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Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Dr. P. Jean Ho, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au
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Introduction |
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In 1984 we first reported the presence of immunoreactive IGF-I together with IGF-binding activity in human milk (4). Both IGF-I and IGF-II have subsequently been reported in the milk of many species (5, 6, 7, 8, 9, 10), and IGF-I is present at considerably higher concentrations in colostrum than in mature milk (4, 6, 8, 11). Insulin and type I and type II IGF receptors are all present in the gastrointestinal epithelium (12, 13), and recent studies suggest that milk-borne IGFs may have a role in stimulating neonatal gut growth (14, 15, 16), although IGF-I was used at very high concentrations in these studies.
The IGF-binding proteins (IGFBPs) are a family of six structurally related proteins that bind IGF-I and IGF-II specifically and modify IGF-mediated effects at both tissue and cellular levels (17, 18). IGFBP-1 through -3 have been reported in milk (7, 19). In particular, total immunoreactive IGFBP-2 levels in human milk can be 10 times higher than those in maternal serum (20). The biological role of IGFBP-2 in the gut is unclear. Whether it protects luminal IGFs from digestion or has a role in neonatal gut growth has not been investigated.
In the course of purifying IGFBP-2 from human milk, we identified several related peptides with molecular masses lower than that of the intact protein. In this paper we describe the purification and binding properties of truncated forms of IGFBP-2 from human milk.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I affinity chromatography
Pooled skim milk (790 and 900 ml in two separate preparations)
was pumped at 0.5 ml/min through a 2.5 x 5-cm Sephadex G-15
(Pharmacia, Uppsala, Sweden) guard column and then onto a 1 x
10-cm agarose-IGF-I affinity column. This column contained 8 mg
recombinant human IGF-I (donated by Genentech, South San Francisco, CA)
that had been coupled to activated agarose (Affi-Gel 10, Bio-Rad,
Hercules, CA) according to the manufacturer’s instructions. Before
loading the milk, the column was prewashed with 25 ml 0.5 M
acetic acid, pH 3.0, followed by 75 ml 0.5 M acetic acid
and 1 M NaCl, pH 3.0, then equilibrated with 100
mM NaCl, 50 mM sodium phosphate, and 0.2
g/liter sodium azide, pH 6.5. After loading the sample, the column was
washed with 100 ml equilibration buffer. Bound protein was eluted with
0.5 M acetic acid, pH 3.0, at 0.5 ml/min, and 1-ml
fractions were collected in siliconized glass tubes. The concentration
of IGFBP-2 in flow-through and eluted fractions was monitored using a
previously described IGFBP-2 RIA (21). The antibody used in the assay,
raised against rhIGFBP-2, had less than 0.1% cross-reactivity with
hIGFBP-1 and -3 and rhIGFBP-4 and -5 and had approximately 0.6%
cross-reactivity with hIGFBP-6 (21).
HPLC
Fractions eluted from the IGF-I affinity column containing
maximal IGFBP-2 immunoreactivity were further purified by reverse phase
HPLC on a C18 column (RP-318, Bio-Rad) eluted with a
15–60% acetonitrile gradient in 0.1% trifluoroacetic acid at 1.5
ml/min over 30 min. These elution conditions were adjusted for further
purification (see Results).
Peptides and radioactive tracers
rhIGFBP-2 was donated by Sandoz (Basel, Switzerland) and was
radioiodinated as previously described (21). Radioiodination of protein
A (Sigma Chemical Co., St. Louis, MO), IGF-I (donated by Kabi,
Stockholm, Sweden), and IGF-II (donated by Eli Lilly, Indianapolis, IN)
was performed by reacting 5 µg pure protein with 1 mCi
Na125I and 10 µg chloramine-T in a final volume of 70
µl 0.5 M sodium phosphate, pH 7.4, for 20 sec at 22 C.
The reaction was stopped with 10 µl 5 mg/ml sodium metabisulfite. The
iodinated protein was separated from unreacted Na125I by
size chromatography in a 1 x 30-cm column of Sephadex G-100 (for
protein A) or Sephadex G-50 (for IGF-I and IGF-II). Fractions of 1 ml
were collected, and the peak fractions of radioiodinated protein, as
measured by 
-counting, were pooled for use. The column buffers used
were 50 mM sodium phosphate and 2.5 g/liter BSA (Sigma), pH
6.5, for protein A tracer; 50 mM sodium phosphate and 0.5
g/liter BSA, pH 6.5, for IGF-I tracer; and 25 mM HEPES and
2.0 g/liter BSA, pH 7.0, for IGF-II tracer.
SDS-PAGE
HPLC fractions with IGFBP-2 immunoreactivity were lyophilized.
The dried samples were reconstituted in Laemmli sample buffer (22) and
subjected to nonreducing 12% SDS-PAGE at 100 V overnight. Molecular
mass standards of 14–97 kDa (Bio-Rad) were used. The gels were
equilibrated with 25 mM Tris (BDH, Poole, UK), 192
mM glycine (Ajax, Auburn, Australia), and 20% methanol for
30 min at 22 C, then transferred to Hybond-C Extra nitrocellulose
(Amersham, Little Chalfont, UK) at 250 mA over 2 h using a semidry
electrophoretic transfer unit (Multiphor II Novablot, Pharmacia
Biotech, Bromma, Sweden). The nitrocellulose membrane was blocked with
10 g/liter BSA in 10 mM Tris and 150 mM NaCl,
pH 7.4, for 4 h at 37 C.
IGFBP-2 immunoblotting
Electroblotted nitrocellulose membrane was incubated with
rhIGFBP-2 antiserum (as used for the RIA) diluted to 1:1000 in
Tris-buffered saline (TBS; 10 mM Tris and 150
mM NaCl) containing 10 g/liter BSA, pH 6.5, overnight at 22
C. The nitrocellulose membrane was washed four times with TBS, pH 6.5;
once in TBS containing 0.5 g/liter Nonidet P-40 (ICN, Costa Mesa, CA),
pH 6.5; then three times in TBS, pH 6.5. The nitrocellulose membrane
was incubated with biotinylated donkey antirabbit Ig (Amersham) in TBS
containing 10 g/liter BSA and 0.5 g/liter Nonidet P-40, pH 7.5, for
1 h at 37 C, washed as before, then incubated with
streptavidin-alkaline phosphatase conjugate (Boehringer Mannheim,
Mannheim, Germany) at a final dilution of 0.5 U/ml in TBS containing 10
g/liter BSA and 0.5 g/liter Nonidet P-40, pH 7.5, for 30 min at 37 C;
and washed again as before. Bands were developed using a
bromochloroindolyl phosphate/nitro blue tetrazolium substrate system
according to the manufacturer’s instructions. The nitrocellulose
membrane was incubated at 22 C in the dark in 50 ml TBS, pH 9.5,
containing 10 mM MgCl2, 0.4 M nitro
blue tetrazolium (Sigma; added as 16.5 mg predissolved in 200 µl
dimethylformamide), and 0.38 M bromochloroindolyl phosphate
(Sigma; added as 8.25 mg predissolved in 200 µl dimethylformamide).
As soon as the desired band intensity developed, the reaction was
stopped using 10 mM Tris and 1 mM EDTA, pH
7.5.
Alternatively, after incubation with rhIGFBP-2 antiserum, IGFBP-2-immunoreactive bands were visualized using radioiodinated protein A. The nitrocellulose membrane was washed three times in TBS, pH 6.5, containing 0.5 g/liter Nonidet P-40, once in TBS without Nonidet P-40, and twice in TBS with Nonidet P-40, then incubated with radioiodinated protein A (2.5 x 106 cpm) diluted in 50 ml TBS containing 10 g/liter BSA, pH 6.5, for 2 h at 22 C. The blots were washed again as before and dried at 22 C. Radioactive bands were detected by autoradiography of the dried blots using Hyperfilm MP autoradiographic film (Amersham) for 3 h at -80 C.
Ligand blotting
Electroblotted nitrocellulose membrane was washed three times in
TBS, pH 6.5, containing 0.5 g/liter Nonidet P-40, once in TBS, and
twice in TBS with Nonidet P-40, then incubated with radioiodinated
rhIGF-II (1 x 106 cpm) diluted in 50 ml TBS
containing 10 g/liter BSA, pH 6.5, overnight at 22 C or for 3 days at 4
C, washed again as described above, dried at 22 C, and subjected to
autoradiography as described above.
[125I]IGF-I and
[125I]IGF-II binding assays
These binding assays were performed in 0.1 M sodium
phosphate, 0.1 g/liter Triton X-100, 1 g/liter gelatin, and 0.2 g/liter
sodium azide, pH 6.5. Incubations consisted of standard or sample (50
µl), buffer (150 µl), and [125I]IGF-I or
[125I]IGF-II (10,000 cpm in 100 µl). After 2 h at
22 C, 2 µl anti-IGFBP-2 Ig previously purified from rabbit antihuman
IGFBP-2 antiserum on a column of protein A-Sepharose CL-4B (Sigma) was
added in 25 µl buffer. After an additional hour at 22 C, 8 µl goat
antirabbit Ig in 25 µl buffer were added. After another hour at 22 C,
bound radioactivity was precipitated by adding 1 ml cold 6 g/100 ml
polyethylene glycol 6000 (BDH, Kilsyth, Australia) in 0.15
M NaCl, waiting 15 min, then centrifuging for 20 min at
4,200 rpm at 2 C in a J6 centrifuge (Beckman Instruments, Fullerton,
CA). Supernatants were decanted, and radioactivity in the pellet was
measured by -counting.
Competitive binding curves and pH dependence
These were performed in 50 mM sodium phosphate, 100
mM Tris, 50 mM acetic acid, 0.1 g/liter Triton
X-100, 1 g/liter gelatin, and 0.2 g/liter sodium azide, adjusted to the
specified pH. Incubations consisted of intact rhIGFBP-2 or truncated
human milk IGFBP-2, [125I]IGF-II (10000 cpm), and
unlabeled rhIGF in a total volume of 300 µl. After 2 h at 22 C,
2 µl protein A affinity-purified IGFBP-2 antiserum in 25 µl buffer
were added. Further steps were the same as those in the
[125I]IGF-I and [125I]IGF-II binding assays
described above. Des(1, 2, 3, 4, 5, 6)-IGF-II used in the competitive binding
curves was obtained from GroPep (Adelaide, Australia).
Other protein analyses
The amino-terminal sequences of the HPLC fractions with IGFBP-2
immunoreactivity were determined by Dr. M. I. Tyler (Deakin
Research, CSIRO, Ryde, Australia) using Edman degradation. Electrospray
mass spectrometry was performed by Dr. A. Kortt (CSIRO Division of
Biomolecular Engineering, Parkville, Australia).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. A major
split peak (peak A) was eluted at 15–16 min, and a smaller single peak
(peak B) was eluted at 16–17 min. Substantially greater IGFBP-2
immunoreactivity was detected in peak B than in peak A (Fig. 1c). Peak
A was further purified by elution with a 15–32.4% acetonitrile
gradient in 0.1% trifluoroacetic acid over 15 min, then isocratically
at 32.4% acetonitrile at 1.5 ml/min, allowing resolution into peaks
A-1 and A-2. Fractions corresponding to these peaks were run separately
with a 15–33.6% acetonitrile gradient in 0.1% trifluoroacetic acid
over 15 min, then isocratically at 33.6% acetonitrile (Fig. 2). Peaks A-1 and A-2 each gave a single
peak of absorbance at 280 nm in the approximate ratio of 3:2. Table 1 summarizes the stepwise recoveries of
IGFBP-2 immunoreactivity during the course of purification.
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shows the composite results of
SDS-PAGE of the starting material and IGFBP-2 peptides purified from it
from two separate preparations. On immunoblotting using antiserum to
rhIGFBP-2, skim milk showed a band at 34 kDa, similar to that obtained
using intact rhIGFBP-2 (Fig. 3a, lane 1), as well as bands at 24 and
14–16 kDa (Fig. 3a, lane 2). On ligand blot, only the 34-kDa band was
detected (Fig. 3b, lane 2). On immunoblot, peak A was detected
principally as two bands between 14–16 kDa (Fig. 3a, lane 3). There
were also several faint bands between 16–30 kDa that were more obvious
in another preparation on immunoblot, but were not seen on IGF-II
ligand blot (result not shown). Both major bands at 14–16 kDa were
also detected by IGF-II ligand blotting (Fig. 3b, lane 3). Peak A had a
separate band at 19 kDa and a faint band at 24 kDa as found by ligand
blot, but neither was detected by immunoblot. These two bands were not
further identified, as they were not immunologically related to
IGFBP-2. Peak A-1 was detected as a single band at about 15 kDa by
immunoblot (Fig. 3a, lane 5). Peak A-2 was detected as a band of
slightly lower molecular mass (Fig. 3a, lane 6). Both peaks A-1 and A-2
were visualized on ligand blotting using radioiodinated IGF-II (Fig. 3b, lanes 5 and 6, respectively). Peak B mainly had material at 24 kDa,
which was detected on both immuno- and ligand blotting (Fig. 3a, lane
4, and Fig. 3b, lane 4), and a smaller amount of 34-kDa intact IGFBP-2,
which was only visible on the immunoblot in one experiment (Fig. 3a, lane 4), but was also weakly visible by ligand blot in another
preparation (data not shown).
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The amino-terminal sequences of peaks A-1, A-2, and B are shown in Fig. 4. Peak B yielded two amino-terminal
sequences in equal amounts: one identical to IGFBP-2, and the other
identical to des(1, 2, 3)-IGFBP-2 (23). It is not known whether the intact
or truncated terminal corresponds to the 24- or 34-kDa component or
both. Peak A-1 contained approximately 94% of an amino-terminal
sequence starting at Gly169 of hIGFBP-2 (sequence a) and
6% of a sequence starting at Lys181 (sequence b), whereas
peak A-2 consisted of a mixture of approximately 71% sequence a and
29% sequence b. The predicted molecular masses of sequences a and b of
peaks A-1 and A-2 were 13,786 and 12,502 Da, respectively, if both
sequences extended to Gln289 of the hIGFBP-2 sequence and
all six cysteine residues were disulfide bonded. All sequences were
confirmed in two separate preparations. Mass spectrometry of peak A-1
material yielded two major species of 13,840 and 13,425 Da,
respectively. No species was detected at 12,502 Da, the predicted
molecular mass of sequence b in peak A-1.
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shows the quantitation of peaks A-1 and A-2 using the IGFBP-2 RIA.
Peak A-1 had minimal [125I]IGF-I-binding activity and
weak [125I]IGF-II-binding activity, whereas peak A-2 had
approximately 25% of the binding activity for
[125I]IGF-II and less than 10% of that for
[125I]IGF-I compared with rhIGFBP-2 (Fig. 5, b and
c).
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). Binding of [125I]IGF-II
by rhIGFBP-2 was negligible at pH 3.0, but rose sharply between pH 3.5
and pH 4.5, and remained at a plateau level between pH 4.5 and pH 9.0.
A similar profile was seen for [125I]IGF-I (result not
shown). In one experiment, there was a peak of binding activity for
both [125I]IGF-I and [125I]IGF-II by
rhIGFBP-2 at pH 4.0 that declined to a plateau of binding between pH
6.0 and pH 9.0 (result not shown). In contrast, peaks A-1 and A-2 were
devoid of any [125I]IGF-II-binding activity at pH 4.0,
and both displayed maximal binding for [125I]IGF-II in
the physiological pH range. The pH dependence of
[125I]IGF-I binding by peaks A-1 and A-2 was similar to
that of [125I]IGF-II binding (results not shown).
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compares the results of
competitive binding between [125I]IGF-II and unlabeled
IGF-II for rhIGFBP-2 at pH 4.5 and 7.0 (Fig. 7a). Scatchard analyses
indicated 3- to 4-fold higher binding affinity at pH 4.5
(Ka = 2.1 ± 1.5 x 1011
M-1; mean ± SD; n = 2)
than at pH 7.0 (Ka = 5.8 ± 2.7 x
1010 M-1; n = 2). The binding
activities of peaks A-1 and A-2 were also compared (Fig. 7b) at pH 7.0
only, since binding was negligible at pH 4.5. Scatchard analysis showed
that the IGFBP-2 fragment preparations had similar binding affinities,
1 order of magnitude lower than that of intact IGFBP-2 [4.5 ±
2.8 x 109 M-1 (n = 3)
for peak A-1 and 6.5 ± 4.5 x 109
M-1 (n = 3) for peak A-2].
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, des(1, 2, 3, 4, 5, 6)-IGF-II displaced IGF-II
tracer from intact rhIGFBP-2 in a nonparallel manner, showing
approximately 10% the activity of IGF-II. In contrast,
des(1, 2, 3, 4, 5, 6)-IGF-II was totally inactive in displacing IGF-II tracer from
the peak A-2 IGFBP-2 preparation, indicating that the amino-terminal
hexapeptide of IGF-II, although clearly involved in the binding to
intact IGFBP-2, is an absolute requirement for binding to the
carboxy-terminal IGFBP-2 peptides.
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Discussion |
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IGFBP-2 appears to be by far the predominant IGFBP in human milk, as detected by ligand blot in this and previous studies (11, 25). At present, its biological significance in this fluid is not well understood. The principal origin of IGFBP-2 in milk is not known. In goats, plasma IGFBP-2 appears to account for a very small proportion of milk-borne IGFBP-2, suggesting that most of the IGFBP-2 in milk is synthesized in the mammary gland (28). The present study shows that apart from intact IGFBP-2, several truncated forms of the binding protein are also present in human milk. The failure to detect any proteolysis of IGFBP-2 tracer after incubation with milk suggests that the fragments are generated before secretion from the mammary gland. A report of proteolysis of IGFBP-2 to fragments of 14 and 22 kDa by porcine vascular smooth muscle cells (29) suggests that smooth muscle cells within mammary gland ducts may be involved in the formation of the IGFBP-2 fragments secreted in human milk. These fragments include amino-terminal peptides of approximately 24 kDa and carboxy-terminal peptides of approximately 14 kDa, which retain partial IGF-binding activity. One sequence identified in a 24-kDa peptide, which was not further characterized, represents des(1, 2, 3)-IGFBP-2, i.e. an amino-terminal truncation of three residues. Interestingly, an IGFBP-2 sequence representing truncation of the amino-terminal tripeptide was also reported for the rat protein isolated and sequenced from BRL-3A rat liver-derived cells (30). The functional consequence of this truncation, if any, remains to be determined.
Proteolysed fragments of other IGFBPs appear to have variable effects on IGF-I bioactivity. Potentiation of IGF-I bioactivity in some studies has suggested a gradual release of IGF-I from truncated IGFBPs with lowered IGF-I binding affinities or other mechanisms independent of IGF-I binding (31, 32). A 22-kDa IGFBP-5 fragment was found to have lost any effect on IGF-I-stimulated cell growth (33). More recently, a 16-kDa IGFBP-3 fragment was reported to inhibit IGF-I bioactivity to a similar extent as intact IGFBP-3 despite a total lack of IGF-I affinity (34).
Minor IGF-binding bands detected in peak A-1 and A-2 material at approximately 24 kDa by ligand blotting were not detected by IGFBP-2 immunoblot under conditions where the bands of approximately 14 kDa were intensely visible. Although, after more prolonged exposure, the 24-kDa bands did become faintly visible by immunoblot, it is clear that the IGFBP-2 immunoreactivity in the fragment preparations is virtually exclusively attributable to the peptides of approximately 14 kDa. As the IGF binding assays in solution depend on immunoprecipitation of bound complexes with the same IGFBP-2 antiserum, we conclude that only these major immunoreactive species contribute to detectable IGF-binding activity.
Mass spectrometry of a preparation that predominantly contained a peptide with a sequence beginning at Gly169 revealed species of 13,840 and 13,425 Da. The former value is close to the predicted size of 13,786 Da for IGFBP-2-(169–289), i.e. a fragment extending to the carboxy-terminal Gln residue, whereas the latter value suggests a species with a deletion or truncation of three or four residues. Based on the amino-terminal sequencing result, a fragment corresponding to IGFBP-2-(181–289), i.e. extending from Lys181 to the carboxy-terminus, with a predicted molecular mass of 12,502 Da was expected to be present at much lower abundance, but was not detected by mass spectrometry. Insufficient peak A-2 material was available for a similar analysis of this preparation. Interestingly, the peptide commencing at Lys181, which was relatively more abundant in peak A-2 than in peak A-1, appeared to have higher IGF-binding activity, as more IGF binding was detected in the peak A-2 preparation. This activity differs from that of intact IGFBP-2 in having a narrower pH optimum (unlike IGFBP-2, it shows no binding at pH 4–4.5), a reduction in IGF-II binding, and a considerable decrease in IGF-I binding. The lack of binding in the acidic pH range suggests that amino-terminal residues of IGFBP-2 must be responsible for this relatively high affinity IGF binding to the intact protein, whereas the optimal binding activity observed near neutral pH values suggests that the carboxy-terminal fragments may be active in biological fluids.
The binding affinities of the carboxy-terminal fragments for IGF-II, although 10-fold lower than that of intact IGFBP-2, are 7- to 20-fold higher than the binding affinities of the type I IGF receptor for IGF-I and IGF-II (35). Thus, the IGFBP-2 fragments could potentially compete with the type I IGF receptor for IGF-I and IGF-II. Although any IGF-IGFBP-2-fragment complexes would be expected to dissociate in the acidic conditions of the stomach, it is possible that they may reassociate in the small intestine where the pH is higher. There is also evidence that intragastric pH is near neutrality in the newborn, and gastric acid secretion may take weeks to mature (36). It is thus possible that milk-borne IGFBP-2 and its fragments may retain some IGF-binding activity in the stomach of young infants.
The milk peptides identified in this study resemble an IGFBP-2 peptide identified in culture medium conditioned by the rat liver-derived cell line, BRL-3A (37). This peptide had a sequence identical to that of a rat IGFBP-2 peptide commencing at Met148 of the mature protein, equivalent to Met166 of hIGFBP-2 (23, 38, 39), indicating a maximal size of 14 kDa if it extends to the carboxy-terminal Gln270. However, its apparent size on nonreduced electrophoretic analysis was reported to be greater than 20 kDa, suggesting possible dimerization and perhaps also accounting for the minor component of apparent molecular mass 24 kDa seen with prolonged exposure in some of our electrophoretograms. Like the milk IGFBP-2 fragments, the rat peptide retained significant IGF-I and IGF-II binding, although with a reported apparent affinity loss of 5- to 15-fold (37). In contrast, IGFBP-2 has been shown to be proteolyzed to fragments of 14 and 22 kDa in porcine vascular smooth muscle cells; IGF binding to these peptides was not observed by ligand blot analysis of unconcentrated cultured medium (40).
This study provides novel information about interacting domains of IGF-II and IGFBP-2. Although the importance of amino-terminal residues of IGF-I and IGF-II interacting with IGFBP-2 and other IGFBPs is well recognized (24, 41, 42, 43), the binding determinants on the IGFBPs are not well delineated. We observed that whereas des(1, 2, 3, 4, 5, 6)-IGF-II shows a somewhat decreased binding to intact IGFBP-2, its binding to the carboxy-terminal IGFBP-2 peptide is unmeasurably low. This indicates that the amino-terminal hexapeptide of IGF-II is essential for interaction with the carboxy-terminal domain of IGFBP-2. Given the considerable structural conservation among the members of the IGFBP family, this observation suggests that, as a generalization, amino-terminal B domain residues of the IGFs are likely to interact with the carboxy-terminal domains of the IGFBPs.
In summary, we have described naturally occurring carboxy-terminal peptides derived from IGFBP-2 that retain considerable affinity for IGF-I and IGF-II at neutral pH, although they have lost the pH 4 binding activity observed for intact IGFBP-2. IGF-II binding to the truncated IGFBP-2 shows an absolute requirement for the amino-terminal IGF hexapeptide, indicating that these IGF residues interact with the IGFBP-2 carboxy-terminal domain. The presence of biologically active, low molecular mass IGF-binding species in milk may represent a means by which the growth and function of the neonatal gut could be regulated. Whereas intact IGFBP-2 inhibits IGF-I-stimulated growth of intestinal epithelial cells (44), the effect of amino-terminal truncation of the binding protein remains to be determined.
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Footnotes |
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Received March 18, 1997.
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 C. K. Buckway, E. M. Wilson, M. Ahlsen, P. Bang, Y. Oh, and R. G. Rosenfeld Mutation of Three Critical Amino Acids of the N-Terminal Domain of IGF-Binding Protein-3 Essential for High Affinity IGF Binding J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4943 - 4950. [Abstract] [Full Text] [PDF]
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G. R. Devi, D.-H. Yang, R. G. Rosenfeld, and Y. Oh Differential Effects of Insulin-Like Growth Factor (IGF)-Binding Protein-3 and Its Proteolytic Fragments on Ligand Binding, Cell Surface Association, and IGF-I Receptor Signaling Endocrinology, November 1, 2000; 141(11): 4171 - 4179. [Abstract] [Full Text] [PDF]
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 R. C. Baxter Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E967 - E976. [Abstract] [Full Text] [PDF]
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V. Hwa, Y. Oh, and R. G. Rosenfeld The Insulin-Like Growth Factor-Binding Protein (IGFBP) Superfamily Endocr. Rev., December 1, 1999; 20(6): 761 - 787. [Abstract] [Full Text]
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V. C. Russo, G. Rekaris, N. L. Baker, L. A. Bach, and G. A. Werther Basic Fibroblast Growth Factor Induces Proteolysis of Secreted and Cell Membrane-Associated Insulin-Like Growth Factor Binding Protein-2 in Human Neuroblastoma Cells Endocrinology, July 1, 1999; 140(7): 3082 - 3090. [Abstract] [Full Text]
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800
ml) was pumped at 0.5 ml/min onto an agarose-IGF-I affinity column.
Bound protein was eluted using 0.5 M acetic acid, pH 3, at
0.5 ml/min. Fractions of 1 ml were assayed by IGFBP-2 RIA. b,
Absorbance profile at 280 nm of reverse phase HPLC fractionation of
human milk IGFBP-2. The IGF-I affinity column eluant containing peak
IGFBP-2 immunoreactivity was purified by reverse phase HPLC on a
C18 column eluted with a 15–60% CH3CN
gradient in 0.1% trifluoroacetic acid over 30 min at 1.5 ml/min.
Fractions of 1 ml were collected. Peaks A and B, as indicated, were
subjected to further analysis. c, IGFBP-2 immunoreactivity of peak
fractions from HPLC of human milk. The fractions from panel b were
assayed for IGFBP-2 by RIA. Most of the IGFBP-2 immunoreactivity RIA
was detected in peak B, which contained substantially less protein than
peak A.
, peak A-1; 
, peak A-2.
, rhIGFBP-2
at pH 7.0; 
, A-2, unlabeled
des(1–3)-IGF-II.