The uteroglobin fold.

Uteroglobin (UTG) forms a fascinating homodimeric structure that binds small- to medium-sized ligands through an internal hydrophobic cavity, located at the interface between the two monomers. Previous studies have shown that UTG fold is not limited to the UTG/CC10 family, whose sequence/structure relationships are highlighted here, but can be extended to the cap domain of Xanthobacter autotrophicus haloalkane dehalogenase. We show here that UTG fold is adopted by several other cap domains within the alpha/beta hydrolase family, making it a well-suited "geode" structure allowing it to sequester various hydrophobic molecules. Additionally, some data about a new crystal form of oxidized rabbit UTG are presented, completing previous structural studies, as well as results from molecular dynamics, suggesting an alternative way for the ligand to reach the internal cavity.

Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy.

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)

Regulation of binding proteins for insulin-like growth factors (IGF) in humans. Increased expression of IGF binding protein 2 during IGF I treatment of healthy adults and in patients with extrapancreatic tumor hypoglycemia.

UNLABELLED: Insulin-like growth factors (IGFs) in blood form two complexes with specific binding proteins (BPs): a large, growth hormone (GH)-dependent complex with restricted capillary permeability, and a smaller complex, inversely related to GH, with high turnover of its IGF pool and free capillary permeability. The distribution of BPs and of IGFs I and II between these complexes was studied in sera from healthy adults treated with IGF I or/and GH and from patients with extrapancreatic tumor hypoglycemia. Like GH, IGF I administration raises IGF I and two glycosylation variants of IGFBP-3 in the large complex, but unlike GH drastically reduces IGF II. During IGF I infusion, IGFBP-3 appears in the small complex whose IGFBP-2 and IGF I increase three- to fivefold and fivefold, respectively. GH treatment, associated with elevated insulin levels, suppresses IGFBP-2 and inhibits its increase owing to infused IGF I. The small complex of tumor sera contains increased amounts of IGFBP-2 and -3, and two- to threefold elevated IGF II. CONCLUSIONS: low GH and/or insulin during IGF I infusion and in extrapancreatic tumor hypoglycemia enhance expression of IGFBP-2 and favor partition of IGFBP-3 into the small complex. Free capillary passage and high turnover of its increased IGF I or II pools may contribute to compensate for suppressed insulin secretion during IGF I infusion or to development of tumor hypoglycemia.

Molecular forms of serum insulin-like growth factor (IGF)-binding proteins in man: relationships with growth hormone and IGFs and physiological significance.

Insulin-like growth factor-I (IGF-I) and IGF-II are associated in the blood with specific binding proteins (BPs), forming complexes that elute in gel filtration with estimated mol wt around 40 and 150 kD. The latter appears to be under GH control. Five molecular forms of BP (41.5, 38.5, 34, 30, and 24 kD) have been identified by Western blotting using 125I-labeled IGF. All five forms are present in the smaller complexes, but only the 41.5- and 38.5-kD forms are found in the larger complexes. In this study immunoblotting showed that the 41.5- and 38.5-kD forms were recognized by antibodies directed against the GH-dependent BP purified from human plasma, and the 30-kD form was recognized by antibodies directed against the BP purified from amniotic fluid. The 34- and 24-kD forms proved to be immunologically unrelated to the other three. In sera with large quantities of the 41.5- and 38.5-kD forms, an additional band was often observed immediately ahead of the migration front of the 30 kD band. This was recognized by the anti-GH-dependent BP antibody and probably corresponds to a degradation product of the 41.5- and 38.5-kD BPs. Serum 41.5- and 38.5-kD BPs have been found to be elevated in acromegaly, where GH hypersecretion causes increased IGF-I levels, and diminished in cases of genetic or idiopathic GH deficiency and defects of the GH receptor (Laron's syndrome), where both IGF-I and IGF-II are decreased, as well as in Pygmy adults and children who have isolated IGF-I deficiency. In all of these conditions, the proportions of the 34- and 30-kD forms were inversely related to those of the 41.5- and 38.5-forms. Under treatment, the BP profiles tended to return to normal. In cases of GH deficiency caused by a tumor, the BP profiles resembled those of hypopituitary or normal serum, depending on whether IGF levels were diminished or normal. It, therefore, seems that BP synthesis is coordinated with IGF-I synthesis and may not be directly GH dependent. The results of neutral pH gel filtration analysis of hypopituitary (idiopathic and tumoral) and normal sera point to a relationship between the levels of circulating IGFs and those of the 150-kD IGF-BP complex whose binding units are the 41.5- and 38.5-kD BPs. It, therefore, seems that the 150-kD complex controls the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation from human cerebrospinal fluid of a new insulin-like growth factor-binding protein with a selective affinity for IGF-II.

In biological fluids IGF-I and IGF-II are bound to specific, high-affinity binding protein (BPs). Two human BPs have been isolated, one from serum, which is GH-dependent, the other from amniotic fluid (AF BP), and their cDNAs have recently been cloned. We report here the isolation of another, new species from cerebrospinal fluid (CSF) where this BP predominates. The protein was purified to homogeneity by a four-step procedure: gel filtration, chromatofocusing, hydrophobic-interaction chromatography and reverse-phase chromatography. Thereafter, SDS-polyacrylamide gel electrophoresis gave an Mr of 34,000 (non-reduced), chromatofocusing gave an isoelectric point of 5.0m and its affinity for IGF-II (3 x 10(10) M-1) was 10 times that for IGF-I. The N-terminal amino acid sequence of the first 15 residues determined in a BP preparation from the CSF of children was Leu-Ala-Pro-Gly-(/)-Gly-Gln-Gly-Val-Gln-Ala-Gly-Ala-Pro-Gly. A similar sequence was found for adult CSF, apart from residues 12 and 13 (-Leu-Leu-). These are highly analogous with the sequences starting from residue 69 of the GH-dependent BP, and from residue 61 of the AF BP. The new BP isolated is therefore related to, but distinct from, the other human BPs.

Purified preparations of the amniotic fluid-derived insulin-like growth factor-binding protein contain multimeric forms that are biologically active.

The insulin-like growth factors (IGFs) appear to be secreted into interstitial fluids by many cell types, along with specific, high affinity binding proteins (IGF-BPs). These proteins, therefore, have the potential to bind IGF-I and -II and modify their ability to interact with specific cell surface receptors In these studies we report the detection of high mol wt, multimeric forms of one form of IGF-BP that has been purified from human amniotic fluid. The multimeric forms, which are either not or barely detectable in native amniotic fluid, are the result of intermolecular disulfide bond formation and can be reduced to a monomeric form by exposure to dithiothreitol. After reduction, the multimers are reduced to either monomeric or dimeric forms, as detected by Western blotting. The multimers can be separated from monomeric and dimeric forms by gel filtration chromatography. The purified multimers were fully biologically active in potentiating the effect of IGF-I on porcine aortic smooth muscle cell DNA synthesis. The monomeric form was also bioactive. No significant differences in the affinity of the monomeric and multimeric forms for IGF-I or -II could be detected. In summary, multimeric forms of this form of IGF-BP are detected during purification. The formation of these multimers is through intermolecular disulfide bonds and does not disrupt IGF binding or potentiation of the cellular growth response to IGF-I. These findings indicate that these higher mol wt forms may be fully active in biological test systems.

Production of insulin-like growth factors and their binding proteins by rabbit articular chondrocytes: relationships with cell multiplication.

Articular chondrocytes from 2- to 3-month-old rabbits were cultured in serum-free medium supplemented with fibroblast growth factor. The effects were studied of GH, insulin-like growth factors (IGFs), and insulin on the production of IGF-I, IGF-II, and their binding proteins (BPs) and on cell multiplication. In the control culture medium, IGF-I levels were about one fifth those of IGF-II. Western blot analysis of the BPs revealed a predominant 30K form and 24K and 20K forms which appeared inconsistently and in small quantities. Ten to 100 ng/ml human GH had no mitogenic effect, and even had a slightly inhibitory effect. IGF-I at 10 ng/ml stimulated cell multiplication above the control level by 41% and at 50 ng/ml by 74%, whereas the mean increase obtained with IGF-II (10 and 50 ng/ml) was only 19%. At the same doses, insulin had no effect, but at 5 micrograms/ml it stimulated cell multiplication by a mean of 67%. There was a positive correlation between cell number and release into the medium of both IGF-I (r = 0.86) and IGF-II (r = 0.77). Neither IGF-I nor IGF-II production was affected by GH. Insulin (5 micrograms/ml) increased IGF-I production by a factor of 2.6, but increased IGF-II production by a factor of only 1.4. Under the various conditions of culture with different doses of GH and insulin, cell multiplication, relative to the control value was positively correlated to the IGF-I/IGF-II production ratio (r = 0.77). It would, therefore, seem that IGF-I secreted by the chondrocytes may stimulate their own proliferation. When IGFs or insulin were added to the culture medium, changes in the electrophoretic profiles of the BPs included an increase in the 30K form and an increase in or the appearance of the 24K and 20K forms. Ten and 50 ng/ml IGF-I or IGF-II had effects equal to or greater than those induced by 5 micrograms/ml insulin. These results indicate that the syntheses of BPs and IGFs are coordinated and that IGFs may be implicated in the control of the synthesis of their BPs.

Insulin-like growth factor (IGF) and IGF-binding proteins: comparison of human serum and lymph.

The extent to which the association between insulin-like growth factors (IGFs) and their specific binding proteins (BPs) prevents their crossing the capillary barrier was studied by comparing their distribution in serum with that in samples of lymph collected from the lower leg of five subjects undergoing radiographical investigation of the lymphatic system. The IGF concentrations in lymph were 10-30% of the corresponding serum levels, and in each subject the ratios of IGF-I and IGF-II in the lymph to those in the serum were similar. Western blot analysis of the BPs revealed that the five molecular forms identified in serum also were present in lymph, but in significantly smaller quantities. The 41.5K and 38.5K forms, which constitute the binding units of the large complex (approximately 150K) of serum and are also capable of binding IGFs in monomeric form, were present in smaller amounts than the 34K, 30K and 24K forms, which belong specifically to the small complex (approximately 40K) of serum. The BPs extracted from lymph were similar to those of the small complex, with a preferential affinity for IGF-II and only half of the affinity for IGF-I of the BPs extracted from serum. With neutral pH gel filtration of lymph, more than 90% of IGFs and binding activity eluted with the material in the area of the 40K zone. These data indicate that the 150K IGF-BP complexes do not cross the capillary barrier, whereas the 40K complexes do. The function of the former may be to provide a reservoir and buffering action of the IGFs, whereas the latter may be involved in the transport of the IGFs to their target cells.

Heterogeneity of insulin-like growth factor binding proteins and relationships between structure and affinity. 1. Circulating forms in man.

Circulating insulin-like growth factors (IGFs) are bound to specific, high-affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150,000 ('large' complex) and 40,000 ('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 polyacrylamide 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 150,000, 120,000, 49,000, 40,000 and 37,000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49,000-37,000-Mr species may constitute the subunits of 150,000-Mr and/or 120,000-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, 34,000, 30,000 and 24,000. 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 34,000, 30,000 and 24,000-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 34,000-Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30,000-Mr form was augmented. With chromatofocusing, the 34,000, 30,000 and 24,000-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.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of insulin-like growth factor binding proteins between structure and affinity. 2. Forms released by human and rat liver in culture.

Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins. In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes. In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40,000, which is similar to that of the 'small' complex of serum. Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S. Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF I revealed molecular heterogeneity. Three specific bands corresponding to Mr 46,000, 40,000 and 37,000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum. SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000, identical to those seen in serum. The relative concentrations of the 41,500 and 38,500-Mr forms varied from one medium to another, but the 34,000 and 30,000-Mr forms were regularly more abundant in the liver culture media than in normal serum. The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources). The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41,500 and 38,500-Mr forms, which are the only binding units of the serum 'large' complex (150,000 Mr), indicates that these two binding proteins are capable of binding IGFs to form 'monomeric' IGF-BP complexes. Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43,000 and 39,000 and three between 32,000 and 24,000. Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41,500 and 38,500-Mr forms were more abundant in the adult and the 34,000 and 30,000-Mr forms more abundant in the foetus.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of an insulin-like growth factor-binding protein in human cerebrospinal fluid with a selective affinity for IGF-II.

Human cerebrospinal fluid (CSF) has been found to contain several different molecular forms of IGF-specific binding proteins (BPs). Qualitatively, they are similar to those present in serum, although their relative proportions are very different, as well as to those present in the culture media of brain tissue from which these BPs presumably arise. One particular form of BP is predominant in CSF. It has an Mr of 34,000, as estimated by SDS-polyacrylamide gel electrophoresis followed by transfer onto nitrocellulose, and an isoelectric point around 5.0 based on chromatofocusing. It has a selective affinity for IGF-II (approximately 4 X 10(10) M-1) as shown by competitive binding experiments in which biosynthetic IGF-I was about 40-times less potent than native IGF-II in displacing 125I-labelled IGF-II. These findings are in agreement with the preponderance of IGF-II in nervous tissue and in CSF and suggest that this BP plays an important role in the interaction of IGF-II with its target cells.

Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies.

A nitrocellulose gel transfer technique has been adapted to study the insulin-like growth factor (IGF) binding proteins of human serum. Normal and hypopituitary sera were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose or nylon membrane. Nonidet-P40 (3%) and Tween 20 (0.1%) were required for quenching and to allow detection of the IGF binding proteins by autoradiography after overlay with either 125I-labeled IGF I or IGF II. Several forms of IGF binding protein have been identified with molecular weights of 41,500, 38,500, 34,000, 30,000, and 24,000. Titration and competitive binding studies with IGF were performed on the transferred IGF binding proteins, indicating that binding proteins isolated by this technique can be characterized.

Somatomedin (insulin-like growth factors)-binding proteins. Molecular forms and regulation.

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 approximately 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.

Evidence for production by the liver of two IGF binding proteins with similar molecular weights but different affinities for IGF I and IGF II. Their relations with serum and cerebrospinal fluid IGF binding proteins.

Studies of the competitive binding of IGF I and IGF II and 125I-labelled IGF I and IGF II to the specific binding proteins (BPs) produced by the liver in culture suggest that two BPs exist, one with a selective affinity for IGF I and the other with a selective affinity for IGF II. The ratio of the former BP to the latter appeared to be higher in the culture medium of young rat livers and of fetal human livers than that in the culture medium of adult human livers. The two BPs form complexes with their IGFs with the same apparent molecular weight of approximately 40K. With gel filtration of adult human serum both types of BP eluted with the greater than 100K and the 40-70K molecular weight material. The BP with the selective affinity for IGF II predominated in the 40-70K material as well as in the cerebrospinal fluid of adult human subjects.

[Biosynthesis and hormonal regulation of IGF (insulin-like growth factors, or somatomedins). Experimental and clinical studies (author's transl)]. / Biosynthèse et régulation hormonale des IGF ("insulin-like growth factors" ou somatomédines). Etudes expérimentales et cliniques.

Rat liver explants in culture release into the culture medium IGF and their carrier which form a complex with an apparent molecular weight of similar to or approximately 40,000. With gel filtration in acetic acid the IGF are separated from their carrier and each can then be measured by radioligand assay. In terms of their hormonal control "in vitro", GH, insulin and cortisol have an independent action on each. Pituitary and various brain tissues in organ culture also produce IGF and their carrier. The "in vitro" regulation of the IGF and their carrier has been investigated through assays of their levels in human serum under normal and pathological conditions, such as hypopituitarism, acromegaly, coeliac disease, Cushing's syndrome and idiopathic short or tall stature.