Endocrinology Vol. 141, No. 1 138-145
Copyright © 2000 by The Endocrine Society
Characterization of a Low Affinity Binding Protein for Growth Hormone in Rat Serum1
Kin-Chuen Leung,
Nathan Doyle and
Ken K. Y. Ho
Pituitary Research Unit, Garvan Institute of Medical Research, St.
Vincent’s Hospital, Sydney, New South Wales 2010, Australia
Address all correspondence and requests for reprints to: Prof. Ken K. Y. Ho, Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail: k.ho{at}garvan.unsw.edu.au
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Abstract
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GH forms a high Mr complex in rat serum distinct from that
with GH-binding protein (GHBP). The present study investigates the
nature of this complex. When subjected to AcA44 filtration
chromatography, 125I-labeled human GH (hGH) in rat serum
eluted in four peaks. Peak 1 eluted at the void volume, whereas peaks
2, 3, and 4 corresponded to the GHBP complex, free hGH, and iodide,
respectively. Stripping of GHBP in serum by immunoaffinity
chromatography depleted peak 2 but did not affect peak 1. Peak 1
accounted for 11.4 ± 1.2% of the total radioactivity (mean
± SEM; n = 6) in stripped serum. Addition of
unlabeled hGH (0.9–9 µM) demonstrated the binding of
[125I]hGH to be specific, with Scatchard analysis
revealing an affinity of 0.88 ± 0.03 x 105
M-1 (n = 3) and a capacity of 2.46
± 0.14 µM. Sepharose CL-6B filtration chromatography
showed the complex to be 260 kDa in size. The distribution of GH
binding to GHBP and this high Mr serum factor was
investigated by incubating [125I]hGH in sera containing a
low (5 nM) and a high (35 nM) concentration of
GHBP over a range of physiological GH concentrations. In sera
containing a low concentration of GHBP, the proportion of GH complexed
in peak 1 increased with increasing GH concentrations. In sera with a
high concentration of GHBP, GH was complexed mainly in peak 2. Studies
with normal rat sera revealed that more GH was complexed in peak 1 in
male than in female rats (3.4 ± 0.4% and 1.4 ± 0.1%,
respectively; P < 0.006), in contrast to that of
peak 2 (1.1 ± 0.2% and 7.6 ± 0.4%, respectively;
P < 0.002).
In summary, we provide strong evidence for the existence of a factor in
rat serum that binds GH with low affinity and high capacity. It has a
Mr of approximately 240 kDa, assuming a 1:1 binding
stoichiometry, and is immunologically distinct from GHBP. This factor
may provide supplementary capacity for GH binding when binding to GHBP
is saturated.
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Introduction
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SPECIFIC BINDING proteins for GH (GHBPs) have
been identified in the circulation of all species examined to date (1).
A major component corresponds to the extracellular, ligand-binding
domain of the GH receptor in tissues, which binds GH with an affinity
(Ka) of
108–109
M-1 and capacity in the nanomolar
range, forming a complex approximately 80 kDa in size (2, 3). By
complexing GH, it modulates the pharmacokinetics and distribution of
the hormone in blood (4, 5). It also plays a role in regulating GH
bioactivity by competing with tissue receptors for GH binding
(6, 7, 8).
In addition to the GH receptor-related, high affinity GHBP, GH
associates with other circulating proteins (9, 10, 11, 12, 13). In humans, GH forms
a 120- to 170-kDa complex with a plasma protein, which is not related
to the high affinity GHBP (10, 12). The binding is of low affinity
(105–106
M-1) and high capacity (2–15
µg/ml). Despite the high capacity, the low affinity GHBP complexes
only 1–15% of the circulating GH, compared with 40–50% by the high
affinity GHBP (2, 12, 14). This protein is detectable in all plasma
samples from subjects with various physiological and disease states and
shows considerable individual variation (14). It appears to be
regulated differently from the high affinity GHBP, as there is no
correlation between the concentrations of the two GHBPs (14, 15). The
nature and physiological function of the low affinity GHBP are
unknown.
Similar to human serum, GH forms a high Mr
complex in rat serum, with the binding only partially saturated by
excess GH (11, 13). In this study we investigated the nature of this
complex and its relation to the high affinity GHBP.
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Materials and Methods
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Reagents
Recombinant human GH (hGH) was produced as previously described
(16). Recombinant Met-rat GH was obtained from Bresatec (Adelaide,
Australia), ovine GH (NIAMDD oGH-12) was obtained from the NIH
(Bethesda, MD), bovine (USDA bGH-B-1) and porcine GH (USDA pGH-B-1)
were obtained from the USDA Reproduction Laboratory (Beltsville, MD).
Recombinant rat GHBP (rGHBP) with a Mr of 30 kDa
(17) was provided by Dr. William Baumbach (American Cyanamid, Princeton, NJ). MAb263, an anti-GH receptor/GHBP
monoclonal antibody (18), was a gift from Prof. Michael Waters
(University of Queensland, Brisbane, Australia). Ultrogel AcA44 and
AcA54 were purchased from Sepracor/IBF (Villeneuve la Garenne, France).
Sepharose CL-6B, cyanogen bromide-activated Sepharose 6MB, and the high
mol wt gel filtration calibration kit (158–669 kDa) were purchased
from Pharmacia Biotech (Uppsala, Sweden). Iodogen,
disuccinimidyl suberate, and the GF5 column were obtained from
Pierce Chemical Co. (Rockford, IL).
Na[125I] was obtained from ARI (Sydney,
Australia), trichloroacetic acid was obtained from Sigma
(St. Louis, MO), precast SDS-polyacrylamide gels (4–15% gradient)
were obtained from Bio-Rad Laboratories, Inc. (Hercules,
CA), and SDS-PAGE Rainbow Mr markers (14.3–200
kDa) were obtained from Amersham Pharmacia Biotech
(Aylesbury, UK).
Iodination of hGH
hGH was radiolabeled with Na[125I] by
the Iodogen method to a specific activity of 25–40 µCi/µg.
Briefly, 10–20 µg hGH in 100 µl 0.5 M sodium phosphate
buffer, pH 7.4, were incubated with 1 mCi
Na[125I] in a vial coated with 2 µg Iodogen.
The reaction was carried out at 23 C for 10 min and was terminated by
the addition of 200 µl 0.2% BSA in PBS. Radiolabeled hGH
was separated from free iodide with a GF5 filtration column
and was further purified on an AcA54 column before use.
Preparation of GHBP-depleted rat serum
GHBP-depleted rat serum was prepared by immunoaffinity
chromatography using MAb263. The antibody was coupled to cyanogen
bromide-activated Sepharose 6MB using procedures recommended by the
supplier and was packed in a 0.8 x 3.5-cm column. Ten milliliters
of a serum pool from male rats with undetectable GH level (as
determined by RIA) were applied to the column at 4 C. The high affinity
GHBP content was measured by a ligand immunofunctional assay (19) to
confirm complete removal of the binding protein in the eluate.
Fractionation of rat serum
Rat serum fractions of defined Mr ranges
were prepared by gel chromatography. Accordingly, 1 ml GHBP-depleted
serum was applied to the AcA44 column (0.8 x 25 cm), 800-µl
fractions were collected, and protein content was monitored by
spectrophotometry at 280 nm. Appropriate fractions were pooled and
concentrated to the original sample volume by centrifugal
ultrafiltration (Centricon-10, Amicon, Beverly, MA). The protein
content in the fractions was analyzed by SDS-PAGE on 4–15% gradient
gel under nonreducing conditions by the method of Laemmli (20). The gel
was stained with Coomassie blue, and the sizes of the proteins were
determined against the Mr markers.
Gel filtration chromatography
The binding of [125I]hGH to serum
samples was studied by AcA44 gel chromatography using either a standard
column (0.8 x 25 cm) or, where appropriate, a longer column
(0.8 x 52 cm) for better resolution. Briefly, 20 µl of samples
(or 120 µl for the longer column) were incubated with 30 µl PBS
containing 0.2% BSA and [125I]hGH (5 x
104 cpm) at 4 C for 18 h in the presence or
absence of unlabeled hGH at predetermined concentrations. After
incubation, the samples were applied to the AcA44 column at 23 C, and
fractions with a volume of 400 µl (or 670 µl for the longer column)
were collected for radioactivity measurement. Bound and free
[125I]hGH were quantified from the
corresponding peaks in the elution profiles.
The binding of [125I]hGH to serum was also
investigated by Sepharose CL-6B chromatography (resolution range,
10–4000 kDa) to determine the size of the GH complex. One hundred
microliters of sample were incubated with
[125I]hGH (5 x 104
cpm) in 30 µl PBS with and without 20 µg unlabeled hGH at 4 C for
18 h before chromatographic analysis (0.8 x 52 cm). The
column was calibrated with blue dextran 2000 (void volume,
Vo), thyroglobulin (669 kDa), ferritin (440 kDa),
catalase (232 kDa), aldolase (158 kDa), BSA (67 kDa), hGH (22 kDa), and
bromophenol blue (total volume, Vt).
Covalent cross-linking
Three microliters of GHBP-depleted serum, serum fractions, or
PBS with 10% BSA were added to 100 µl PBS with
[125I]hGH (5–10 x
105 cpm) and unlabeled hGH (9 µM)
or rGHBP (7 µM). After incubation at 4 C for 18 h,
the samples were diluted with 150 µl PBS, and 10 µl 10
mM disuccinimidyl suberate were added. The cross-linking
reaction was carried out at 23 C for 15 min and terminated by the
addition of 25 µl 1 M Tris-HCl, pH 7.5. Proteins in the
samples were precipitated with 5% trichloroacetic acid at 23 C for 30
min and centrifuged at 104 x g for 5
min. The pellets were washed once with 600 µl ice-cold deionized
water before being resuspended in 50 µl SDS-PAGE sample buffer (0.03
M Tris-HCl, pH 6.8, with 3% SDS, 10% glycerol,
and 60 µg/ml bromophenol blue) with 10% ß-mercaptoethanol. After
heating in boiling water for 10 min, the samples were applied to
SDS-PAGE on 4–15% gradient gels. The gels were dried and subjected to
autoradiography at -70 C using Kodak Bio Max x-ray films
(Eastman Kodak Co., Rochester, NY) and DuPont intensifying
screens (NEN Life Science Products, Boston, MA). The sizes
of the bands were determined against the Mr
markers.
Statistical analysis
All experiments were performed three times or as otherwise
stated, and results are expressed as the mean ± SEM.
Differences between groups with P < 0.05 by ANOVA
(StatView 4.02, Abacus Concepts, Berkeley, CA) were considered
significant.
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Results
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[125I]hGH binding to rat serum
When subjected to AcA44 chromatography,
[125I]hGH in rat serum eluted in four peaks,
with the first peak (peak 1) appearing at Vo (Fig 1
). Peak 2 corresponded to the complex formed
with the high affinity GHBP and was completely eliminated by the
addition of excess unlabeled hGH (not shown). Peaks 3 and 4
corresponded to free hGH and iodide, respectively. Stripping of serum
of its high affinity GHBP content resulted in the disappearance of peak
2 but not peak 1 (Fig 1). Peak 1 accounted for 11.4 ± 1.2% of
the total radioactivity (n = 6) in GHBP-depleted serum.
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Figure 1. AcA44 gel chromatography of
[125I]hGH in rat serum. AcA44 elution profiles (0.8
x 52 cm) of [125I]hGH in rat serum before (•) and
after ( ) removal of endogenous GHBP by immunoaffinity
chromatography. The Vo and Vt of the column are
indicated.
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Addition of unlabeled hGH at concentrations of 0.9–9 µM
to GHBP-free serum reduced peak 1 and increased peak 3 in a
concentration-dependent manner (Fig 2).
Scatchard analysis revealed a linear plot with a
Ka of 0.88 ± 0.03 x
105 M-1 and a
binding capacity of 2.46 ± 0.14 µM (n = 3;
Fig. 2b, inset). In contrast to the binding properties
reported for the high affinity GHBP (19), the binding of GH to this
serum factor was of low affinity but high capacity.
Species specificity for GH binding to the serum factor was examined
using unlabeled rat, bovine, ovine, and porcine GHs to compete for
[125I]hGH binding. As shown in Fig. 3, all of the nonprimate GHs displaced peak 1
in a similar manner as hGH, but with 5- to 7-fold lower potencies.
[125I]hGH binding to rat serum
fractions
To characterize further the nature of the serum factor,
GHBP-depleted serum was resolved arbitrarily into three fractions by
gel filtration chromatography in accordance with the elution pattern of
its protein content (not shown). SDS-PAGE analysis illustrated that
fraction I mainly contained proteins with Mr of
160 kDa or greater, with a trace amount at 69 kDa (Fig. 4a). The majority of proteins in fraction II
were 50–65 kDa in size, with some at 97–200 kDa. Fraction III
contained small proteins at around the Vt in the
gel filtration chromatography, which appeared at 20 kDa or less in the
SDS-PAGE analysis.
Peak 1 was readily detectable in fraction I (Fig. 4b
), which accounted
for 28.7 ± 0.9% of the total radioactivity (n = 7). A small
but prominent high Mr peak was also found in
fraction II, probably formed from a trace amount of the serum factor
due to incomplete separation of high Mr proteins
in this fraction. No high Mr peak was detected in
fraction III. Similar to what was observed with unfractionated serum,
Peak 1 in fraction I was specifically displaced by unlabeled hGH (data
not shown), with Scatchard analysis revealing a
Ka of 1.12 ± 0.08 x
105 M-1 and a
binding capacity of 7.58 ± 0.25 µM (n =
2).
Because the molecular size of peak 1 could not be determined by AcA44
chromatography, Sepharose CL-6B chromatography was performed, which has
a higher exclusion limit (>4000 kDa) than that of AcA44 (200 kDa). As
shown in Fig. 5, peak 1 in fraction I eluted
as a broad peak with a median size of 260 kDa (range, 150–400 kDa).
Similar to the AcA44 chromatography, the peak was only partially
displaced by excess unlabeled hGH.
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Figure 5. Sepharose CL-6B chromatography of
[125I]hGH in fraction I. Elution profiles on a Sepharose
CL-6B column (0.8 x 52 cm) of [125I]hGH in fraction
I with () and without (•) 20 µg unlabeled hGH. The positions of
Mr markers (in kilodaltons) are indicated at the
top of the profiles.
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Covalent cross-linking
To investigate the nature of the GH complex with the serum factor,
[125I]hGH was covalently cross-linked to
GHBP-depleted serum, serum fractions, or PBS containing 10% BSA and
studied by SDS-PAGE and autoradiography.
[125I]hGH in buffer migrated as a major band at
22 kDa (Fig. 6a). The addition of
unfractionated serum and fraction I, but not fraction II, resulted in
the appearance of a second band approximately 280 kDa in size. This
band was not detected if the samples were not cross-linked, and its
mobility was not affected by carrying out SDS-PAGE under nonreducing
conditions (data not shown).
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Figure 6. Cross-linking of [125I]hGH to rat
serum and serum fractions. a, [125I]hGH was covalently
cross-linked to PBS with 10% BSA (control), unfractionated rat serum,
and fractions I and II and analyzed by SDS-PAGE and autoradiography. b,
Effects of unlabeled rGHBP (7 µM) and hGH (9
µM) on the cross-linking of [125I]hGH to
rat serum and fraction I.
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The specificity of the high Mr band was evaluated
by adding unlabeled hGH (9 µM) or rGHBP (7
µM) to [125I]hGH in rat serum,
fraction I, or PBS with BSA. Addition of unlabeled hGH markedly reduced
the intensity of the high Mr band in rat serum
and fraction I (Fig. 6b). The free hGH band in buffer was not affected
by the addition of unlabeled hGH. On the other hand, rGHBP diminished
the high Mr band in rat serum and fraction I,
suggesting that the high affinity GHBP competed with the serum factor
for GH binding. Rat GHBP also profoundly reduced the free hGH band in
all samples, with the formation of two bands at 55 and 100 kDa
corresponding to the GH-GHBP and GH-(GHBP)2
complexes, respectively (21). There were also a 35-kDa band in all
samples with rGHBP and a 45-kDa band in fraction I with hGH. These
bands probably represented cross-linked products of the degraded
radioligand which appeared as a 14-kDa band in the same gel.
Combined effects of GH and GHBP on [125I]hGH binding
to rat serum
In light of the findings from the cross-linking experiments, we
examined by AcA44 chromatography how the complexing of GH was
partitioned between the high affinity GHBP and the serum factor.
Studies were performed by adding increasing amounts of unlabeled hGH to
GHBP-free serum reconstituted with varying concentrations of rGHBP. hGH
was added at concentrations of 0.5, 4.6, and 13.6 nM to
mimic the nadir and peak GH levels reported for female and male rats,
respectively (22). Similarly, rGHBP was added to GHBP-free serum to
achieve concentrations of 5 and 35 nM to mimic the GHBP
levels in male and female rats, respectively (19). In the absence of
rGHBP, the addition of unlabeled hGH slightly reduced peak 1 from
13.7 ± 0.8% to 11.3 ± 1.0% at 13.6 nM hGH and
increased peak 3 from 86.3 ± 0.8% to 88.7 ± 1.0% (Fig. 7a). In sera containing 5 nM
rGHBP only, [125I]hGH appeared mainly as peak 2
(Fig. 7b). The addition of increasing amounts of unlabeled hGH resulted
in a reduction in peak 2 and parallel increases in peaks 1 and 3. In
sera containing 35 nM rGHBP, most
[125I]hGH was associated in peak 2 (Fig. 7c),
and addition of unlabeled hGH caused only small changes in all three
peaks.
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Figure 7. Combined effects of hGH and rGHBP on
[125I]hGH binding to rat serum. GHBP-depleted rat serum
was incubated with [125I]hGH in the absence or presence
of unlabeled hGH (0.5, 4.6, and 13.6 nM) and with rGHBP at
0 (a), 5 (b), and 35 nM (c). The samples were then
subjected to AcA44 chromatographic analysis, and peak 1 (white
bars), peak 2 (gray bars), and peak 3
(black bars) were measured.
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Peaks 1 and 2 in normal rat serum
We have previously demonstrated that male rats have lower serum
levels of the high affinity GHBP than female rats (19). Accordingly, we
investigated whether GH binding to the serum factor also exhibited
sexual dimorphism. Figure 8 illustrates the
scattergram plot of GH binding activities in sera from 10 normal rats
(6 males and 4 females), with peak 1 plotted against peak 2. There was
a clear separation of both peaks 1 and 2 between male and female rats.
The level of peak 1 in male rats (3.4 ± 0.4% of the total
radioactivity) was significantly higher than that in the females
(1.4 ± 0.1%; P < 0.006). In contrast, the level
of peak 2 was lower in male (1.1 ± 0.2%) than female (7.6
± 1.8%; P < 0.002) rats.
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Figure 8. Scattergram of peaks 1 and 2 in normal rat sera.
Peaks 1 and 2 in random sera from normal male () and female (•)
rats were plotted.
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Discussion
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We confirm previous findings that GH in rat serum forms a complex
much larger than that with the high affinity GHBP, but extend these
findings by demonstrating that the complex is formed from a high mol wt
serum factor immunologically unrelated to the high affinity GHBP. The
binding of the serum factor for GH was of low affinity and high
capacity, forming a complex 260 kDa in size. It bound approximately
10% of GH in the absence of the high affinity GHBP, and this level
decreased with increasing concentration of the high affinity GHBP. The
level of GH binding to the serum factor in male rats was significantly
higher than that in female rats, a phenomenon opposite that of the high
affinity GHBP.
The serum factor had an affinity for GH of 0.9 x
105 M-1,
approximately 3 orders lower than that of the high affinity GHBP (19),
and therefore will be referred to as the low affinity GHBP. The binding
capacity of the low affinity GHBP was at the micromolar level, compared
with the nanomolar concentration of the high affinity GHBP in rat blood
(19). These values are comparable to those reported for a low affinity
GHBP in human plasma (10). On the other hand, the rat low affinity GHBP
showed a broad species specificity for GH binding, with hGH a better
ligand than nonprimate GHs. This is very different from the human low
affinity GHBP, which binds only hGH (10).
A single species of GH complex for the low affinity GHBP with a size
greater than 200 kDa was detected in the cross-linking and SDS-PAGE
studies. As no commercial Mr marker covering this
region is available, precise determination of the size is not possible.
Nevertheless, the complex is estimated to be 280 kDa, which is in good
agreement with the result from Sepharose CL-6B chromatography. The rat
low affinity GHBP complex thus is much larger than its human
counterpart, which is around 120 kDa in size (10). To exclude the
possibility that the high Mr band detected in the
present study was an aggregation artifact, milder conditions for
cross-linking, including shorter incubation time, lower concentrations
of disuccinimidyl suberate, and less sera, were examined. The
cross-linked samples were also boiled in 10% SDS to enhance complete
dissociation of aggregates. However, in none of these conditions was a
complex band with a smaller size detected (data not shown), strongly
suggesting that the 280-kDa band was a genuine cross-linked GH complex
of the low affinity GHBP.
The identity of the low affinity GHBP remains to be defined. It is
unlikely to be derived from the high affinity GHBP, as the latter was
undetectable after stripping serum with an antibody to GH
receptor/GHBP. However, we cannot exclude the possibility that it may
be an anti-hGH antibody in rat serum, although this possibility is
considered unlikely. In the cross-linking experiments, the complex had
similar mobilities in SDS-PAGE under reducing and nonreducing
conditions. This finding is not compatible with antibody complexes that
are dissociated to smaller subunits by reducing agents. Another
possible candidate of the low affinity GHBP is rat
2-macroglobulin. Recently, GH has been shown
to bind human 2-macroglobulin (23), which is a
720-kDa glycoprotein consisting of four identical, noncovalently
associated subunits (24). GH binds human
2-macroglobulin with an affinity of
0.02–2 x 106
M-1 (23), forming a complex between
180–360 kDa in size as detected by gel electrophoresis (25). These
findings are similar to those of the rat low affinity GHBP identified
here. However, because rat 2-macroglobulin is
not commercially available, we are unable to examine its possible
identity as the low affinity GHBP in rats.
Our data reveal that the level of GH binding to the low affinity GHBP
depends on both the concentrations of GH and the high affinity GHBP. To
evaluate how GH complexing is partitioned between the two GHBPs, we
have derived an algebraic method, based on the law of mass action (see
Appendix), to calculate the levels of free GH and the two
complexes as a function of the concentrations of GH and the high
affinity GHBP. In the absence of the high affinity GHBP, about 18% of
GH is bound to the low affinity GHBP, and this proportion does not
change with increasing GH concentration (Fig. 9a). The theoretical level of the low
affinity GHBP complex is higher than that obtained from chromatographic
studies (
11%), suggesting that the proportion of GH complexed in
this form was underestimated under the experimental conditions. A
likely explanation is dissociation occurring during the column
separation. The presence of the high affinity GHBP reduces the
proportions of the low affinity GHBP complex (Fig. 9a) and free GH
(Fig. 9c) by complexing GH (Fig. 9b). This effect is dependent not only
on the concentration of the high affinity GHBP, but also on the GH
concentration. When the GH concentration increases to a level
saturating the high affinity GHBP, the proportion of the high affinity
GHBP complex falls, with parallel increases in levels of free GH and
the low affinity GHBP complex. These predicted changes in the
distribution of free and complexed GH are consistent with the
experimental findings (Fig. 7). The collective data thus suggest that
although the high affinity GHBP is the primary binding protein for GH,
the low affinity GHBP provides supplementary GH binding capacity in
serum.
In a previous study on the dynamics of interaction of GH with the high
affinity GHBP, we demonstrated that in male rats, most GH exists in the
free form during secretory peaks, as the concentration of the high
affinity GHBP is low (19). In female rats, as GH secretion is
continuous and the high affinity GHBP level is high, most GH circulates
in the complexed form. The identification of a second GHBP adds a
further level of complexity to the distribution of free and complexed
GH during spontaneous secretion in the rat. Our findings show evidence
of sexual dimorphism in the partitioning of GH complexing between the
two GHBPs, with the proportion of GH binding to the low affinity GHBP
higher and that to the high affinity GHBP lower in male rats. The low
affinity GHBP may modulate the extent of free GH during secretory
spikes in male rats when the high affinity GHBP is saturated and reduce
the clearance of GH between secretory episodes. This effect would be
less in female rats.
The physiological function of the low affinity GHBP is not known. There
is strong evidence that the high affinity GHBP alters the
pharmacokinetics and distribution of circulating GH (4, 5) and inhibits
GH actions by competing with tissue GH receptors for GH binding (6, 7, 8).
Whether the low affinity GHBP can modulate the circulating half-life
and bioavailability of GH warrants future studies.
In conclusion, we provide strong evidence that rat serum contains a
high Mr factor that binds GH with low affinity
but high capacity. The high affinity GHBP is the major binding protein
for GH in rat serum, whereas the low affinity GHBP provides additional
GH binding capacity when binding to the high affinity GHBP is
saturated.
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Appendix
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The equilibrium concentrations of free GH
(Hf) and GH complexes with the high
(C1) and low (C2) affinity
GHBP are calculated based on the following equations:
Ht = Hf +
C1 + C2,
B1t = C1 +
B1f, B2t =
C2 + B2f,
K1 =
C1/(Hf x
B1f), and K2 =
C2/(Hf x
B2f), where Ht
is the total GH concentration, B1t and
B1f are the concentrations of total and
free high affinity GHBP, B2t and
B2f are the concentrations of total and
free low affinity GHBP, and K1 and
K2 are the association constants for the
complexes of the high and low affinity GHBPs. By solving these
equations simultaneously, Hf,
C1, and C2 are determined
as follows:
With nominal values for K1 = 2.5 x 108
M-1 (19), K2 = 0.9 x
105 M-1 and
B2t = 2.5 µM (from the present
study), Eq I and II become:
As Hf3 and
C23 are much smaller than the other terms in
the respective equations, Eq IV and V can be approximated to and solved
as binomial equations. Thus, Hf and
C2 are determined as follows:
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Acknowledgments
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We thank Dr. William Baumbach for the recombinant rat GHBP,
Prof. Michael Waters for MAb263, and Ms. Irit Markus for her excellent
technical assistance. We also thank Dr. Christopher Ormandy for the
useful discussion about the chromatographic work.
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Footnotes
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1 This work was supported by a grant from the National Health and
Medical Research Council of Australia. 
Received July 13, 1999.
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Top
Abstract
Introduction
), 1.8
(•), and 9.1 (
), bovine (
), and porcine GHs (
), 5 (