Vol. 90, No. 4, 1979 October
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
29, 1979
QUANTIFICATION
OF PITUITARY
1249-1256
MEMBRANE RECEPTOR SITES TO LHRH:
USE OF A SUPERACTIVE ANALOG AS TRACER B.S. Endocrine
Conne, M.L. Unit,
30, boulevard Received
July
Aubert
Department University de la Cluse,
and P.C.
Sizonenko
of Pediatrics and Genetics, of Geneva, 1211 Geneva 4, Switzerland
9, 1979
SUMMARY DesGlyl"-DTrph-LHRH Ethylamide, which possesses a 144 fold increased biological activity over native LHRH and is very resistant to proteolytic enzymes, was used as radioiodinated tracer, for the detection of LHRh' receptor sites in preparations of ovine, bovine and rat pituitary plasma membranes. Up to 50% of [125I]-DesGlylODTrp6-LHRH-EA added could be bound by plasma membranes and displaced by LHRH analogs or native LHRH in relation to biological activity. Scatchard analysis showed only one class of binding sites (KA = 1010 M-l). The use of this LHRH analog as tracer offexs a very convenient and precise method for the quantification of LHRH receptors in small amounts of tissues.
INTRODUCTION The secretion
of LH and FSH by the pituitary
gland
is
stimulated
by the hypothalamic releasing hormone tion
is
receptors affinity, terized (4-5), have
decapeptide called luteinizing hormone (LHRH). The first step involved in this stimularecognition of LHRH by specific binding sites or
the at
the
surface
of
the gonadotroph
low capacity receptor sites in plasma membrane preparations or rat pituitary glands (5-9).
also
binding of preparation amount of
been reported [125 I]-LHRH is usually tracer
added.
in non pituitary obtained with low, averaging Analogs
for
cells
Low affinity tissues
LHRH (ll),
great affinity use as a tracer proposed.
can be easily
High
pituitary
sites for LHRH (10). Specific plasma membrane
only 1 to 4% of the total of LHRH bearing a D-amino-acid
position 6 possess a greatly increased biological 10 Since DesGly -DTrp' -LHRH-EA, which is 144 times native
(1).
LHRH have been characof bovine (2-3), ovine
radioiodinated
activity (1). more potent than
and exhibits
a
for pituitary plasma membrane binding sites, for the detection of LHRH receptor sites is
MATERIAL AND METHODS Hormone: Native LHRH and all analogs of LHRH used in this as well as somatostatin were synthesized by Dr. J. Rivier
1249
in
its
study at the
0006-291X/79/201249-08$01.00/0 Copyright @ 1979 by Acndemic Press. All rights of reproducrion in anyform
Inc. reserved
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Salk Institute for Biological Research, La Jolla, California Carrier free Iodine 125 was obtained from the Swiss Reactor Institute at Wiirenlingen.
(11).
Radioiodination: Native LHRH and analogs of LHRH were radioiodinated by the lactoperoxidase-glucose oxidase technique (12) using the solid phase modification proposed by Bio Rad Laboratories. Briefly, 5 ug LHRH was reacted with 50 ~1 0.5 M phosphate buffer, 1.0 mCi 1251, 25 ul of Enzymo-bead TM, an immobilized preparation of lactoperoxidase and glucose oxidase covalently bound to hydrophilic spheres, and 25 ~1 of 1% D-glucose. The reaction was stopped after 20 min. by addition of 100 r~l of a transfer buffer containing 0.1% sodium aside, 16% sucrose and 1% sodium iodide. Purification of radioiodinated peptides was performed on Sephadex G25 using 0.1 N acetic acid supplemented with 0.25% BSA as eluant, as already described (13). In this chromatographic system, radioiodinated DesGlylO-DTrp6-LHRH-EA is greatly retarded and elutes largely after the radioactive Iodine peak (Kd 2.0), whereas unlabeled LHRH analog elutes slighly after the 1251 peak, therefore the radioiodinated LHRH analog is virtually free of unlabeled hormone, similarly to what was observed in the case of native LHRH (13), and has a constant specific activity of 1660 mCi/mg, provided that carrier-free 1251 was used for radioiodination (17 C/mg). Preparation of membrane: Bovine and ovine pituitary glands were obtained from a local slaughter-house within 20 minutes of the animal's death. They were kept on ice in a 1 mM sodium bicarbonate, 2 mM dithiothreitol buffer, pH 10, during transport to the laboPituitary plasma membranes were prepared according to the ratory. method of Poirier et al. (14), as modified by Clayton et al. (2). Rat pituitary membranes were prepared according to Park et al. (7). Binding studies: A 0.01 M Tris-HCl, 1 mM MgC12, 0,25% BSA buffer, pH 7.4 was used as diluent. Pituitary membranes (usually 200 ug) were incubated with radioiodinated LHRH or LHRH analog (5-10 PM) at 4OC, 23OC, or 37OC for variable times, in the absence or presence of various concentrations of unlabeled LHRH or LHRH analog. The incubation was terminated by addition of 2.0 ml ice cold buffer to each tube followed by immediate centrifugation at 5000 RPM for 30 min. In each assay condition, non specific binding was assessed in parallel by addition of excess of LHRH or LHRH binding was the analog (1000 ng) to the medium, and specific difference between total and non specific binding. Statistical analysis of radioreceptor assay data was performed on a Cyber 170 by Dr David CDC computer using the V'SCATFIT" program established Rodbard and his colleagues (15). RESULTS DesGlylO-DTrp6 specifically
-LHRH-EA could
be easily
radioiodinated
to pituitary membrane preparation usually 30-50% of tracer origin.
and bound
of either added (3-5
bovine, fmole)
ovine or rat was bound and non specific binding averaged only about 10% of max. with a maximum observed at 7.4. binding. Binding was pH dependent, At 4OC and 23OC binding equilibrium was reached after about 4 was 1.03 x 10 8 M-1 hours incubation (fig. 1); rate of association
1250
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
o’, . , . , . , . , , , . , a::, 0 2 I 6 8 10 12 HOURS OF INCUBATION Fig.
1
24
0 2 L 6 8 10 12 HOURS OF INCUBATION
Rate of association and dissociation of radioiodinated [1251]DesGlylO-DTrp6-LHRH-Ed to ovine pituitary membranes. 230 ug membrane were incubated with 3.4 pmles tracer for different times at 4OC and 23OC. Total binding (e) and non specific binding (--O--j are indicated for each time point. After 3 and 6 hours at 4OC and 2 hours at 23OC, dissociation of bound tracer was induced by addition of excess unlabeled LHRH analog in 1 ml buffer I-::-). 29% binding of tracer represents 0.1 fmle of LHRH analog.
-1 -1 min and 4.3 x lo7 M-lmin at 4OC and 23OC, respectively. At 37OC, maximum binding was reached after 30 minutes but represented only lo-20% of the binding obtained at lower temperature shown). Non specific binding did not change significantly incubation, ing ing data,
representing
5-10% of the
added tracer.
(data not during
Maximum bind-
achieved at 4OC was consistently higher than at 23OC, indicatfaster dissociation rate at higher temperature. Based on these subsequent
assays were run at
addition of an excess of unlabeled hour association provoked a rapid, membrane bound tracer,
reaching
the
0-4OC for LHRH analog
6 hours. after
At 4OC, either
3 or 6
time-dependent decrease of level of non-specific binding
after 24 h (fig. 1). The rate of dissociation (kl-) was 4.4 x t; -2 -1 min when dissociation was started after 3 hours and 2.7 x 10 -1 min when started after 6 hours. At 23OC, dissociation was very fast and was complete after 2 hours (kl- = 4.3 x lo-'min -5 . Affinity Saturation
constant and binding capacity analysis of pituitary membrane receptor sites were 10 performed using DesGly -DTrp6 -LHRH-EA both as tracer and standard; figure 2 exhibits a typical saturation curve and its Scatchard analysis. Similar curves were obtained with bovine and rat pituitary membranes. Maximum binding was repeatedly above 40%. There was only one class of high affinity binding sites detectable on the Scatchard analysis; an asymptote binding was seen in each case. Affinity
1251
corresponding constant
to non-specific (KA) and binding
Vol. 90, No. 4, 1979
BIOCHEMICAL
oJ+,
,,
0
0.01 Amount
Fig.
2
,I,
AND BIOPHYSICAL
,I, 0.1
.I
0.512
of Pcptidc
5
added
+I1000
RESEARCH COMMUNICATIONS
25
(ng 1
50
75
Bound
100
( pM )
Saturation analysis of ovine pituitary membrane receptor sites (190 ug) with DesGlylO-DTrp6-LHRH-EA. Tracer (7.4 fM) was incubated for 6 hours at 4OC with increasing amounts of unlabeled LHRH analog. Maximum binding was 42.2% non specific binding in presence of excess 7.5%, as seen on the left panel. peptide, The Scatchard analysis is shown on the right panel. The asymptote for non specific binding represents a B/F value of 0.039. Affinity COnstant (KA) was 2.1 x 1010~-1 and binding capacity, 33 pM or 87 fmole/mg membrane. The closed circle represents the value for maximum binding Ino unlabeled peptide).
TRH, hGH,oPRL MELATONINE
.-.
A
E!! IL4
.---.
0
66
I.-
- .. c
32
.-.
D
lb
x--x
E
3.6
o--_--v
F
0.6
D-O
LHRH
90-1
z 60 45 70 0 1
5 30. w 2 20L
10OJ
Fig.
3
1
b. 0.01
.I,
VI. 0.1
-1.
-1.
I
IO
UNLABELED
PEPTIDE
II 100
1000
I ng 1
Displacement of [125I]-DesGlylG-DTrp 6-LHRH-Ed from the receptor sites of bovine pituitary membranes by different superactive analogs of LHRH as well as by native LHRR. Biological activity (BA) is indicated on the right for each substance in terms of the biological activity of LHRH. For the coding of LHRH analogs, see Table 1. Maximum binding was
45%.
capacity were 2.1 x 101' M-1 and 87 fmole/mg, respectively, for ovine pituitary membranes (fig. 2), 1 . 3 x 101'M-1 and 91 fmole/mg for bovine membranes, 1.0 x 10 10M-1 and 96 fmole/mg for female
1252
BIOCHEMICAL
Vol. 90, No. 4, 1979
Table
AND BIOPHYSICAL
BIOLOGICAL AND RECEPTOR ACTIVITY LHRH ANALOGS
1.
NAME
RESEARCH COMMUNICATIONS
OF SEVERAL
BIOLOGICAL ACTIVITY*
SUPERACTIVE
RECEPTOR ACTIVITY Half displ. Normal(ng) ized **
A
DesGly" -DTrp6-LHRH-EA
144
0.15
-144
B
DesGly 10 -DTy+LHRH-EA DesGly 10 -DAla6(N-Me)Leu'-
68
0.32
68
LHRH-EA
32
0.32
68
D
D-Tyr6-LHRH
14
0.97
22
E
D-Ly&LHRH
3.8
6.7
3.2
F
Trp'-LHRH
0.8
19
1.1
LHRH
1
109
0.2
C
* **
adult
In terms of native Analog A arbitrarily
rats
and 1.0
LHRH activity set at 144,
(11) to match
biological
activity
x 10 10 M-1 and 206 fmole/mg
for
male adult
rats,
respectively. Specificity The specificity
of
the binding
of
radioiodinated
DesGly 10 -DTrp'-
LHRH-EA to receptor sites of pituitary membranes was established by testing the binding affinity of a great variety of peptides either related or unrelated to LHRH. Fig. 3 exhibits the displacement curves obtained by these different peptides. The greatest sensitivity was obtained with the homologous peptide DesGlylODTrp6-LHRH-EA, with a half displacement of 0.15 ng. Other analogs of LHRH with a D-amino acid in position 6 showed binding affinities in relation to their known biological activities as shown on Table 1. Native
LHRH had a weak activity
in this
system
since
half
dis-
placement was achieved with about a thousand times more peptide than needed for the DesGlylO-DTrp6-LHRH-EA analog. No difference was found when Bacitracin (21) was present in the incubation buffer. Substances not related to LHRH such as thyrotropin releasing hormone (TRH), somatostatin, human growth hormone (hGH), ovine prolactin (oPRL) or melatonin did not inhibit the binding of DesGlylO-DTrp6-LHRH-EA
to pituitary
1253
membranes.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION Superactive analogs of LHRH exhibit great binding affinity for preparations of pituitary plasma membranes which are likely to contain LHRH receptors. 4-8 fmole of [1251]-DesGly10-DTrp6-LHRH-EA bound up to 60% to pituitary plasma membranes. This contrasts particularly with the low binding observed when native LHRH is used as tracer. In the unlabeled form, all superactive analogs of LHRH tested competed with [12S11-DesGly10-DTrp6-LHRH-EA for receptor sites in direct relation to their biological activity (Table 1). Complete displacement of the tracer was achieved with either 2 ng of DesGlylO-DTrp6 -LHRH-EA or 1000 ng LHRH. This contrasts markedly with the enormous amounts of peptides necessary to completely displace [12SI]-LHRH from pituitary receptor sites observed in other studies (2,6,9,10). No low affinity binding sites (KA = 105M'1) were found in pituitary membranes in contrast to the finding of Heber et al. (10). It is possible that the LHRH analog used in this study could not detect such low affinity sites. The markedly increased biological activity of DesGlylO-DTrp6-LHRHEA and other DesGlylO- LHRH-EA analogs carrying an aminoacid of D anfigura tion in position 6 results from two distinct properties of these molecules: 1) increased binding affinity for the receptor site 2) markedly reduced susceptibility to enzymatic degrada(1,111; tion (1). Introduction of any D-amino-acid in position 6 consistently increases biological activity, but the extent of this increase is. variable, ranging from 3.2 times for D-Leu6-LHRH to 36 times for D-Trp6-LHRH (11). It has been postulated that a dextrogyre amino-acid in position 6 might provoke a restriction in the conformational degree of freedom of LHRH, which results in the stabilization of conformations offering a higher affinity for the LHRH receptor (1,ll). This stabilization is more important with this modification in position 6 aromatic D-amino-acids. Conversely, might protect the LHRH molecule from enzymatic splitting between 6 and 7, a cleavage that was postulated to be one of the possible mechanisms of inactivation of the LHRH molecule (17). The Fujino modification (18) at the C-terminal end of LHRH molecules carrying a D-amino-acid in position 6 provides additional protection against the great stability of DesGlylO-DTrp6enzymatic degradation. Thus, LHRH-EA in regard to enzymatic degradation is probably the most relevant advantage for its use as tracer in binding studies.
1254
Vol. 90, No. 4, 1979
Similar in vitro D-Ala6-LHRH-EA
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
binding studies to this one, using (19) or DesGlylO-[D-Ser(TBu)6]-LHRH-EA
either DesGlylO(20) as
radioiodinated tracer have been recently reported. In both studies, great resistance of the tracer to enzymatic degradation was documented. Although the binding affinity of the different proportional to biological activity (Table l), variations: DesGlylO-D-Ala6(N-Me)Leu7-LHRH-EA more receptor binding affinity than expected activity.
for example exhibited from its biological
In contrast,
lower relative to enzymatic peptides
analogs tested was there were some
native LHRH had a binding affinity 5 times to its biological activity. Variable susceptibility degradation during in vivo and in vitro assays of these
may explain
these
differences;
the binding
affinity
of
native LHRH however was not found different in absence or presence of Bacitracin, an agent which has been described as an effective inhibitor
of
homogenates
in vitro
degradation
of LHRH by hypothalamic
or brain
(21).
Quantification glands which
of LHRH receptor represents another
concentration approach for
in rat testing
pituitary pituitary
responsiveness to LHRH is of great relevance in reproductive endoPark et al. (7) described a peak concentration of LHRH crinology. receptors
in the proestrus
described
technique,
in
female
we observed
rats.
a significant
Using the
above
increase
of LHRH
receptor sites at 40 days of life during puberty in the male rat; furthermore, LHRH receptor concentration is increased after castration, an observation which is in agreement with the striking increases of LH and FSH secretion after removal of the androgen suppressive
action
(22).
ACKNOWLEDGEMENTS We are very much indebted to Dr Jean Rivier, the Salk Institute, La Jolla, California, for his generous gifts of the various LHRH peptides which made this study possible. We are grateful to Dr David Rodbard (NIH, Bethesda, Md) for making his program "SCATFIT" available for this study. The secretarial assistance of Mrs Monique Jaccoud and Mrs Martine Pasche and the illustration work of Mr Daniel Furrer are greatly appreciated. Grants from the Swiss National Science Foundation (Nos 3.496.0-75 and 3.926.0-78). REFERENCES 1.
Vale, W., Rivier, C., Brown, M., Leppaluoto, J., Ling, N., Monahan, M., and Rivier, J. (1976) Clin. Endocrinol. 5 (suppl.) 2615-2735.
1255
Vol. 90, No. 4, 1979
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Clayton, R.N., Shakespear, R.A., and Marshall, J.C. (1978) Molecul. Cellul. Endocrinol. 11, 63-78. Zolman, J.C., and Valenta, L. (1978) Acta Endocrinol. 89, 232-239. Wagner, T.O.F., Adams, T.E., and Nett, T.M. (1979) Biol. Reprod. 20, 140-149. Theoleyre, M., Berault, A., Garnier, J., and Jutisz, M. (1976) Molec. Cell. Endocrinol. 5, 365-377. Marshall, J.C., Shakespear, R.A., Odell, W.D. (1976) Clin. Endocrinol. 5, 671-677. Park, K.R., Saxena, B.B., and Gandy, H.M. (1976) Acta Endocrinol. 82, 62-70. Pedroza, E., Vilchez-Martinez, J.A., Fishback, J., Arimura, A and Schally, A.V. (1977) Biochem. Biophys. Res. Comm. 79; 234-238. Grant, G., Vale, W., and Rivier, J. (1973) Biochem. Biophys. Res. Comm. 50, 771-778. Heber, D., Marshall, J.C., and Odell, W.D. (1978) Am. J. Physiol. 235, E227-E-230. Rivier, J., Brown, M., Rivier, C., Ling, N., and Vale, W. (1976) "Peptides 1976", A. Loffet (ed.), pp. 427-445, Editions de l'universite de Bruxelles, Belgium. Marshall, J.C., and Odell, W.D. (1975) Proc. Sot. Exptl. Biol. Med. 149, 351-355. Aubert, M.L., Grumbach, M.M., and Kaplan, S.L. (1977) J. Clin. Endocrinol. Metab. 44, 1130-1141. Poirier, G., De Lean, A., Pelletier, G., Lemay, A., and Labrie, F. (1974) J. Biol. Chem. 249, 316-322. De Lean, A., Munson, P.J., and Rodbard, D. (1978) Am. J. Physiol. 235, E97-E102. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Koch, Y., Baram, T., Chobsieng, P., and Fridkin, M. (1974) Biochem. Biophys. Res. Commun. 61, 95-103. Fujino, M., Kobayashi, S., Obayashi, M., Shinagawa, S., Fukuda, T ., Kitada, C., Nakayama, R., Yamazaki, 11, White, W.F., and Res. Commun. 49, 698-705. Rippel, R.H. (1972) Biochem. Biophys. Frager, M., Duncan, J., Pieper, D., Tonetta, S., and Marshall, J.C. (1979) The Endocrine Society 61st Annual Meeting, Anaheim, Abstract 432. Society 61st Annual Meeting, Clayton, R.N. (1979) The Endocrine Anaheim, Abstract 438. McKelvy, D.F., Leblanc, P., Laudes, C., Perrie, S., GrimmJorgensen, Y., and Kordon, C. (1976) Biochem. Biophys. Res. comm. 73, 507-515. Aubert, M.L., Conne, B.S., and Sizonenko, P.C. (1979) The Endocrine Society 61st Annual Meeting, Anaheim, Abstract 501.
1256
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
29, 1979
QUANTIFICATION
OF PITUITARY
1249-1256
MEMBRANE RECEPTOR SITES TO LHRH:
USE OF A SUPERACTIVE ANALOG AS TRACER B.S. Endocrine
Conne, M.L. Unit,
30, boulevard Received
July
Aubert
Department University de la Cluse,
and P.C.
Sizonenko
of Pediatrics and Genetics, of Geneva, 1211 Geneva 4, Switzerland
9, 1979
SUMMARY DesGlyl"-DTrph-LHRH Ethylamide, which possesses a 144 fold increased biological activity over native LHRH and is very resistant to proteolytic enzymes, was used as radioiodinated tracer, for the detection of LHRh' receptor sites in preparations of ovine, bovine and rat pituitary plasma membranes. Up to 50% of [125I]-DesGlylODTrp6-LHRH-EA added could be bound by plasma membranes and displaced by LHRH analogs or native LHRH in relation to biological activity. Scatchard analysis showed only one class of binding sites (KA = 1010 M-l). The use of this LHRH analog as tracer offexs a very convenient and precise method for the quantification of LHRH receptors in small amounts of tissues.
INTRODUCTION The secretion
of LH and FSH by the pituitary
gland
is
stimulated
by the hypothalamic releasing hormone tion
is
receptors affinity, terized (4-5), have
decapeptide called luteinizing hormone (LHRH). The first step involved in this stimularecognition of LHRH by specific binding sites or
the at
the
surface
of
the gonadotroph
low capacity receptor sites in plasma membrane preparations or rat pituitary glands (5-9).
also
binding of preparation amount of
been reported [125 I]-LHRH is usually tracer
added.
in non pituitary obtained with low, averaging Analogs
for
cells
Low affinity tissues
LHRH (ll),
great affinity use as a tracer proposed.
can be easily
High
pituitary
sites for LHRH (10). Specific plasma membrane
only 1 to 4% of the total of LHRH bearing a D-amino-acid
position 6 possess a greatly increased biological 10 Since DesGly -DTrp' -LHRH-EA, which is 144 times native
(1).
LHRH have been characof bovine (2-3), ovine
radioiodinated
activity (1). more potent than
and exhibits
a
for pituitary plasma membrane binding sites, for the detection of LHRH receptor sites is
MATERIAL AND METHODS Hormone: Native LHRH and all analogs of LHRH used in this as well as somatostatin were synthesized by Dr. J. Rivier
1249
in
its
study at the
0006-291X/79/201249-08$01.00/0 Copyright @ 1979 by Acndemic Press. All rights of reproducrion in anyform
Inc. reserved
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Salk Institute for Biological Research, La Jolla, California Carrier free Iodine 125 was obtained from the Swiss Reactor Institute at Wiirenlingen.
(11).
Radioiodination: Native LHRH and analogs of LHRH were radioiodinated by the lactoperoxidase-glucose oxidase technique (12) using the solid phase modification proposed by Bio Rad Laboratories. Briefly, 5 ug LHRH was reacted with 50 ~1 0.5 M phosphate buffer, 1.0 mCi 1251, 25 ul of Enzymo-bead TM, an immobilized preparation of lactoperoxidase and glucose oxidase covalently bound to hydrophilic spheres, and 25 ~1 of 1% D-glucose. The reaction was stopped after 20 min. by addition of 100 r~l of a transfer buffer containing 0.1% sodium aside, 16% sucrose and 1% sodium iodide. Purification of radioiodinated peptides was performed on Sephadex G25 using 0.1 N acetic acid supplemented with 0.25% BSA as eluant, as already described (13). In this chromatographic system, radioiodinated DesGlylO-DTrp6-LHRH-EA is greatly retarded and elutes largely after the radioactive Iodine peak (Kd 2.0), whereas unlabeled LHRH analog elutes slighly after the 1251 peak, therefore the radioiodinated LHRH analog is virtually free of unlabeled hormone, similarly to what was observed in the case of native LHRH (13), and has a constant specific activity of 1660 mCi/mg, provided that carrier-free 1251 was used for radioiodination (17 C/mg). Preparation of membrane: Bovine and ovine pituitary glands were obtained from a local slaughter-house within 20 minutes of the animal's death. They were kept on ice in a 1 mM sodium bicarbonate, 2 mM dithiothreitol buffer, pH 10, during transport to the laboPituitary plasma membranes were prepared according to the ratory. method of Poirier et al. (14), as modified by Clayton et al. (2). Rat pituitary membranes were prepared according to Park et al. (7). Binding studies: A 0.01 M Tris-HCl, 1 mM MgC12, 0,25% BSA buffer, pH 7.4 was used as diluent. Pituitary membranes (usually 200 ug) were incubated with radioiodinated LHRH or LHRH analog (5-10 PM) at 4OC, 23OC, or 37OC for variable times, in the absence or presence of various concentrations of unlabeled LHRH or LHRH analog. The incubation was terminated by addition of 2.0 ml ice cold buffer to each tube followed by immediate centrifugation at 5000 RPM for 30 min. In each assay condition, non specific binding was assessed in parallel by addition of excess of LHRH or LHRH binding was the analog (1000 ng) to the medium, and specific difference between total and non specific binding. Statistical analysis of radioreceptor assay data was performed on a Cyber 170 by Dr David CDC computer using the V'SCATFIT" program established Rodbard and his colleagues (15). RESULTS DesGlylO-DTrp6 specifically
-LHRH-EA could
be easily
radioiodinated
to pituitary membrane preparation usually 30-50% of tracer origin.
and bound
of either added (3-5
bovine, fmole)
ovine or rat was bound and non specific binding averaged only about 10% of max. with a maximum observed at 7.4. binding. Binding was pH dependent, At 4OC and 23OC binding equilibrium was reached after about 4 was 1.03 x 10 8 M-1 hours incubation (fig. 1); rate of association
1250
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
o’, . , . , . , . , , , . , a::, 0 2 I 6 8 10 12 HOURS OF INCUBATION Fig.
1
24
0 2 L 6 8 10 12 HOURS OF INCUBATION
Rate of association and dissociation of radioiodinated [1251]DesGlylO-DTrp6-LHRH-Ed to ovine pituitary membranes. 230 ug membrane were incubated with 3.4 pmles tracer for different times at 4OC and 23OC. Total binding (e) and non specific binding (--O--j are indicated for each time point. After 3 and 6 hours at 4OC and 2 hours at 23OC, dissociation of bound tracer was induced by addition of excess unlabeled LHRH analog in 1 ml buffer I-::-). 29% binding of tracer represents 0.1 fmle of LHRH analog.
-1 -1 min and 4.3 x lo7 M-lmin at 4OC and 23OC, respectively. At 37OC, maximum binding was reached after 30 minutes but represented only lo-20% of the binding obtained at lower temperature shown). Non specific binding did not change significantly incubation, ing ing data,
representing
5-10% of the
added tracer.
(data not during
Maximum bind-
achieved at 4OC was consistently higher than at 23OC, indicatfaster dissociation rate at higher temperature. Based on these subsequent
assays were run at
addition of an excess of unlabeled hour association provoked a rapid, membrane bound tracer,
reaching
the
0-4OC for LHRH analog
6 hours. after
At 4OC, either
3 or 6
time-dependent decrease of level of non-specific binding
after 24 h (fig. 1). The rate of dissociation (kl-) was 4.4 x t; -2 -1 min when dissociation was started after 3 hours and 2.7 x 10 -1 min when started after 6 hours. At 23OC, dissociation was very fast and was complete after 2 hours (kl- = 4.3 x lo-'min -5 . Affinity Saturation
constant and binding capacity analysis of pituitary membrane receptor sites were 10 performed using DesGly -DTrp6 -LHRH-EA both as tracer and standard; figure 2 exhibits a typical saturation curve and its Scatchard analysis. Similar curves were obtained with bovine and rat pituitary membranes. Maximum binding was repeatedly above 40%. There was only one class of high affinity binding sites detectable on the Scatchard analysis; an asymptote binding was seen in each case. Affinity
1251
corresponding constant
to non-specific (KA) and binding
Vol. 90, No. 4, 1979
BIOCHEMICAL
oJ+,
,,
0
0.01 Amount
Fig.
2
,I,
AND BIOPHYSICAL
,I, 0.1
.I
0.512
of Pcptidc
5
added
+I1000
RESEARCH COMMUNICATIONS
25
(ng 1
50
75
Bound
100
( pM )
Saturation analysis of ovine pituitary membrane receptor sites (190 ug) with DesGlylO-DTrp6-LHRH-EA. Tracer (7.4 fM) was incubated for 6 hours at 4OC with increasing amounts of unlabeled LHRH analog. Maximum binding was 42.2% non specific binding in presence of excess 7.5%, as seen on the left panel. peptide, The Scatchard analysis is shown on the right panel. The asymptote for non specific binding represents a B/F value of 0.039. Affinity COnstant (KA) was 2.1 x 1010~-1 and binding capacity, 33 pM or 87 fmole/mg membrane. The closed circle represents the value for maximum binding Ino unlabeled peptide).
TRH, hGH,oPRL MELATONINE
.-.
A
E!! IL4
.---.
0
66
I.-
- .. c
32
.-.
D
lb
x--x
E
3.6
o--_--v
F
0.6
D-O
LHRH
90-1
z 60 45 70 0 1
5 30. w 2 20L
10OJ
Fig.
3
1
b. 0.01
.I,
VI. 0.1
-1.
-1.
I
IO
UNLABELED
PEPTIDE
II 100
1000
I ng 1
Displacement of [125I]-DesGlylG-DTrp 6-LHRH-Ed from the receptor sites of bovine pituitary membranes by different superactive analogs of LHRH as well as by native LHRR. Biological activity (BA) is indicated on the right for each substance in terms of the biological activity of LHRH. For the coding of LHRH analogs, see Table 1. Maximum binding was
45%.
capacity were 2.1 x 101' M-1 and 87 fmole/mg, respectively, for ovine pituitary membranes (fig. 2), 1 . 3 x 101'M-1 and 91 fmole/mg for bovine membranes, 1.0 x 10 10M-1 and 96 fmole/mg for female
1252
BIOCHEMICAL
Vol. 90, No. 4, 1979
Table
AND BIOPHYSICAL
BIOLOGICAL AND RECEPTOR ACTIVITY LHRH ANALOGS
1.
NAME
RESEARCH COMMUNICATIONS
OF SEVERAL
BIOLOGICAL ACTIVITY*
SUPERACTIVE
RECEPTOR ACTIVITY Half displ. Normal(ng) ized **
A
DesGly" -DTrp6-LHRH-EA
144
0.15
-144
B
DesGly 10 -DTy+LHRH-EA DesGly 10 -DAla6(N-Me)Leu'-
68
0.32
68
LHRH-EA
32
0.32
68
D
D-Tyr6-LHRH
14
0.97
22
E
D-Ly&LHRH
3.8
6.7
3.2
F
Trp'-LHRH
0.8
19
1.1
LHRH
1
109
0.2
C
* **
adult
In terms of native Analog A arbitrarily
rats
and 1.0
LHRH activity set at 144,
(11) to match
biological
activity
x 10 10 M-1 and 206 fmole/mg
for
male adult
rats,
respectively. Specificity The specificity
of
the binding
of
radioiodinated
DesGly 10 -DTrp'-
LHRH-EA to receptor sites of pituitary membranes was established by testing the binding affinity of a great variety of peptides either related or unrelated to LHRH. Fig. 3 exhibits the displacement curves obtained by these different peptides. The greatest sensitivity was obtained with the homologous peptide DesGlylODTrp6-LHRH-EA, with a half displacement of 0.15 ng. Other analogs of LHRH with a D-amino acid in position 6 showed binding affinities in relation to their known biological activities as shown on Table 1. Native
LHRH had a weak activity
in this
system
since
half
dis-
placement was achieved with about a thousand times more peptide than needed for the DesGlylO-DTrp6-LHRH-EA analog. No difference was found when Bacitracin (21) was present in the incubation buffer. Substances not related to LHRH such as thyrotropin releasing hormone (TRH), somatostatin, human growth hormone (hGH), ovine prolactin (oPRL) or melatonin did not inhibit the binding of DesGlylO-DTrp6-LHRH-EA
to pituitary
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membranes.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION Superactive analogs of LHRH exhibit great binding affinity for preparations of pituitary plasma membranes which are likely to contain LHRH receptors. 4-8 fmole of [1251]-DesGly10-DTrp6-LHRH-EA bound up to 60% to pituitary plasma membranes. This contrasts particularly with the low binding observed when native LHRH is used as tracer. In the unlabeled form, all superactive analogs of LHRH tested competed with [12S11-DesGly10-DTrp6-LHRH-EA for receptor sites in direct relation to their biological activity (Table 1). Complete displacement of the tracer was achieved with either 2 ng of DesGlylO-DTrp6 -LHRH-EA or 1000 ng LHRH. This contrasts markedly with the enormous amounts of peptides necessary to completely displace [12SI]-LHRH from pituitary receptor sites observed in other studies (2,6,9,10). No low affinity binding sites (KA = 105M'1) were found in pituitary membranes in contrast to the finding of Heber et al. (10). It is possible that the LHRH analog used in this study could not detect such low affinity sites. The markedly increased biological activity of DesGlylO-DTrp6-LHRHEA and other DesGlylO- LHRH-EA analogs carrying an aminoacid of D anfigura tion in position 6 results from two distinct properties of these molecules: 1) increased binding affinity for the receptor site 2) markedly reduced susceptibility to enzymatic degrada(1,111; tion (1). Introduction of any D-amino-acid in position 6 consistently increases biological activity, but the extent of this increase is. variable, ranging from 3.2 times for D-Leu6-LHRH to 36 times for D-Trp6-LHRH (11). It has been postulated that a dextrogyre amino-acid in position 6 might provoke a restriction in the conformational degree of freedom of LHRH, which results in the stabilization of conformations offering a higher affinity for the LHRH receptor (1,ll). This stabilization is more important with this modification in position 6 aromatic D-amino-acids. Conversely, might protect the LHRH molecule from enzymatic splitting between 6 and 7, a cleavage that was postulated to be one of the possible mechanisms of inactivation of the LHRH molecule (17). The Fujino modification (18) at the C-terminal end of LHRH molecules carrying a D-amino-acid in position 6 provides additional protection against the great stability of DesGlylO-DTrp6enzymatic degradation. Thus, LHRH-EA in regard to enzymatic degradation is probably the most relevant advantage for its use as tracer in binding studies.
1254
Vol. 90, No. 4, 1979
Similar in vitro D-Ala6-LHRH-EA
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
binding studies to this one, using (19) or DesGlylO-[D-Ser(TBu)6]-LHRH-EA
either DesGlylO(20) as
radioiodinated tracer have been recently reported. In both studies, great resistance of the tracer to enzymatic degradation was documented. Although the binding affinity of the different proportional to biological activity (Table l), variations: DesGlylO-D-Ala6(N-Me)Leu7-LHRH-EA more receptor binding affinity than expected activity.
for example exhibited from its biological
In contrast,
lower relative to enzymatic peptides
analogs tested was there were some
native LHRH had a binding affinity 5 times to its biological activity. Variable susceptibility degradation during in vivo and in vitro assays of these
may explain
these
differences;
the binding
affinity
of
native LHRH however was not found different in absence or presence of Bacitracin, an agent which has been described as an effective inhibitor
of
homogenates
in vitro
degradation
of LHRH by hypothalamic
or brain
(21).
Quantification glands which
of LHRH receptor represents another
concentration approach for
in rat testing
pituitary pituitary
responsiveness to LHRH is of great relevance in reproductive endoPark et al. (7) described a peak concentration of LHRH crinology. receptors
in the proestrus
described
technique,
in
female
we observed
rats.
a significant
Using the
above
increase
of LHRH
receptor sites at 40 days of life during puberty in the male rat; furthermore, LHRH receptor concentration is increased after castration, an observation which is in agreement with the striking increases of LH and FSH secretion after removal of the androgen suppressive
action
(22).
ACKNOWLEDGEMENTS We are very much indebted to Dr Jean Rivier, the Salk Institute, La Jolla, California, for his generous gifts of the various LHRH peptides which made this study possible. We are grateful to Dr David Rodbard (NIH, Bethesda, Md) for making his program "SCATFIT" available for this study. The secretarial assistance of Mrs Monique Jaccoud and Mrs Martine Pasche and the illustration work of Mr Daniel Furrer are greatly appreciated. Grants from the Swiss National Science Foundation (Nos 3.496.0-75 and 3.926.0-78). REFERENCES 1.
Vale, W., Rivier, C., Brown, M., Leppaluoto, J., Ling, N., Monahan, M., and Rivier, J. (1976) Clin. Endocrinol. 5 (suppl.) 2615-2735.
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Vol. 90, No. 4, 1979
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Clayton, R.N., Shakespear, R.A., and Marshall, J.C. (1978) Molecul. Cellul. Endocrinol. 11, 63-78. Zolman, J.C., and Valenta, L. (1978) Acta Endocrinol. 89, 232-239. Wagner, T.O.F., Adams, T.E., and Nett, T.M. (1979) Biol. Reprod. 20, 140-149. Theoleyre, M., Berault, A., Garnier, J., and Jutisz, M. (1976) Molec. Cell. Endocrinol. 5, 365-377. Marshall, J.C., Shakespear, R.A., Odell, W.D. (1976) Clin. Endocrinol. 5, 671-677. Park, K.R., Saxena, B.B., and Gandy, H.M. (1976) Acta Endocrinol. 82, 62-70. Pedroza, E., Vilchez-Martinez, J.A., Fishback, J., Arimura, A and Schally, A.V. (1977) Biochem. Biophys. Res. Comm. 79; 234-238. Grant, G., Vale, W., and Rivier, J. (1973) Biochem. Biophys. Res. Comm. 50, 771-778. Heber, D., Marshall, J.C., and Odell, W.D. (1978) Am. J. Physiol. 235, E227-E-230. Rivier, J., Brown, M., Rivier, C., Ling, N., and Vale, W. (1976) "Peptides 1976", A. Loffet (ed.), pp. 427-445, Editions de l'universite de Bruxelles, Belgium. Marshall, J.C., and Odell, W.D. (1975) Proc. Sot. Exptl. Biol. Med. 149, 351-355. Aubert, M.L., Grumbach, M.M., and Kaplan, S.L. (1977) J. Clin. Endocrinol. Metab. 44, 1130-1141. Poirier, G., De Lean, A., Pelletier, G., Lemay, A., and Labrie, F. (1974) J. Biol. Chem. 249, 316-322. De Lean, A., Munson, P.J., and Rodbard, D. (1978) Am. J. Physiol. 235, E97-E102. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Koch, Y., Baram, T., Chobsieng, P., and Fridkin, M. (1974) Biochem. Biophys. Res. Commun. 61, 95-103. Fujino, M., Kobayashi, S., Obayashi, M., Shinagawa, S., Fukuda, T ., Kitada, C., Nakayama, R., Yamazaki, 11, White, W.F., and Res. Commun. 49, 698-705. Rippel, R.H. (1972) Biochem. Biophys. Frager, M., Duncan, J., Pieper, D., Tonetta, S., and Marshall, J.C. (1979) The Endocrine Society 61st Annual Meeting, Anaheim, Abstract 432. Society 61st Annual Meeting, Clayton, R.N. (1979) The Endocrine Anaheim, Abstract 438. McKelvy, D.F., Leblanc, P., Laudes, C., Perrie, S., GrimmJorgensen, Y., and Kordon, C. (1976) Biochem. Biophys. Res. comm. 73, 507-515. Aubert, M.L., Conne, B.S., and Sizonenko, P.C. (1979) The Endocrine Society 61st Annual Meeting, Anaheim, Abstract 501.
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