ABSTRACT'. IGF-I, IGF-11, and their binding proteins (BP) were studied in sera obtained by direct puncture of umbilical cords in utero between 20 and 37 wk of gestation in 103 normal fetuses and in 16 fetuses with intrauterine growth retardation, as well as in the cord blood of 37 normal newborns of 38-to 42-wk pregnancies. In normal fetuses, IGF-I levels were approximately 50 ng/mL and IGF-I1 levels approximately 350 ng/mL up to the 33rd wk of pregnancy. Thereafter, both increased to reach values two to three times higher at term. Correlations were found between fetal placental lactogen levels and those of IGF-I and IGF-11, which is consistent with the hypothesis that placental lactogen is involved in the regulation of IGF synthesis in the fetus. With weight (either measured at birth or deduced from echographical data) as index of fetal size, IGF-I levels were significantly ( p < 0.001) higher in fetuses with weights above the mean for gestational age than in fetuses with weights below the mean, whereas IGF-I1 levels were similar in the two groups. Similarly, IGF-I (but not IGF-11) levels in fetuses with intrauterine growth retardation were significantly lower than those in normal fetuses of the same age ( p < 0.01). These findings suggest that, during the latter months of intrauterine life, IGF-I (but not IGF-11) is involved in the control of fetal size. Total fetal BP concentrations were approximately '/J those of adults. The fetal electrophoretic profile obtained by Western-ligand blotting bore a strong resemblance to that of subjects with growth hormone deficiency. In newborns, the proportions of IGF-I and IGF-I1 associated with BP to form 150-kD complexes were considerably lower than those in adults, but similar to those in hypopituitary patients. It may be deduced from these findings that during fetal life, BP synthesis is adapted to increase the bioavailability of the IGF at a time when growth is at a maximum.

Western ligand blot analysis of the different molecular forms of insulin-like growth factor-binding protein IGF-BP) in serum and plasma samples from 89 pregnant women has revealed a marked decrease, after the second month of pregnancy, in the 41.5 and 38.5K species (which are the binding units of the 150K complex) as well as in the 24K form. There was also a slight decrease in the 34K form, the 30K form was unaffected, and additional 21.5 and 20K bands appeared. Cross-linking experiments demonstrated the disapperance of a 49K band which is characteristic of the 150K complex. The alterations of the electrophoretic profile of the BPs were accompanied by a decrease in binding activity of up to 90%. Gel filtration at pH 7.4 confirmed that the decrease was essentially attributable to changes in the 150K complex BPs: 1) material eluting in the 150K zone contained only one third of the binding activity, as opposed to three quarters in reference material; 2) radiocompetition experiments illustrated the loss of affinity for IGF-I and IGF-II of the BPs extracted from the 150K complex; 3) ligand blot analysis revealed, in contrast with the virtual disappearance of the 41.5 and 38.5K forms, the appearance of a broad indistinct band at 30K and additional bands at 21.5 and 20K. With immunoblotting, the anti-IGF-BP-3 antibody, which specifically recognizes the 41.5 and 38.5K species, cross-reacted with this 30K material. The alterations of the BPs appeared to be enzymatic. When pregnancy serum was mixed with reference serum, the 41.5, 38.5, and 24K forms contributed by the reference serum were markedly reduced after 30 min of incubation at 37 C. However, these alterations could be prevented by incubation at either 0 or at 37 C in the presence of EDTA or aprotinin and could be curbed in the presence of high concentrations of phenylmethylsulfonylfluoride. Unmixed reference serum incubated at 37 C yielded an unchanged BP profile. Incubation of pregnancy serum with hypopituitary serum, which has elevated levels of the 34 and 30K BPs, resulted in a marked decrease in the 41.5 and 38.5K forms, a slight alteration of the 34K form, and no change in the 30K form. These findings suggest that during pregnancy, enzymatic (probably protease) activity either appears or is significantly increased in the circulation, which specifically degrades some of the IGF-BPs.(ABSTRACT TRUNCATED AT 400 WORDS)

Circulating insulin‐like growth factors (IGFs) are bound to specific, high‐affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150000 (‘large’ complex) and 40000 (‘small’ complex). The large complex appears to be under growth‐hormone control and its proportions vary with those of the IGFs. Molecular heterogeneity among the binding proteins was revealed by polydcrylamide gel electrophoresis (SDS‐PAGE) of serum in which they were cross‐linked to 125I‐labelled IGF I or II. Out of the six specific bands observed, of 150000, 120000, 49000, 46000, 40000 and 37000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49000–37000‐Mr species may constitute the subunits of 150000‐Mr and/or 120000‐Mr IGF‐BP complexes. With electrophoresis followed by transfer onto nitrocellulose and incubation with either 125I‐labelled IGF I or II (western blot), the different binding proteins were identified per se. There were five molecular forms with Mr of 41 500, 38 500, 34000, 30000 and 24000. In normal serum the 41 500 and 38 500‐Mr forms were the major binding proteins. They appeared in both complexes, but were predominant in the large complex where they constitute the elementary binding units. These two proteins therefore bind to IGFs to form both ‘monomeric’ IGF‐BP and ‘oligomeric’ (IGF‐BP)n complexes. The 34000, 30000 and 24000‐Mr forms, by contrast, were visible only in the small complex. Different mechanisms appear to regulate the different binding proteins: in acromegalic serum the 41 500 and 38 500‐Mr forms were augmented and the 34000‐Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30000‐Mr form was augmented. With chromatofocusing, the 34000, 30000 and 24000‐Mr forms eluted in three peaks between pH 6.0 and 4.0, whereas the 41 500 and 38 500‐Mr forms eluted throughout the gradient, principally at pH 7.5 and 7.0. Competitive binding studies, done on binding proteins separated either by chromatofocusing or by SDS‐PAGE and transfer onto nitrocellulose, revealed different affinities for the IGFs among the different molecular forms. The 41 500 and 24 000‐Mr binding proteins preferentially bound IGF I and the 38 500, 34 000 and 30 000‐Mr proteins preferentially bound IGF II. Our findings demonstrate the molecular heterogeneity of the binding proteins and the existence of a relationship between their structure and their affinities for the IGFs. They also suggest that, apart from their function as IGF carriers, the binding proteins also play a modulating role in the interaction between IGFs and their target cells.

We have studied the relationships between the structure and affinity of two insulin-like growth factor-binding proteins (IGFBPs) purified from human cerebrospinal fluid (CSF). Competitive binding studies were performed using preparations of human recombinant IGF (rhIGF-I, rhIGF-II, and their labeled homologs) and the truncated variant form of IGF-I, rh-Des-(1-3)-IGF-I. One of these BPs, which is the most consistently detected in CSF, corresponds to IGFBP-2. The other is a new form whose N-terminal sequence we reported earlier, which we call the 32-30K BP on the basis of its electrophoretic migration. Comparisons were made with an IGFBP-1 preparation purified from amniotic fluid and with two BPs purified from human serum, which are homologous to the CSF BPs. The CSF BPs have particularly strong affinities for IGF-II. The estimated affinity constants (Ka) were 2 X 10(10) M-1 for IGFBP-2 and 10(11) M-1 for the 32-30K BP. These affinities were 15-20 and 70 times stronger than the respective affinities for IGF-I. The affinity of the 32-30K BP is the strongest among the BPs identified to date. The two BPs isolated from serum, which correspond to the 32-30K CSF BP and IGFBP-2, had affinities for IGF-II and IGF-I similar to those of the CSF BPs. IGFBP-1 had nearly identical affinities for the two IGFs of approximately 10(10) M-1. Des-(1-3)-IGF-I failed to bind to the CSF BPs, but bound to IGFBP-1, although with a 40-fold weaker affinity than IGF-I. From our data it would seem that IGFBP-1 has two classes of IGF-binding site, one of high and one of low (less than 10(9) M-1) affinity for both IGFs. The other two BPs, by contrast, each possess a predominant class of high affinity binding site for IGF-II. A second class of lower affinity (greater than 10(9) M-1) sites bind both IGF-I and IGF-II. In the case of the 32-30K BP, these preferentially bind IGF-II; in the case of IGFBP-2, their binding of the two IGFs is similar. These different types of binding site may play an important role in controlling the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

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In a variety of physio-pathological conditions, but also in the normal state, calcium-dependent serine protease(s) partially proteolyze proportions of circulating insulin-like growth factor binding protein-3 (IGFBP-3). This occurs without disrupting the 150-kilodalton (kDa) complexes in which IGFBP-3 carries of most of the IGF-I and IGF-II in serum. In this work we show that the 150-kDa complex is functionally altered during pregnancy, which is when the largest proportion of IGFBP-3 is proteolyzed. After preincubation at 4 C with [125I]IGF-I or -II with or without 1 microgram unlabeled IGF-I or -II, pooled plasma samples were gel filtered at pH 7.4. Larger proportions of labeled IGFs were found in the 150-kDa complexes of the third trimester pregnancy plasma pool than in those of the normal plasma pool, suggesting increased binding capacity. Nevertheless, competitive binding experiments using [125I]IGF-I and -II and IGFBP-3 preparations from each of the plasma pools showed that the competitive potencies of IGF-II and especially IGF-I were lower in pregnancy IGFBP-3 than in normal IGFBP-3. Scatchard analysis revealed a 2-fold loss of affinity for IGF-II and a 10-fold loss for IGF-I compared with that for normal plasma IGFBP-3. In studies at 37 C of the kinetics of dissociation of [125I]IGF-I and -II bound to IGFBP-3, IGF-II was dissociated 6 times faster, and IGF-I 10 times faster from pregnancy plasma IGFBP-3 than from normal plasma IGFBP-3. After incubation of individual plasma samples at 37 C and gel filtration at room temperature (in the presence of EDTA), IGFs were assayed in the three circulating pools (150-kDa and 40-kDa complexes and free IGFs), revealing a redistribution of pregnancy plasma IGFs. The proportion of total IGF-I in free form was nearly three times greater in pregnancy than in normal plasma (11.4% vs. 4.1%, P < 0.005), whereas that of free IGF-II was slightly smaller (1.5% vs. 2%). In the 150-kDa complexes, the proportion of total IGF-I was significantly smaller in pregnancy than in normal plasma, and that of IGF-II was greater. In the 40-kDa complexes, the proportion of total IGF-II was significantly smaller. The mean ratios of molar concentrations of free IGF-I/IGF-II were 0.43 in normal plasma and 2.23 in pregnancy plasma (P < 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)

The insulin-like growth factor (IGF)-binding proteins present in the human serum and various biological media have been characterized by several methods: gel filtration, sucrose gradient sedimentation, polyacrylamide gel electrophoresis and chromatofocusing. Several forms have been identified with molecular weights of ∼ 42,000, 39,000, 34,000, 30,000 and 24,000 daltons. Results of competitive binding studies suggest that the different forms of binding proteins have different affinities for IGF-I and IGF-II. The influence of various hormones and pathophysiological conditions on the biosynthesis of the binding proteins has been investigated.