PURIFICATION OF GONADOTROPIN FROM RAINBOW
TROUT (SALMO GAIRDNERII RICHARDSON)
PITUITARY GLANDS
B. BRETON
B. JALABERT
P. REINAUD
Labovatoive de Physiologie des Poissons,
Centre national de Recherches zootechniques, 1. N. R. A.,
78350 Jouy
en
Josas
SUMMARY
A method of purifying trout gonadotropic hormone is discussed. The technique consists of
o and
5
following stages : alcoholic extraction, two successive gel filtrations on Sephadex GG-ioo, chromatography on DEAE-cellulose and ultrogel ACA
¢. The biological activity of
S
fractions obtained is measured using as assay the maturation in vitro of trout oocytes.
The preparation obtained has a molecular weight of about 27500
, determined by behavior on
Sephadex-G 100 and analytical ultracentrifugation for a KS
. Polyacrylamide gel electro5
$ow of z.¢
8. Analysis of the amino acid
5
.
phoresis shows two strips of electrophoretic mobility,
50 and 0
.
0
0 AA residues per mole.
6
composition of the preparation indicates a number of 1
These different values are compared to the corresponding ones for c-GtH, s-GtH and
mammalian gonadotropins.
the
INTRODUCTION
The
purification of fish gonadotropin has encountered a major problem up
namely, a lack of sufficiently sensitive and specific biological assays for a
given species (F
ONTAINE
et al., zg!2 ; BR!TON et al., ig
). However, a Carp pituitary
73
has
been
for
the
first
time in a highly purified state
gonadotropin (c-GtH)
prepared
U
B
A
W
A
RARD
-GE
RZ
the
by
frog spermiation assay, criteria of little spe1971 using
(
)
ONDCHAROV )
cificity. The same assay was also used by G
1972 to prepare sturgeon
(
a
variation
Oet
D
DSON
NA al. )
ONAL
D
GtH, while I,
1972 employed
(
AMAZAKI and (Y
, 19
DSON
4 a et b)
6
68) of the goldfish gonad hydratation assay (Cr,!M!rrs et al., 19
to obtain gonadotropic hormone from salmon pituitary. In all cases, gametogenesis
promoting activity in fish was only controlled a posteriori (BW!,ARD et al., 1970
;
DorrAr,nsoN et al., ig72 ; SUNDAxAxAJ et al., ig!z).
In the present work, the purification of a rainbow Trout gonadotropin was
undertaken using as bioassay the in vitro intrafollicular maturation of Trout oocytes
to now,
(to avoid species specificity), which can be considered as representative of a typical
gonadotropic activity (JAr,AS!R’r et al., 1974
) since, as far as now, only one gonadotropin seems to promote both follicular growth (vitellogenesis) maturation and ovulation in fishes (S
WA and FONTAINE, 1072 ;
A
RZ
BU
D
J et al., I(!72 ; -GERAR
A
UNDARAR
URZ 1973).
B
AWA-GERARD,
MATERIAL AND METHODS
Pituitaries
were taken from adult (
3 years minimum) rainbow trout during spawning.
The following preparation techniques were used :
alcohol extraction according to S
CHMIDT et al. (i
) ;J
5
6
9
gel filtration on Sephadex Go and G-ioo ;
5
ion-exchange chromatography on DEAE-cellulose Whatman DE 23 with ionic force
-
-
-
’
gradient ;
-
I,KB
chromatography
on
acrylamide gel ACA
q (1.
S
agarose
B. F.
ultrogel
commercialized
by
France) ;
concentration of protein solutions between each purification stage was done on Amicon
UM 05 membrane in a Diaflo chamber under nitrogen pressure, followed by equilibrium dialysis
against the proper buffers for subsequent chromatography ;
protein concentration of chromatographic effluents was determined by Folin reaction
OWRY et al. (ig
using the technique of L
i).
5
The preparations obtained were analyzed using the following physico-chemical techniques :
determination of electrophoretic mobilities by polyacrylamide gel electrophoresis in
8 p. 100 gel, using ’
RNSTEIN technique (ig6q), and Isco gel scanner reading ;
O
S
study of comportment on Sephadex G ioo (exclusion volume-molecular weight), accorAURANT and ’
ILLANDER technique 6
K
S
ding to L
19
(
)
4
;
analysis of ultraviolet absorption spectra on a Carry spectrophotometer ;
measurement of sedimentation coefficient KS
ZOw and molecular weight (equilibirium
-
-
-
-
-
-
sedimentation) by analytical ultracentrifugation (Beckman Spinco analytic ultracentrifuge,
Model E, equipped with an ultraviolet scanning device) ;
o°C.
analysis of amino acid composition after 24
-hours acid hydrolysis with HCl 6N at II
Free amino acid assay was done using the technique of M
OORE et al. (i
B
20
8) with a Beckman T
95
ENEZO technique )
M
, and ’s
analyzer for preparation I
1973 with an Optica a aminolyzer for pre(
.
paration 2
-
RESULTS
.
I
-
Alcohol extraction
Immediately after collection, pituitaries are frozen into liquid nitrogen, lyophilized and reduced to acetonic powder, according to the technique described for
mammalian glycoprotein hormones by T
OUR 0).
C
E
197
(
The acetonic powder is then extracted for 24 hours at 40
C with a solution
oo NaCI, (
40 p. 100 ethanol and 2 p. I
5 g of acetonie powder).
.
100 ml of solution for 2
After 20 minute centrifugation at 5 500 g, the precipitate is extracted twice again in
the same volume of alcoholic solution. The supernatants are pooled and adjusted to
pH 5
2 with glacial acetic acid, and the alcohol content brought to 8
.
5 p. 100 by a
slowly addition of absolute alcohol. After precipitation for 24 hours at 4°
C, the
suspension obtained is centrifuged 20 minutes at 3
o g. The dry centrifuged residue
00
6
constitutes the pituitary protein extract, EPH.
In two successive year preparations the ponderal and activity yields are similar
(tabl I
).
.
2
-
Gel filtration
on
Sephadex
0 X 2
EPH is fractionated by Sephadex G.5 cm column with
50 gel filtration on a 6
Tris-HCI 0
for
first
the
same
buffer of o.oim
the
M
0
.
5 buffer, pH 7
.8
preparation;
is
used
for
the
second
,
0
8.
dithiothreitol,
mM
5
.
molarity at pH
containing 0
preparation.
Figure i shows the elution diagram obtained in the first purification. Biological
activity is found between fractions 37 and 45
, in the drop of the protein peak coming
out in the exclusion volume of the column. Employing the second buffer
not change the yield or biological activity.
system does
After concentration, the active fractions are re-chromatographed on Sephadex
G-ioo in the same two buffer systems on a 100 X 2
5 cm column. Activity peaks are
.
found between fractions 30 and 37 (fig. 2
for
the
first preparation (Kd : ),
29 and
.
0
)
between 29 and 39 for the second (Kd : o.2g). In this case, yields are higher than those
obtained at pH 7
.8 in absence of dithiothreitol, and there is no notable variation of
mean activity per mg of protein.
.
3
-
Chromatography
on
DEAE-cellulose DE 23
shows results obtained in different buffer systems of fixation and elution.
are obtained by using a Tris-HCI o.oim fixation system, pH 8.q.,
5 mM dithiothreitol, elution is carried out using a linear gradient of
containing o.
the buffer and NaCI o.5M (goo cc
o cc) (fig. 3
0
5
). Protein yield is similar at this
pH in absence of dithiothreitol and a shorter gradient o
M).
S
400
(
4
0 starting at o.o
However, protein activity drops as compared to that obtained at the preceding stage
.8, besides loss of biological activity (
(table a). At pH 7
7 U/ml), yields become very
.
0
due
to
fixation
of
active
material
on the ion exchanger
incomplete
poor
4 p. ioo)
(5
and to considerable dilution of activity on the whole chromatogram (fig. 4
). The
of
the
is
at
this
1
t-GtH
purification
stage.
stopped
Table
2
Best results
-
.
4
-
Chromatography
on
ultrogel
ACA54
gel
2was submitted to this chromatography in ammonium bicarbonate
Only t-GtH
buffer o.oim, pH 8.
. Activity is found in the first elution peak (Kd : 0
0
85). No
2
.
activity is found in the second peak, which includes a protein complex of about 15
to 17
).
ooo in molecular weight (fig. 5
Table 3 shows the plan used for purification of trout (t-GtH) gonadotropin :
04 mg active material presenting an activity
.
3
o
0
5 mg pituitary acetonic powder give 5
to
the
acetonic
of
as
powder.
gain 6!. j,
compared
.
5
-
Physico-chemical
characteristics
Overall results are shown in table 4
. The Kd and molecular weight, determined
G-ioo are identical for the two t-GtH preparations and comparable
to those of the c-GtH prepared by zAWA-GE
U
B
A
R
RD
R ).
1973 On the other hand,
(
sedimentation coefficients and molecular weight, determined by equilibrium ultracentrifugation, are different for the two trout preparations, but comparable for the
2 and c-GtH. The equilibrium sedimentation peak is apparently homogenous.
t-GtH
In polyacrylamide gel electrophoresis at 8 p. 100 pH 9
4 (at concentration of
.
i nig/ce), the protein obtained seems to be heterogenous with two electrophoretic
Rf strips 5
0.
o,
8
on
Sephadex
Amino acid compositions of t-GtH, and t-GtH
2 are given in tableand compared
URZAWA
B
R
A
R
D )
by -GE
1973 on c-GtH.
(
Compositions, expressed in residues/ioo amino acid residues, are comparable from
one preparation to another.
Comparison of the numbers of residues/mole for t-GtH,
and c-GtH (
shows
68)
0
6
1
large differences, mainly in valine and tyrosine, and
many similarities : arginine, glutamic acid, glycine, alanine, hemicystine, phenylalanine. Between the two preparations of t-GtH the number residues per mole increases from 125 for t-GtH, to 1
0 for t-GtH
6
.
2
The ultraviolet absorption spectra presents a maximum at 27
i, characteristic
[
6m
of tyrosinized protein.
to values obtained
DISCUSSION
With the techniques described, a gonadotropic hormone (t-GtH) has been obtained from trout pituitary which is 6
7 times more active than the crude pituitary
acetonic powder. The ponderal yield is 0
. This technique resembles those
14 p. IOO
.
used by -GE
OV
D
L
A
N
URZAWA )
B
RARD
1971 for carp gonadotropic hormone and by (
N
so
et al. )
1972 for the salmon one (s-GtH).
(
The physico-chemical characteristics of the three hormones are similar as regards
molecular weight determined on Sephadex G-ioo, sedimentation coefficients and molecular weight determined by equilibrium ultracentrifugation. The constants are compa-
rable to those of o. LH and o.FSH (P
AHILL and HART, 1969). As do
, 1971
APKOFF
; C
and
salmon
hormones, trout gonadotropin gives two strips of similar mobility
carp
in polyacrylamide gel electrophoresis characteristic of a molecular polymorphism
OURTE ig
C
, obtained after chromaI
(B!s!E!,!corr and McSxnrr, 1970
;,
o). If the t-GtH
7
on
in
absence
of
DEAE-cellulose
dithiothreitol, and t-GtH! (the most
tography
have
the
same
molecular
completely purified)
weight determined on Sephadex G- Ioo,
they behave very differently with ultracentrifugation. Biological activity of the t-GtH,
decreases after chromatography on Db;AE-C in comparison to the preceding purification stage. Mammalian hormones, LH and FSH, may be dissociated into two
APKOFF and ExBr,aD, 1970
sub-units, « andp (LI and ,
TARMAN ig6q.; P
S
), having a
molecular weight of about 15 000
, possessing KS of about 1
5 without biological
.
URZAWA )
B
ERARD
activity. -G
1971 also observed that after i5 hours of ultracentrifuga(
tion in saccharose gradient at 6
o r.p.m., the sedimentation peak at TS z.6
5 00
7 of native
c-GtH gives a second fraction of TS 2
.6 without biological activity, having a molecu1
lar weight of 15 8oo. This author thus suspected spontaneous dissociation of c-GtH,
which was confirmed by her later studies (B
ARD, ig75).
R
-Gt
URZAWA
The characteristics of t-GtH
1 (KS : i.4 ; equilibrated molecular weight of
sedimentation i
oo ; drop in biological activity) leads us to suppose that this trout
.
5
gonadotropic preparation was obtained in a partially dissociated state, dissociation
being completed during ultracentrifugation. The t-GtH would present no differences
in quaternary structure (dissociation into sub-units) as compared to other glycoprotein hormones, such as TSH, LH, FSH, and c-GtH.
It also seems that dissociation could be spontaneous for both t-GtH and c-GtH.
Fish gonadotropic hormones are thus more fragile than mammalian gonadotropins.
Ending purification at the DEAE-cellulose chromatography stage (DONA!,DSON
et al., 1972
) seems insufficient to obtain pure hormone. Amino acid composition
analysis of the preparation obtained after this stage gives an overall composition of
125 residues per mole, corresponding to 50 p. 100 of the molecular weight. On the
other hand, after ultrogel ACA
54 chromatography, the number of residues per mole
increases to 1
, representing 70 p 100 of hormone weight. This values is similar
0
6
to that obtained by -GE
URZAWA )
B
RARD
68 residues/mole). We
1
1973 for c-GtH (
(
have equivalent results (B
two
c-GtH preparations
al.,
RETON
et
) comparing
1975
obtained according to -G
URZAWA technique after DEAE-cellulose (i)
B
S
’
ERARD
and G-ioo post-Db;A!-cellulose (
) chromatographies.
2
A comparison of the numbers of residues of each amino acid for t-GtH and
c-GtH reveals notable differences which, besides a different sequence, may explain
immunological and biological specificity of fish gonadotropins. These values are
also different from those obtained for mammalian LH and FSH. Analysis of carbohydrate and sialic acid contents in the protein obtained could not be done, the
amounts procured being too small.
In conclusion, trout pituitary gonadotropin was obtained with a sufficiently
good degree of homogeneity to allow the radioimmunological determination of this
hormone in the plasma of the Trout (B
RETON
et al., unpublished data). This hormone
has physico-chemical characteristics comparable to corresponding fish c-GtH and
s-GtH hormones and to mammalian LH and FSH.
Reçu pour publication
en
.y’uillet 1975.
ACKNOWLEDGEMENTS
We wish to thank Mr. P
ERNOLLET and Mr. G
ARNIER (Laboratoire de Biochimie physique,
I. N. R. A.) who performed analytical ultracentrifugations, and Mr. M
ARCHELIDON (Laboratoire
de Physiologie comparée du Museum d’Histoire Naturelle) and Mr. M
ENEZO (Laboratoire de Biologie, 1. N. S. A., Villeurbanne). who helped us in assays of amino acid composition of our preparations.
Mrs. D
AIFUKU is acknowledged for the translatation of this manuscript.
We also wish to thank I. N. R. A. and the C. N. R. S. (A. T. P. Physiologie ecologique,
contract n° 4!9909) who gave funds for this work.
RÉSUMÉ
PURIFICATION
D’UN!
HORMONE GONADOTROPE
HYPOPHYSAIRE DE LA TRUITE ARC-!N-CIEI.
!SAGMO
GAIRDNERII
RICHARDSON)
Une méthode de purification d’une hormone gonadotrope de Truite est proposée. La technique comprend les étapes suivantes : une extraction alcoolique, deux gel filtrations successives sur
o et G-ioo, une chromatographie sur DEAE-cellulose et une chromatographie sur
5
Séphadex Gultrogel ACA
. L’activité biologique des fractions obtenues a été mesurée en utilisant le test de
54
maturation des ovocytes de Truite in vitro.
La préparation obtenue présente un poids moléculaire d’environ 27500 déterminé par comportement sur Séphadex G-ioo et ultracentrifugation analytique pour un KS
45 L’élec,
2
w de .
20
trophorèse sur gel de polyacrylamide révèle la présence de deux bandes de mobilité électrophorétique o,
o et o,
5
8. L’analyse de la composition en acides aminés de la préparation indique un
5
nombre de résidus 1
0 AA par mole.
6
Ces différentes valeurs sont comparées
et les gonadotropines mammaliennes.
aux
valeurs
correspondantes
pour la c-GtH, la s-GtH
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