High molecular weight insulin-like growth factor binding protein complex. Purification and properties of the acid-labile subunit from human serum

In the human circulation, the insulin-like growth factors (IGFs) circulate as part of a growth hormone-dependent 125- to 150-kDa complex. This complex has been postulated to contain, in addition to IGFs and one or more IGF-binding proteins, an acid-labile subunit (ALS) which does not itself bind IGFs. In this study, the ALS has been purified 1600-fold from human serum, and its binding properties have been examined. Fresh serum was fractionated on DEAE-Sephadex, and active fractions (determined by radioimmunoassay) were purified by affinity chromatography on an IGF-agarose column saturated with the plasma IGF-binding protein BP-53. After further high performance anion exchange chromatography, an ALS preparation was obtained which contained only an 84-86-kDa protein doublet, converting to a single 70-kDa band on N-glycanase treatment, and having an amino-terminal sequence unrelated to IGF-binding proteins or receptors. Pure ALS formed a complex with BP-53 (Ka approximately 5 x 10(8) M-1), immunoprecipitable by anti-BP-53 antiserum, only in the presence of IGF-I or IGF-II. This complex appeared at approximately 150 kDa on high performance gel chromatography. Pure ALS had no intrinsic IGF-binding activity and no effect on the binding of IGF-I or IGF-II to BP-53. These studies suggest that formation of the high molecular weight IGF-binding protein complex requires ALS, BP-53, and IGF.     

Zebrafish IGF Genes: Gene Duplication,Conservation and Divergence,and Novel Roles in Midline and Notochord Development

Insulin-like growth factors (IGFs) are key regulators of development, growth, and longevity. In most vertebrate species including humans, there is one IGF-1 gene and one IGF-2 gene. Here we report the identification and functional characterization of 4 distinct IGF genes (termed as igf-1a, -1b, -2a, and -2b) in zebrafish. These genes encode 4 structurally distinct and functional IGF peptides. IGF-1a and IGF-2a mRNAs were detected in multiple tissues in adult fish. IGF-1b mRNA was detected only in the gonad and IGF-2b mRNA only in the liver. Functional analysis showed that all 4 IGFs caused similar developmental defects but with different potencies. Many of these embryos had fully or partially duplicated notochords, suggesting that an excess of IGF signaling causes defects in the midline formation and an expansion of the notochord. IGF-2a, the most potent IGF, was analyzed in depth. IGF-2a expression caused defects in the midline formation and expansion of the notochord but it did not alter the anterior neural patterning. These results not only provide new insights into the functional conservation and divergence of the multiple igf genes but also reveal a novel role of IGF signaling in midline formation and notochord development in a vertebrate model.     

类胰岛素生长因子信号途径及其对脊椎动物生长和发育的调控作用:以斑马鱼为模式动物的新进展

类胰岛素生长因子(包括IGF-I和IGF-Ⅱ)是进化上保守性很强的多肽。IGFs对脊椎动物的生长和早期发育有极其重要的调控作用。IGF的生理作用是由IGF受体中介并受几个分泌性的IGF结合蛋白调节。本文主要介绍了以斑马鱼为模式动物，用基因敲除、转基因动物和培养细胞系等现代实验方法对IGF信号途径的最新研究进展，综述了IGF配体、受体和结合蛋白的结构特点、基因表达和调节和生物学功能。此外，也对斑马鱼作为模式动物的许多优点进行了探讨。     

Binding proteins for insulin-like growth factors in adult rat serum. Comparison with other human and rat binding proteins

Insulin-like growth factor (IGF) binding protein has been purified from adult rat serum by affinity chromatography on agarose-IGF-II and high performance reverse-phase chromatography. The final preparation contains two components, of apparent molecular mass 50 and 56 kDa nonreduced, or 44 and 48 kDa reduced, both of which specifically bind IGF-I and IGF-II. Competitive binding data indicate association constants of 5-10 X 10(10) l/mol for both IGFs, with a slightly higher affinity for IGF-II than IGF-I. Amino-terminal sequence analysis yields a unique sequence, identical in 11 of the first 15 amino acids with that of a human plasma IGF binding protein (Martin, J. L., and Baxter, R. C. (1986) J. Biol Chem. 261, 8754-8760), and with slight homology to other human and rat IGF binding proteins characterized to date. By analogy with the binding protein from human plasma, it is likely that the rat protein is part of the growth-hormone dependent complex which appears to carry most or all of the circulating IGFs.     

Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When 125I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, whilst the remaining three (MW 37 K, 34K and 18 K) appeared as minor bands only. Inhibitor bioactivity, following further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands. These data describe, for the first time, the structural nature of the IGF inhibitor protein and raise important questions regarding the relationship of the inhibitor to the primary IGF-binding subunit of the native high MW IGF carrier protein of serum.     

High levels of "complexed" interleukin-6 in human blood.

The biochemical nature of endogenous interleukin-6 (IL-6) as it exists in human serum or plasma was investigated. Serum from a patient following bone marrow (BM) transplantation and fresh plasma samples from patients with epidermolysis bullosa or psoriasis, as well as from normal volunteers, were fractionated through G-200 columns and each of the eluted fractions assayed for IL (interleukin)-6 content using enzyme-linked immunosorbent assays (ELISAs) based on the monoclonal antibody (mAb) pairs IG61/5IL6 or 4IL6/5IL6 and in the B9 hybridoma growth factor bioassay. The IG61/5IL6 ELISA and the B9 assay detected IL-6 in BM serum almost exclusively of molecular mass approximately 20 kDa. In contrast, the 4IL6/5IL6 ELISA detected strong IL-6 immunoreactivity in complexes of size 100-150 and 400-500 kDa. IL-6 present in the 100-150- and 400-500-kDa complexes was purified by immunoaffinity chromatography through a 5IL6 mAb column. The 5IL6 mAb immunoaffinity column eluate of the respective pools from BM serum contained IL-6 at concentrations approaching 1 microgram/ml as characterized by Western blotting. Sufficient IL-6 and associated proteins were purified by 5IL6 mAb immunoaffinity column chromatography of the 100-150-kDa complex from 0.8 ml of BM serum to allow (i) verification of three of the polypeptides as IL-6 by amino-terminal sequencing (estimate of IL-6 in original serum sample: 5-10 micrograms/ml), (ii) identification by amino acid sequencing of the "associated" proteins as complement factor C3b (carboxyl-terminal of the alpha-chain), complement factor C4b (gamma-chain), C-reactive protein, and albumin, and (iii) detection of an "associated" polypeptide consistent with the soluble IL-6 receptor. Taken together, these data establish that IL-6 is present at unexpectedly high concentrations in human blood in novel biochemical complexes that include other plasma proteins, which in turn, can camouflage IL-6 immunoreactivity and bioactivity as measured in conventional assays.     

Purification and properties of human placental NADPH-cytochrome P-450 reductase

Human placental NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 microM 4-androstene-3,17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35-60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2',5'-ADP-Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolysis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 +/- 0.7 mumol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.     

Presence of IGF-1-like peptides in the neuroendocrine system of the Atlantic hagfish, Myxine glutinosa (Cyclostomata): evidence derived by chromatography, radioimmunoassay and immunohistochemistry

By the use of radioimmunoassay and chromatography peptides related to insulin-like growth factor 1 (IGF-1) have been identified in the cylostomian species Myxine glutinosa. IGF-1-like-immunoreactivity was detected in serum as well as in brain, intestine, pancreas and liver. After acid gel chromatography, the IGF-1-like immunoreactivity eluted as one major peak, with an apparent molecular weight of between 2-4 kDa. When the same antiserum was applied immunohistochemically, IGF-1-like-immunoreactivity was observed in endocrine cells of the mucosal epithelium throughout the primitive intestinal tube. These cells were of the open type and occurred in small clusters. In addition, the majority of the endocrine cells of the pancreas of Myxine displayed IGF-1-like-immunoreactivity. In some of the specimens investigated IGF-1-like-immunoreactive perikarya and fibers were observed on all levels of the brain. Distribution patterns and densities of the IGF-1-like-immunoreactive structures in Myxine correlated with the measurements obtained by radioimmunoassay. Absorption studies with insulin- and IGF-related peptides as well as with crude extracts and the peak material obtained after gel chromatography indicated that the IGF-1-like peptides in Myxine are different from mammalian and non-mammalian insulins as well as from mammalian IGF-1. Generally, the results suggest a long phylogenetic history of IGF-1-like peptides and indicate their fundamental functional impact in all vertebrates.     

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)     

Peptides related to insulin-like growth factor 1 in the gastro-entero-pancreatic system of bony and cartilaginous fish.

Evidence for the presence of peptides, related to insulin-like growth factor 1 (IGF-1) has been obtained in serum and various organs of representatives of osteichthyes and chondrichthyes, i.e., the bony fish Myoxocephalus (Cottus) scorpius and the cartilaginous fish Raja clavata. The peptides were identified by means of gel chromatography and an IGF-1 radioimmunoassay. IGF-1-like immunoreactivity was detected in three different apparent molecular mass forms, i.e., 17 kDa, 6 kDa and 4 kDa, the occurrence of which seemed to depend on the species. When the same antiserum was used immunohistochemically, IGF-1-like immunoreactivity was observed in endocrine cells of the open type in the intestinal mucosal epithelium. These cells exhibited distinct and species-specific distribution patterns. Endocrine cells of the pancreas as well as epithelial cells of the pancreatic duct also showed IGF-1-like immunoreactivity. Occasionally, IGF-1-like immunoreactivity was observed also in interstitial cells. The distribution patterns and densities of the IGF-like immunoreactive cells correlated with the results obtained by radioimmunoassay of the crude extracts. Absorption studies indicated that the IGF-1-like peptides observed differ from mammalian and submammalian insulins as well as from mammalian IGF-1.     

Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells

We have purified recombinant human interleukin 4 (huIL-4), formerly named B-cell stimulatory factor-1, from supernatants of COS-7 monkey kidney and L-929 cells transfected with the cDNA for huIL-4. The purified protein exhibited a specific activity of 2.6 X 10(7) units/mg in a T-cell proliferation assay and consisted of multiple components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibiting Mr values of 15,000, 18,000, and 19,000. All forms of huIL-4 eluted on gel filtration chromatography with an apparent Mr of 22,000. Gas-phase microsequencing identified 26 and 8 amino acid residues at the N and C termini, respectively, all of which were consistent with the cDNA sequence. The site of processing of the signal sequence was found to occur between Gly-24 and His-25. Incubation with N-glycanase converted the 18- and 19-kDa variants to a 15-kDa form. Treatment with endo-beta-N-acetylglucosaminidase H reduced the molecular mass of the 18-kDa variant to 15 kDa, but did not have any apparent effects on the mass of the 19-kDa species. The removal of oligosaccharide by any of these treatments did not affect bioactivity in the T-cell proliferation assay. Neither O-glycanase nor endo-beta-N-acetylglucosaminidase D affected the molecular weight of any of these species. These data suggest that differences in carbohydrate structure account, at least in part, for the observed microheterogeneity.     

Characterization of insulin-like growth factor-binding proteins of porcine ovarian follicular fluid.

Using competitive ligand-binding studies, ligand blotting, and immunoprecipitation, we have characterized the insulin-like growth factor (IGF)-binding proteins (BPs) of porcine follicular fluid. Competitive ligand-binding studies revealed a preference of ovarian IGFBPs for IGF-II over IGF-I. Follicular fluid from small, 1-3-mm follicles had nearly twice the binding capacity for IGFs as that from large, 6-10-mm follicles. Ligand blots of porcine follicular fluid resolved 5 major bands of IGF-binding activity having apparent molecular sizes of 44, 40, 34, 29, and 22 kDa. The 40-44-kDa bands were immunoprecipitated by an antibody to porcine IGFBP-3, the acid-stable subunit of the 150-kDa growth hormone-dependent IGF-binding protein complex of porcine serum. The 34-kDa band was immunoprecipitated by an antibody to rat IGFBP-2, the major IGF-binding protein found in fetal rat serum. To date we have been unable to immunoprecipitate the 29- and 22-kDa bands with any of the antibodies tested, including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGF-binding protein. The 40-44-kDa species (IGFBP-3) was the predominant form and was equally abundant in fluid from large and small follicles. In contrast, the smaller forms, including IGFBP-2 and the 29- and 22-kDa forms were significantly more prominent in fluid from small follicles. In view of other studies indicating a significant effect of IGFBPs on ovarian cell function, follicular IGFBPs may play an important role in the IGF autocrine/paracrine regulatory system of the ovary.     

Characterization of a novel insulin receptor from stingray liver

The insulin receptor from the liver of stingray, a cartilaginous fish, has characteristics which are in marked contrast to those of the mammalian insulin receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cross-linked, affinity-labeled stingray insulin receptor shows an apparent molecular mass of 210 kDa for the intact receptor. Reduction with mercaptoethanol resulted in no alteration in the apparent size of the stingray insulin receptor. Gel filtration studies of Triton X-100 solubilized stingray insulin receptor demonstrated an apparent Stokes radius of 7.6 nm. Ultracentrifugation sucrose gradient studies of cross-linked affinity labeled stingray receptor resulted in determination of a sedimentation coefficient of 13 S. Both of these parameters were greater than simultaneously obtained data for the human insulin receptor (7.2 nm and 11 S, respectively). Unlabeled insulin competed with binding of 125I-insulin and 125I-insulin growth factor (IGF) I with a half-maximal concentration of 1 nM for either. Unlabeled IGF I and II also competed, but were 4-5-fold less potent than insulin. It was found that not only did IGF I bind to the 210-kDa material, but both insulin and IGF I stimulated phosphorylation of a 210-kDa material which was immunoprecipitable by a polyclonal insulin receptor antibody. Elution of this material from the gel followed by hydrolysis and thin layer chromatography demonstrated that the 210-kDa material was specifically phosphorylated on tyrosine only. These data suggest that the insulin receptor from stingray liver is a dimer made up of 2 identical subunits of about 210 kDa size which contain both binding regions and insulin-stimulated tyrosine kinase. Specificity studies suggest that the stingray insulin receptor may represent a phylogenetic position prior to the evolutionary divergence of insulin and the insulin-like growth factors.     

Short-term effects of anastrozole treatment on insulin-like growth factor system in postmenopausal advanced breast cancer patients

Insulin-like growth factors (IGFs) play a fundamental role in cancer development by acting in both an endocrinal and paracrinal manner, and hormone breast cancer treatments affect the IGF system by modifying circulating growth factor levels. We evaluated total IGF-1, IGF-2, IGF binding protein (IGFBP)-1 and IGFBP-3 in the blood of 34 postmenopausal advanced breast cancer patients (median age 63 years, range 41–85) treated with anastrozole, a non-steroidal structure aromatase inhibitor (NSS-AI). The plasma samples were obtained at baseline, and after 2, 4, 8 and 12 weeks of treatment. The IGFs were quantitated by means of sensitive radioimmunoassays (RIAs). IGF-1 significantly increased during anastrozole treatment (baseline versus 12 weeks, P=0.031), IGF-2 showed a trend towards an increase, and IGFBP-1 constantly but not significantly decreased; IGFBP-3 did not seem to be affected at all. The anastrozole-induced changes in IGFs and IGFBP-1 appeared to be different in the patients receiving a clinical benefit from those observed in non-responders. We have previously shown that letrozole (a different type of NSS-AI) modifies blood IGF-1 levels, and the results of this study of the biological effects of anastrozole on the components of the IGF system confirm our previous observations.     

Isolation from adult human serum of four insulin-like growth factor (IGF) binding proteins and molecular cloning of one of them that is increased by IGF I administration and in extrapancreatic tumor hypoglycemia.

We have isolated four insulin-like growth factor binding proteins (IGFBPs) from adult human serum by insulin-like growth factor (IGF) I affinity chromatography and high performance liquid chromatography. A 36-kDa binding protein (BP), not digestible with N-glycanase, is increased in patients with extrapancreatic tumor hypoglycemia and during IGF I administration in healthy adults. Its 38 NH2-terminal amino acids are identical to those of an IGFBP sequence derived from a human cDNA that cross-hybridizes with the rat IGFBP-2 cDNA. With probes encoding a NH2-terminal, COOH-terminal, and a middle region of this protein we have obtained three cDNA clones from a Hep G2 cDNA library; one encodes human IGFBP-2, and the other two presumably represent unspliced heteronuclear and alternatively spliced mRNA, respectively. A 28-30-kDa IGFBP represents a novel BP species in human serum. Its 30 NH2-terminal amino acids are not homologous to IGFBP-1, -2, or -3. It is not digestible with N-glycanase and does not bind 125I-IGF I. The NH2-terminal sequences of a 42/45- and a 31-kDa IGFBP are identical to that of human IGFBP-3. The 42/45-kDa proteins are two glycosylation variants of BP-3. The 31-kDa protein presumably is a degradation product of BP-3 that lacks the COOH terminus. It is likely that the different IGFBPs modulate auto-/paracrine and endocrine effects of IGFs on growth and metabolism in a different and specific manner.     

Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.     

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K_D ∼ 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Escherichia coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP.     

Type I procollagen C-proteinase from mouse fibroblasts. Purification and demonstration of a 55-kDa enhancer glycoprotein

The enzyme procollagen C-proteinase removes the carboxy-terminal propeptide from procollagen. In the present study we describe an improved procedure for the purification of this enzyme. From the medium of cultured mouse fibroblasts, consisting of ammonium sulfate precipitation, gel filtration and affinity chromatography on a lysyl-Sepharose column, followed by chromatography on a column of Sepharose coupled to the carboxy-terminal propeptide of type I procollagen (PP-Sepharose). This procedure yielded a practically homogeneous, 18,500-fold-purified enzyme preparation and the molecular mass of the purified C-proteinase as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 80 kDa. The lysyl-Sepharose step separated the enzyme from the majority of the contaminating proteins, including a 55-kDa protein which was further purified by PP-Sepharose chromatography and identified as an additional form of the 36-kDa and 34-kDa procollagen C-proteinase enhancer proteins described before [Adar et al. (1986) Collagen Relat. Res. 6,267-277]. It enhanced the C-proteinase activity, bound to the carboxyl propeptide of type I procollagen, cross-reacted immunologically with the 36-kDa as well as the 34-kDa enhancer proteins, and in common with the latter proteins, it was glycosylated. In the course of PP-Sepharose chromatography, a large proportion of the 55-kDa protein disappeared with the concomitant appearance of the smaller enhancer proteins. All these findings suggest that the 55-kDa protein is a precursor of the low molecular mass enhancer proteins. Also suggested from this study is that lysyl-Sepharose chromatography is a highly beneficial purification step which may find use in the purification of the C-proteinase from other sources as well.     

Insulin-like growth factor binding protein measurement: sodium dodecyl sulfate-stable complexes with insulin-like growth factor in serum prevent accurate assessment of total binding protein content by ligand blotting.

The possibility that sodium dodecyl sulfate (SDS)-stable complexes of insulin-like growth factor I (IGF-I) and its binding proteins (IGF-BP) exist in rat serum has been examined by using SDS-polyacrylamide gel electrophoresis (PAGE) followed by both [125I]IGF-I ligand blotting and immunoblotting with antisera directed against either IGF-BP3 or IGF-I. While ligand blotting of rat serum only revealed free IGF-BP subunits (Mr approximately 50, 35, and 30 kDa), immunoblotting with either the IGF-BP3 antiserum or IGF-I antiserum revealed major immunoreactive bands with higher molecular weights (greater than 110, approximately 100, and approximately 84 kDa). The IGF-BP3 antiserum also stained the 50-kDa form of the serum IGF-BP. Specifically stained protein bands were identified by comparison with control immunoblots incubated with normal rabbit serum. Treating the serum with 0.1 N HCl prior to electrophoresis reduced the amount of high molecular weight IGF-BP3 immunoreactive species, with a concomitant increase in the amount of the 50-kDa form. A similar result was obtained if the samples were boiled prior to electrophoresis. These data indicate that not all IGF-BP/IGF complexes may dissociate under normal SDS-PAGE conditions. Therefore, data obtained by using ligand blotting alone may underestimate the amount of total IGF-BP present, especially if the mixture being analyzed also contains large amounts of IGF.