From the
According to Burkhardt et al. (Burkhardt, E., Adham, I.
M., Brosig, B., Gastmann, A., Mattei, M. G., and Engel, W.(1994) Genomics 20, 13-19) Leydig cells contain the message for
a protein of the insulin/relaxin family which was named Leydig cell
insulin-like protein (LEY I-L). We have synthesized the human LEY I-L
according to the amino acid sequence deduced from the published cDNA
structure and obtained preliminary results concerning its potential
target organs and its biological activity. Leydig cell insulin-like
protein binds specifically to crude membrane preparations of mouse
uterus and brain and shows cross-reactivity with the relaxin receptor,
but not the insulin receptor. On the basis of these observations,
together with the results of earlier structure-function considerations,
we suggest that the new protein is a relaxin-like factor. By itself the
new factor shows no obvious effect, but when given together with
relaxin it significantly enhances relaxin-mediated widening of the
mouse symphysis pubis.
Relaxin, the Leydig cell insulin-like
protein(1, 2) , and insulin share general structural
features such as molecular weight, the two-chain structure, and the
number and disposition of disulfide links. Large sequence differences
exists between relaxins from different species (3) and between
relaxins, the new factor, and insulins(1) . The fine structural
differences of these two chain proteins, however, depend critically
upon only a few type-specific amino acid residues such as the glycine
in position A14 (human relaxin II and the relaxin-like factor) or the
isoleucine in the equivalent position (A10) in insulin(3) . The
receptor-binding site of relaxin is comprised of a cassette of the
general structure Arg-X-X-X-Arg which is located in the
midsection of the B chain helix in positions B13 to B17 in human
relaxin II and in the same relative position in all relaxins (4). This
cassette is also present in the new protein but offset toward the C
terminus by precisely four residues. In these critical regions the new
factor resembles relaxin rather than insulin. Here we are reporting the
synthesis of the new factor and the experiments that support our
contention that this factor be called relaxin-like factor
(RLF)
L-Amino acid derivatives for peptide synthesis were
purchased either from Bachem Bioscience (Philadelphia, PA) or Bachem
California (Torrance, CA). Solvents for peptide synthesis and
chromatography were distilled in high purity solvent (Burdick and
Jackson; Muscagon, MI), and the chemicals for peptide synthesis were
obtained from Perkin-Elmer Applied Biosystems (Foster City, CA). Other
chemicals of analytical grade were used without further purification.
The A chain (0.25 mmol) was synthesized via Fast-moc
For chain
combination 33.4 mg (11.3 µmol) of the A chain (AcmA10,MBzlA24) was
treated with 4 ml of HF in the presence of 200 µl of m-cresol for 45 min at 0 °C. Thereafter the HF was
evaporated in a stream of nitrogen and the peptide precipitated with
ether. The pellet was collected and dried over KOH in vacuo for 30 min. The monothiol A chain was dissolved in 4 ml of 8 M guanidinium chloride in 0.1 M acetic acid at pH 4.5 and
added to 36.3 mg (9.6 µmol) of the B chain. The disulfide bond
A24-B22 was formed at 37 °C for 24 h and the resulting
product separated first on Sephadex G50-sf in 1 M acetic acid
(column 2.5
The resulting peptide contained
acetamidomethyl groups in positions Cys
Complete deprotection was achieved first by treatment of 11.3 mg of
the peptide with 2 ml of water/piperidine 9:1 (v/v) for 2 min at room
temperature. The base was neutralized with 0.4 ml of acetic acid and
the peptide purified by preparative HPLC, dried (yield: 11.0 mg,
97.5%), and 10 mg of peptide-containing methionine sulfoxide was
reduced with 1 ml of trifluoroacetic acid, 0.5 M
NH
For
preparative HPLC a Waters HPLC system consisting of two pumps (model
6000A) and gradient programmer (model 680) was used in combination with
a Synchropak RP-P column (C18) (SynChrom, Inc.) and an Uvicord S UV
(226 nm) monitor (LKB, Bromma, Sweden). Usually 1-20 mg of
peptide was separated using linear gradients as indicated.
The relaxin-like factor was
assayed either on crude membrane preparations of human placenta under
the conditions described for insulin or on crude membrane preparations
of mouse tissues using the assay system described for relaxin. Tissue
specificity was determined using crude membrane preparations of leg
muscles, kidneys, liver, brain, and uterus (of estrogen-primed mice).
The crude membranes were prepared as described for relaxin. Binding was
based on protein concentration determined by Lowry.
The synthesis of the human Leydig cell-derived protein (Fig. 1) has been achieved by solid phase methodologies in
combination with site-directed sequential disulfide bond formation (Fig. 2). Correctness of the disulfide bonding pattern is an
intrinsic result of the cross-linking technique and has been confirmed
in many previous syntheses of relaxins and
insulins(4, 13, 15, 16, 17, 18) .
The new factor
appears not to be limited to Leydig cells (21) and is certainly
not insulin-like as regards functional affinities. Although we have
demonstrated relaxin-enhancing properties, it is distinctly possible
that the new protein has another independent biological activity as yet
to be discovered. At this time the term RLF may be most appropriate.
The competitive character of the relaxin/relaxin-like factor
interaction with the mouse brain receptor is surprising when one
considers the shift of the receptor interacting region. Comparatively
small changes, i.e. substitution of homoarginine or lysine for
arginine, reduce receptor binding of relaxin much more (3) than
shifting the whole binding region by one helix turn (5.4Å) in
relation to the molecular core.
It seems quite implausible that a
molecule that binds to an extraneous receptor would not be an inhibitor
of the natural ligand of that receptor. Instead of the expected partial
inhibition of relaxin by RLF a distinct stimulation of the relaxin
response was observed. A similar ``cross-talk'' effect is
also observed between relaxin and insulin in the pregnant rat ovarian
fat cell assay(22) . The physiological importance of these
observations require close scrutiny. Human relaxin is not quite as
active in humans as porcine relaxin. The relaxin-like factor may
therefore play an important supportive role for the relaxin action in
humans. In addition, preliminary experiments suggest that RLF may play
a role independent of relaxin in the male gonads.
We thank Barbara Rembiesa and Robert Bracey for their
excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)rather than insulin-like protein.
Materials
Methods
Peptide Synthesis
The B chain of Leydig
cell-derived protein was synthesized by tert-butyloxycarbonyl
chemistry using conventional HF-labile side chain-protecting groups for
all three functional amino acids except cysteines. Cysteine B10 was
protected by the acetamidomethyl group and B22 by the
thiol-protecting/activating group
[S-(3-nitro-2-pyridinesulfenyl)]
(Cys). Methionine was protected by sulfoxide formation
and tryptophan by the N(in)-formyl group. The synthesis was
performed on an Applied Biosystems peptide synthesizer model 430A on
4-(oxymethyl)phenylacetamidomethyl resin loaded with 0.4 mmol of tert-butyloxycarbonyl-alanine. Deprotection and removal from
the solid support was accomplished by HF treatment in the presence of
5% m-cresol. The crude peptide was extracted with 20% acetic
acid and lyophilized (yield 1.387 g). The B chain was purified on
Sephadex G50-sf (2.5
50 cm) in 1 M acetic acid (yield:
840 mg), followed by preparative HPLC on Synchropak RP-P (2.1
25 cm) in portions of 50-70 mg. The column was equilibrated in
20% solvent B and the peptide eluted with a linear gradient of 20%
solvent B to 50% solvent B over 1 h at a flow rate of 5 ml/min (overall
yield: 233 mg). Amino acid composition: Thr, 2.00(2) ; Ser,
0.86(1) ; Glu, 2.90(3) ; Pro, 3.09(3) ; Gly,
3.28(3) ; Ala, 2.16(2) ; Cys, 0.89(2) ; Val,
3.19(3) ; Met, 1.22(1) ; Leu, 1.94(2) ; Phe, 0.99
(1); His, 2.44(2) ; Lys, 0.96(1) ; Arg, 3.81(4) .
chemistry on an ABI peptide synthesizer (model 430A) on p-benzyloxybenzyl resin. All side chains were protected by
trifluoroacetic acid-labile protecting groups except Cys
,
which was Acm-protected, and Cys
which was protected by
the HF-labile p-methylbenzyl group. The A chain was
deprotected with trifluoroacetic acid/thiophenol (10:1, v/v), using 50
mg of peptidyl resin/ml for 90 min at room temperature(5) . The
trifluoroacetic acid was evaporated and the peptide precipitated with
ether. The precipitate was collected by centrifugation, the supernatant
discarded, and the pellet washed twice with ether and air-dried. The
peptide was suspended in water, dissolved by the addition of ammonia,
and desalted on Sephadex G-25m in 50 mM
NH
HCO
. To the eluate (100 ml) 50 ml of
Me
SO was added in order to accelerate the oxidation of the
intrachain disulfide bond A10-A15(6) . The progress of
oxidation was observed by the Ellman reaction(7) . After
completion of the disulfide bond formation the A chain was dialyzed
against water and lyophilized (yield: 372.3 mg). Aliquots of 20 mg were
further purified by preparative HPLC on Synchropak RP-P (10
250
mm). The column was equilibrated in 30% solvent B and the peptide
eluted with a linear gradient of 30% solvent B to 50% solvent B over 30
min at a flow rate of 3 ml/min (overall yield: 166.5 mg). Amino acid
composition: Asp, 2.20(2) ; Thr, 3.00(3) ; Ser,
0.99(1) ; Glu, 1.92(2) ; Pro, 2.25(2) ; Gly,
1.06(1) ; Ala, 4.18(4) ; Cys, 1.62(4) ; Leu,
3.60(4) ; Tyr, 1.82 (2); Arg, 0.98(1) .
50 cm) (yield: 48.7 mg, 78.3%), followed by
preparative HPLC on Synchropak RP-P (10
250 mm). The column was
equilibrated in 30% solvent B and the peptide eluted with a linear
gradient of 30-45% solvent B over 30 min at a flow rate of 3
ml/min (yield: 34.1 mg, 54.8%).
and
Cys
, the N(in)-formyl group in
Trp
, and a sulfoxide in the side chain of
Met
. For the formation of the third disulfide bond the
peptide (9.3 mg) was dissolved in water (3.5 ml) and added to a stirred
solution consisting of acetic acid (3.5 ml) 6 N HCl (19.1
µl) and 3 ml of 50 mM iodine in acetic acid(8) .
The reaction was performed at room temperature for 10 min, quenched
with ascorbic acid, and the product was desalted on Sephadex G25-sf in
1 M acetic acid and lyophilized. After purification by
preparative HPLC (conditions as before) (yield: 3.42 mg, 36.8%) the
protein still contained protecting groups in Trp (B27) and Met (B5).
I in water 9:1 (v/v) for 15 min at 0 °C. Free iodine
was reduced with 0.5 M ascorbic acid in water and the reaction
quenched by dilution with water. The final peptide was recovered by
preparative HPLC (conditions as before) (yield: 7.57 mg, 75.7%). Amino
acid composition: Asp, 2.02(2) ; Thr, 4.79(5) ; Ser,
1.77(2) ; Glu, 4.86(5) ; Pro, 5.17(5) ; Gly,
4.15(4) ; Ala, 6.09(6) ; Cys, 3.51(6) ; Val,
2.86(3) ; Met, 0.70(1) ; Ile, 0(0) ; Leu,
5.74(6) ; Tyr, 2.12(2) ; Phe, 0.98(1) ; His,
2.00(2) ; Lys, 1.26(1) ; Trp, 1.00 (1); Arg, 5.02 (5) (overall yield 14.9%).
HPLC
The mobile phase of all HPLC systems used
consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1%
trifluoroacetic acid in 80% acetonitrile (solvent B).
Analytical HPLC I
Analytical HPLC I was performed
on Aquapore 300 (C; 2.1
30 mm) using an Applied
Biosystems HPLC model 130A. Separation was achieved with a linear
gradient from 23 to 34% solvent B in 60 min at a flow rate of 0.1
ml/min. The peptide was detected by UV absorbance at 230 nm.
Analytical HPLC II
Analytical HPLC II was
performed on Synchropak RP-P (C18, 4.1 250 mm) using a Waters
HPLC system. Separation was achieved with a linear gradient from 20%
solvent B to 50% solvent B in 30 min at a flow rate of 1 ml/min. The
peptide was detected by UV absorbance at 220 nm.
Amino Acid Analyses
Peptides were hydrolyzed in
vapor phase 6 N HCl containing 0.1% phenol for 1 h at 150
°C. The amino acids were detected after pre-column modification
with phenylisothiocyanate and separation by HPLC (PicoTag system,
Waters Millipore).
Sequence Analyses
Sequence analyses were performed
on an ABI 477A pulsed liquid protein sequencer and an in-line ABI 120A
phenylthiohydantoin analyzer (ABI, Applied Biosystems, Foster City CA).
Chains were prepared by reduction of about 10 µg of the
relaxin-like factor in 20 µl of 50 mM dithiothreitol in 3 M guanidinium chloride, 0.2 M Tris/HCl at pH 8.5 for
1 h at 37 °C, diluted with 30 µl of solvent A, followed by
separation on Aquapore 300 (see ``Analytical HPLC I'' and
``Analytical HPLC II'' for conditions).
UV Spectroscopy
UV spectroscopy was performed on
an OLIS Cary 15 spectrophotometer conversion (On-Line Instrument
Systems Inc., Bogart, GA). UV spectroscopy was used to determine the
protein concentrations of Leydig cell-derived protein. The specific
absorption coefficient ( = 1.40
cm
/mg) was obtained by direct comparison of UV absorbance
and the recovery of amino acids after hydrolysis and amino acid
analysis.
Circular Dichroism
CD spectroscopy was performed
on a Jasco J-710 spectropolarimeter using a cell of 0.02-cm path
length. Proteins were dissolved in 25 mM Tris/HCl at pH 7.7,
and concentrations were determined by UV spectroscopy: 0.67 mg/ml for
porcine relaxin, 0.54 mg/ml for relaxin-like factor, and 0.55 mg/ml for
human relaxin. Spectra were measured at a resolution of 0.2 nm, a
bandwidth of 2 nm, and five spectra were averaged. Molar ellipticity
was calculated according to Adler et al.(9) using mean
residual weights of 110.4 for relaxin-like factor, 113.6 for porcine
relaxin, and 112.5 for human relaxin.
Mass Spectrometry
Mass spectra were recorded on a
Jeol HX110/HX110 4 sector tandem mass spectrometer (Jeol, Tokyo,
Japan). Samples were dissolved in 0.1% trifluoroacetic acid at a
concentration of about 0.8 nmol/µl.
For tracer preparation a synthetic precursor of the
relaxin-like factor was used which contained side chain-protected
tryptophan and methionine. The peptide, 10 µg in 5 µl of water,
was placed into a 200-µl Eppendorf vial, 5 µl of phosphate
buffer (250 mM, pH 7.4) was added, followed by 2 µl of I-Labeled Relaxin-like
Factor
I
(1 mCi), and 5 µl of chloramine T
(2 mg/ml in phosphate buffer, pH 7.4). The reaction was performed for 1
min on ice, quenched by addition of 5 µl of sodium thiosulfate (5
H
O) (50 mg/ml in phosphate buffer, pH 7.4), and 5 µl of
NaI (20 mg/ml in phosphate buffer, pH 7.4). The side chain-protecting
group of Trp was removed by addition of 5 µl of piperidine. After 2
min at room temperature the reaction was quenched by the addition of 5
µl of glacial acetic acid, the reaction mixture was diluted with 10
µl of water and loaded onto a Aquapore 300 column for separation.
The protein was detected by UV absorbance, and peaks were manually
collected into 100 µl of 1% bovine serum albumin in water.
Receptor Binding Assays
Insulin-receptor binding
assays were performed on crude membrane preparations of term placenta (10) using
[I]iodo-Tyr
-porcine insulin as
tracer(11) . Assays were performed in HMS buffer (25 mM HEPES, 104 mM NaCl, 5 mM MgCl
, 0.2%
bovine serum albumin, pH 7.4) in a total volume of 100 µl. Labeled
insulin (50,000 cpm/assay, 150 pM), and variable amounts of
insulin were incubated with crude membranes for 1 h at room
temperature. Thereafter 1 ml of buffer was added, the membranes
collected by centrifugation in a microcentrifuge at 14,000 rpm for 5
min, the supernatant discarded, and the tip of the Eppendorf vial cut
off and counted in a
-counter (Minigamma, LKB, Bromma, Sweden). To
determine nonspecific binding, unlabeled insulin was used at a
concentration of 2 µg/ml (0.33 µM), and nonspecific
binding was usually below 10% of the total binding.
Relaxin Binding Assays
Relaxin binding assays were
performed as described (12, 13) using crude membrane
preparations of mouse tissue. Mouse brains of two mice were collected
into 15 ml of chilled buffer (25 mM HEPES, 0.14 M NaCl, 5.7 mM KCl, 0.2 mM phenylmethylsulfonyl
fluoride, and 80 mg/ml soybean trypsin inhibitor, pH 7.5) supplemented
with sucrose (0.25 M, final concentration). The tissue was
homogenized on ice for 10 s with a Polytron homogenizer (Brinkmann
Instruments) at setting 5. The homogenate was centrifuged at 700 rpm
for 10 min at 4 °C, and the supernatants were recentrifuged at
10,000 g for 1 h. The pellet was resuspended in 15 ml
of ice-cold binding buffer, 25 mM HEPES, 0.14 M NaCl,
5.7 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and
80 mg/ml soybean trypsin inhibitor, pH 7.5, supplemented with 1% bovine
serum albumin, and centrifuged for 1 h at 10,000
g.
The crude membrane preparation was suspended in 1 ml of binding buffer
and 40 µl was used per assay. The assay was performed using 40
µl of tracer (about 100,000 cpm of porcine relaxin tracer =
150 pM) and 20 µl of relaxin at various concentrations.
The assay was incubated for 1 h at room temperature, and the suspension
diluted with 1 ml of wash buffer (25 mM HEPES, 0.14 M NaCl, 5.7 mM KCl, 1% bovine serum albumin, 0.01%
NaN
) and centrifuged in an Eppendorf centrifuge at 14,000
rpm for 10 min. The supernatant was discarded and the tip of the vial
cut and counted in a
-counter. Nonspecific binding was determined
in the presence of 2 µg/ml of unlabeled competitor (0.33
µM). In a typical experiment the specific binding was
between 25 and 40% of the total binding.
Mouse Symphysis Pubis Assay
Mouse interpubic
ligament assays were performed essentially as described by Steinetz et al.(14) . Ovariectomized virgin female mice were
primed with 5 µg of estrogen cypionate in 100 µl of sesame oil.
Five days later the mice were injected subcutaneously with human
relaxin(15) , RLF, or mixtures of human relaxin and RLF in 100
µl of 0.1% benzopurpurin 4B. For negative control 100 µl of
0.1% benzopurpurin 4B in water were injected. After 16 h the mice were
killed in an atmosphere of CO the symphysis pubis
dissected free, and the distance between the interpubic bones measured
with a dissecting microscope fitted with transilluminating fiber
optics.
Figure 1:
The primary structure of the
relaxin-like factor is compared with the sequences of human relaxin and
insulin. Note the relative positions of the B chain arginines (highlighted) in RLF versus relaxin. Insulin does not
contain these residues. In the A chain loop of RLF and relaxin the
relaxin-specific glycine is seen. The corresponding residue in insulin
is isoleucine.
Figure 2:
Schematic of the site-directed sequential
disulfide link formation. 1) Trifluoroacetic acid deprotection; 2)
oxidation of the thiols using MeSO/50 mM NH
HCO
(1:2, v/v); 3) HF deprotection of
Cys(MBzl); 4) combination of A and B chains at pH 4.5 in 8 M guanidinium chloride; 5) formation of the third disulfide link by
reaction with iodine in 70% acetic acid; 6) liberation of the
tryptophan side chain with 10% piperidine; 7) reduction of methionine
sulfoxide with 33-fold excess of NH
I in 90% trifluoroacetic
acid. CHO, N(in)formyl; Npys,
[S-(3-nitro-2-pyridinesulfenyl)]; O,
sulfoxide.
The purity of the RLF was verified by reverse phase high performance
liquid chromatography (Fig. 3) and amino acid analysis. Upon
reduction two chains were generated, isolated, and sequenced; both
chains had the correct primary structure. Partial hydrolysis at the
acid-labile Asn-Pro bond was not detected, and mass spectrometry showed
the correct ion for the synthetic RLF (found: m/z 6294.6;
theoretical: m/z 6293.2). Comparative circular dichroic
spectra suggested near identity of the solution structures of RLF and
porcine relaxin (Fig. 4). Human relaxin (Fig. 4) and
insulin (data not shown) have an absolute minimum at a slightly longer
wavelength and, in case of insulin, of lesser intensity(19) .
Figure 3:
HPLC record of the purified RLF.
Chromatography was performed on Synchropak RP-P (4.1 250 mm)
using a linear gradient from 20 to 50% solvent B in 30 min (solvent A:
0.1% trifluoroacetic acid in H
O and solvent B: 0.1%
trifluoroacetic acid in 80% acetonitrile) at a flow rate of 1
ml/min.
Figure 4:
Comparison of the CD spectra of human
relaxin, human RLF, and porcine relaxin. The spectra of porcine relaxin
and RLF are nearly identical.
RLF tracer was prepared from a partially protected intermediate.
This procedure allowed for iodination of one or both of the A chain
tyrosines without damage to the tryptophan side chain. The different
derivatives could be separated by HPLC to yield a carrier-free tracer (Fig. 5). The new factor was tested in the relaxin and insulin
receptor assay, and the results depicted in show relaxin
rather than insulin-like cross-reactivities. A 100-fold excess of human
RLF displaces 50% of the relaxin tracer from a mouse brain relaxin
receptor preparation. The difference in affinity is still within the
range of specific binding, i.e. several orders of magnitude
better than the binding of insulin or guinea pig relaxin to this
receptor (13) so that one can conclude that RLF recognizes the
relaxin receptor. This result was surprising because the critical
Arg-X-X-X-Arg sequence in RLF is offset toward the C-terminal
end of the B chain by exactly one turn of the helix (Fig. 1).
Although the new factor projects the arginines at nearly right angles
away from the molecular surface (as does relaxin(20) ), shifting
of the whole receptor-binding site must present quite a different
binding environment to the receptor. To prove that the relaxin-like
factor competes with relaxin for the relaxin receptor, it was important
to determine whether or not relaxin would compete for an RLF-specific
receptor if one could be detected. Of the tissues tested with I-RLF as tracer such as brain, uterus, skeletal muscle,
kidney, and liver, only the brain and the uterus membrane preparation
showed specific binding (Fig. 6). These are tissues that also
bind relaxin in a competitive and saturable manner. To test for
cross-reactivity the assays were performed with tracers and competitive
cold molecules exchanged. The results shown in suggest
strongly that RLF does have its own receptor in these tissues and that
the relaxin receptor is recognized by RLF, but with a significantly
lower affinity than relaxin. Furthermore, the data support the notion
that the brain and uterine relaxin receptors differ as concerns
cross-reactivity. The uterine relaxin receptor barely recognizes RLF,
whereas the brain receptor shows moderate cross-reactivity. In general,
the RLF receptor binds its substrate with greater affinity than the
relaxin receptor displays toward relaxin.
Figure 5:
RLF tracer preparation. Shown is the
elution record of the HPLC separation of the reaction mixture. The
largest peak is unmodified RLF and the shaded region is the
major radioactive peak used as tracer. Chromatography was performed on
Aquapore 300 (2.1 30 mm) using a linear gradient from 23%
solvent B to 34% solvent B over 60 min (solvent A: 0.1% trifluoroacetic
acid in H
O and solvent B: 0.1% trifluoroacetic acid in 80%
acetonitrile) at a flow rate of 0.1 ml/min.
Figure 6:
The tissue distribution of RLF receptors
in female estrogen-primed mice as measured in vitro in a
receptor-binding assay.
It has been established
that binding to the relaxin receptor requires two arginines in an n, n + 4 position on an -helix, and
therefore, one can assume that RLF binds the relaxin receptor with the
analogous arginine sequence in the n + 4, n + 8 position (relative to relaxin). A mouse bioassay with up
to 20 µg of RLF per mouse shows however that the factor by itself
does not cause widening of the symphysis pubis (Fig. 7A). If RLF is indeed a hormone instead of an
autocrine factor it could in theory act as a competitive inhibitor of
relaxin.
Figure 7:
A, comparative bioassay of relaxin, RLF,
and an optimal dose of both. RLF alone does not cause sympheseal
widening, but the high dose of the mixture still improves upon the high
dose of relaxin alone. B, bioactivity of an increasing amount
of relaxin in the presence and absence of 5 µg of RLF/animal. The
increase in sympheseal width was easily recognized. C,
bioactivity of an increasing amount of RLF in the presence of a uniform
amount of human relaxin again shows relaxin enhancement. hRLX,
human relaxin.
To further explore a possible role for RLF, a bioassay was
performed using ovariectomized estrogen-primed virgin female
mice(14) . Groups of five animals received either human relaxin
at a suboptimal dose or a mixture of 0.2, 0.4, and 0.8 µg of human
relaxin and 5 µg of relaxin-like protein as given in Fig. 7B. The RLF significantly increases the activity of
human relaxin in the mouse bioassay. Increasing RLF concentrations in
the presence of 0.5 µg of human relaxin shows that 5 µg of RLF
is optimal (Fig. 7C). Again the effect of the
relaxin-like factor is clearly recognized. In the next assay relaxin
alone, RLF alone, and a maximal dose of both are compared (Fig. 7A). Although RLF alone has no effect, the relaxin
effect at maximal dose is still augmented by RLF. In light of the lack
of additive effects during receptor binding studies in vitro,
this result can be explained only as receptor ``cross-talk,'' i.e. the intact cell is required to show the cooperative
effect of RLF on the biological activity of relaxin.
Table: Binding of relaxin and RLF to mouse brain and
uterus
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.