1206. I,Joods,--K.R. and Wang, K.T. (1967) Biochim. Biophys. Acta 133, 369-370. D a v i s , D.J. (1965) Ann. N.Y. Acad. S c i . 121, 404. C h r ~ t i e n , M., G i l a r d e a u , C., Seidah, N.G. and L i s , M (1976) Can. J. Biochem. (In P r e s s ) . L i , C.H. and Chung, D. (1976) Nature 260, 622-624. C h r # t i e n , M. and G i l a r d e a u , C. (1970) Can. J. Biochem. 4__~8, 511-515. C h r ~ t i e n , M., L i s , M., G i l a r d e a u , C. and Benjannet, S. (1976) Can. J. Biochem. ( I n P r e s s ) . Lohmar, P. and L i , C.H. (1971) Biochim. Biophys. Acta 147, 381-383. G i l a r d e a u , C. and C h r # t i e n , M. (1970) Can. J. Biochem. 48, 1017-1021. Graf, L . , B a r a t , E., Cseh, G. and Sajgo, Mo (1971) Biochim. Biophys. Acta 229, 276-278. G u i l l e m i n , R., L i n g , N. and Burgus, R. (1976) C.R. Acad. Sc. Paris 282, 783-785. S t e i n e r , D . F . , Kemmler, W., Tager, H.S. and P e t e r s , n , J.D. (1974) Fed. Proc. 3__3_3,2105-2115.
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