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.     

Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities

Insulin-like growth factors I and II (IGF-I and IGF-II) were prepared from fetal calf serum and adult bovine serum by gel filtration, immunoaffinity chromatography, chromatofocusing, and reverse-phase high pressure liquid chromatography, and their complete amino acid sequences determined. IGF-I and -II are found in both adult and fetal serum. The sequence of bovine IGF-I is found to be identical to that of human IGF-I, whereas 3 out of 67 amino acid residues are found to be different between bovine and human IGF-II. The differences are located in the C-peptide region of the molecule. Bovine IGF-II shows less than 10% immunological cross-reactivity with antisera against human and rat IGF-II, but is equipotent to human IGF-II in displacing human 125I-labeled IGF-II from human placental receptor. Bovine IGF-I was equipotent to human IGF-I in both radioimmunoassays and radioreceptor assays within the limits of the assay.     

Characterization of insulin-like growth factor binding proteins from human breast cancer cells

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor-binding protein from human plasma. Purification and characterization

Insulin-like growth factor (IGF)-binding protein (BP) has been purified from Cohn fraction IV of human plasma by acidification, ion exchange to remove endogenous ligands, and affinity chromatography on agarose-IGF-II. The pure protein appeared as a single peak by high performance reverse-phase and gel permeation chromatography (molecular mass, 45-50 kDa), but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band at 53 kDa and a minor band at 47 kDa, unreduced, or 43 and 40 kDa, respectively, reduced. The two bands stained for both protein and carbohydrate. After storage at 2 degrees C for 5 months at pH 3, two additional bands, at 26 and 22 kDa on unreduced gels, were also present. Autoradiography after affinity labeling with IGF-I or IGF-II tracer revealed a single labeled band of 61 kDa. BP, quantitated using a specific radioimmunoassay, was retained by agarose-immobilized IGF-I, IGF-II, concanavalin A, and wheat germ lectin, but not Helix pomatia lectin. Competitive binding curves using pure BP and human IGF-I and IGF-II as both labeled and unlabeled ligands indicated association constants of 2-3 X 10(10) liters/mol for both peptides, with a slightly higher affinity for IGF-II than IGF-I, and 0.9 binding sites for either peptide per 53-kDa protein. The exact relationship of this acid-stable IGF BP to the 150-kDa complex from which it is derived remains to be determined.     

Purification and characterization of an insulin-like growth factor II variant from human plasma

An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.     

Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid

Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three-step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenyl-Sepharose); and third, anion-exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50%. Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35000, determined by exclusion chromatography, and 32000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidic/amidated residues. The N-terminal amino acid sequence was Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala.     

The regulation of GH-dependent hormones and enzymes after feed restriction in dwarf and control chickens.

The principal objective of this study was to examine the GH-dependency of IGF-I and IGF-II changes in the chicken. To this end, the regulation of GH-dependent hormones and enzymes were studied in undernourished normal and dwarf chickens. The dwarf chickens examined exhibit a Laron-type dwarfism and have been shown to be GH receptor deficient. Thus, they provide an interesting model to determine the GH-dependency of IGF-I and IGF-II changes. Short (1 day) and long-term (7 days) feed restriction was imposed on growing normal and dwarf chickens to follow the subsequent endocrine changes. Since short-term feed restriction of dwarf chickens resulted in decreased plasma IGF-I, it appears that this is not a GH-dependent effect. However, with longer term undernutrition, IGF-I was not decreased in dwarf chickens. So, after a longer restriction period, the regulation of these factors appears to become more GH-dependent. IGF-II was not depressed at all by feed restriction in the dwarf chicken, suggesting a degree of GH-dependency.     

Purification of a 31,000-dalton insulin-like growth factor binding protein from human amniotic fluid. Isolation of two forms with different biologic actions

Human amniotic fluid has been shown to contain a protein that binds insulin-like growth factor I and II (IGF-I and IGF-II). Partially purified preparations of this protein have been reported to inhibit the biologic actions of the IGFs. In these studies our laboratory has used a modified purification procedure to obtain a homogeneous preparation of this protein as determined by polyacrylamide gel electrophoresis and amino acid sequence analysis. During purification the ion exchange chromatography step resulted in two peaks of material with IGF binding activity termed peaks B and C. Each peak was purified separately to homogeneity. Both peaks were estimated to be 31,000 daltons by polyacrylamide gel electrophoresis and their amino acid compositions were nearly identical. Amino acid sequence analysis showed that both peaks had identical N-terminal sequences through the first 28 residues. Neither protein had detectable carbohydrate side chains and each had a similar affinity for radiolabeled IGF-I (1.7-2.2 x 10(10) liters/mol). In contrast, these two forms had marked differences in bioactivity. Concentrations of peak C material between 2 and 20 ng/ml inhibited IGF-I stimulation of [3H]thymidine incorporation into smooth muscle cell DNA. In contrast, when peak B (100 ng/ml) was incubated with IGF-I there was a 4.4-fold enhancement of stimulation of DNA synthesis. Additionally, pure peak B was shown to adhere to cell surfaces, whereas peak C was not adherent. The non-adherent peak C inhibited IGF-I binding to its receptor and to adherent peak B. We conclude that human amniotic fluid contains two forms of IGF binding protein that have very similar physiochemical characteristics but markedly different biologic actions. Since both have similar if not identical amino acid compositions, N-terminal sequences, and do not contain carbohydrate, we conclude that they differ in some other as yet undefined post-translational modification.     

Mutation of Arg55/56 to Leu55/Ala56 in insulin-like growth factor-I results in two forms different in disulfide structure and native conformation but similar under reverse-phase conditions

Folding of recombinant human insulin-like growth factor-I (IGF-I) results in two distinct species as resolved by reversed-phase high-performance liquid chromatography (RP-HPLC). The earlier eluting peak (PI) has a nonnative disulfide structure, while the later eluting peak (PII) assumes the native disulfide structure. This folding problem causes a lower yield and requires expensive RP-HPLC separation. In contrast, IGF-II folds mainly into a single form with all three disulfide bonds correctly formed. Sequence comparison of the two molecules revealed that IGF-I has arginine at residues 55 and 56, while IGF-II has alanine and leucine, respectively, at these positions. Two analogs of IGF-I, IGF-I (Ala55/Leu56) and IGF-I (Leu56), behave similarly to IGF-II upon refolding and RP-HPLC; that is, a single peak eluted from the RP-HPLC column. However, when the peaks isolated by RP-HPLC were subjected to hydrophobic interaction chromatography, circular dichroism, and peptide mapping, they were found to be a mixture of PI and PII. It was then concluded that factors other than just these two residues contribute to correct folding of IGF-II and that the PI and PII of the above two IGF-I mutants assume different conformation at neutralpH but similar conformation under the RP-HPLC condition.     

Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein

N-terminal as well as internal amino acid sequence data were obtained from the GH dependent, insulin-like growth factor (IGF) binding protein, BP-53, purified from human plasma. Based on these sequence data, full-length cDNA clones of BP-53 have been isolated, and the complete deduced sequence of BP-53 determined. This sequence contains a 27 amino acid putative signal sequence followed by a mature protein of 264 amino acids containing 18 cysteine residues clustered near the N- and C-terminus. The deduced protein sequence of BP-53 has 33% amino acid identity including conservation of all 18 cysteine residues with the recently cloned BP-28, a smaller human IGF-binding protein identified in amniotic fluid and also secreted by the cell line HEP G2. Expression of the cloned BP-53 cDNA in mammalian tissue culture cells results in secretion of the protein into the culture medium. This expressed protein is identical to plasma-derived BP-53 in its immunoreactivity, high affinity binding of IGF-I and IGF-II, and mobility on sodium dodecyl sulfate gel electrophoresis.     

Regulation of breast cancer growth by insulin-like growth factors

The IGFs may be important autocrine, paracrine or endocrine growth factors for human breast cancer. IGF-I and II stimulate growth of cultured human breast cancer cells. IGF-I is slightly more potent, paralleling its higher affinity for the IGF-I receptor. Antibody blockade of the IGF-I receptor inhibits growth stimulation induced by both IGFs, suggesting that this receptor mediates the growth effects of both peptides. However, IGF-I receptor blockade does not inhibit estrogen (E₂)-induced growth suggesting that secreted IGFs are not the major mediators of E₂ action. Several breast cancer cell lines express IGF-II mRNA by both Northern analysis and RNase protection assay. IGF-II activity is found in conditioned medium by radioimmuno and radioreceptor assay, after removal of somatomedin binding proteins (BP) which are secreted in abundance. IGF-I is undetectable. BPs of 25 and 40 K predominate in ER-negative cell lines while BPs of 36 K predominate in ER-positive cells. Blockade of the IGF-I receptor inhibits anchorage-independent and monolayer growth in serum of a panel of breast cancer cell lines. Growth of one line (MDA-231) was also inhibited in vivo by receptor antibody treatment of nude mice. The antibody had no effect on growth of MCF-7 tumors. These data suggest the IGFs are important regulators of breast cancer cell proliferation and that antagonism of this pathway may offer a new treatment strategy.     

Identification of insulin-like growth factor-II in human seminal and follicular fluids

Antisera raised in rabbits against synthetic insulin-like growth factor-II (IGF-II) were used to develop a specific radioimmunoassay (RIA) for IGF-II. Affinity purified antibodies showed 6% cross-reactivity with IGF-I but failed to recognize insulin even at 10 micrograms/tube. Utilizing this RIA system, immunoreactive IGF-II was identified in the pooled samples of human follicular fluid and seminal plasma. The acid-ethanol precipitates of human seminal and follicular fluids were chromatographed on Sephadex G-50 column and the IGF-II immunoreactive fractions were subjected to reversed-phase high performance liquid chromatography. It was found that immunoactive IGF-II was eluted in the same location as that of synthetic IGF-II. The data indicate for the first time that human seminal plasma and follicular fluid contain significant amounts of IGF-II.     

Purification and characterization of heparinase from Flavobacterium heparinum

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.     

Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-II

Human skeletal growth factor (human SGF) extracted from human bone has been purified to homogeneity by hydroxyapatite chromatography and gel filtration under dissociative conditions followed by FPLC heparin-Sepharose affinity chromatography and reverse phase HPLC. Human SGF was homogeneous except that in each preparation about 30% of SGF molecules lacked the N-terminal alanine. 75% of the human SGF sequence has been determined. The amino acid sequences of the N-terminal 20 amino acids and of several tryptic fragments were identical to the corresponding sequences of human insulin-like growth factor-II (IGF-II) purified from serum. However, since the C-peptide (variable region) of human SGF has not yet been sequenced, we cannot conclude that SGF is identical to IGF-II. Comparison of the amino acid sequence of human SGF with that of IGF-II variants that have been described in the literature revealed that human SGF is not one of the known IGF-II variants. IGF-I was also found in human bone extract but was several-fold less abundant than SGF/IGF-II. The relative abundance of SGF/IGF-II and IGF-I in bone corresponded to the relative rates of production of these two mitogens by human bone cells in vitro. Regarding the physiological significance of IGF-II in bone, previous studies on the biological actions of SGF in vitro suggest that this growth factor can have both paracrine and autocrine functions on cells of the osteoblast line. In addition, we have proposed the concept that SGF is a mediator of the coupling of bone formation to bone resorption, an important bone volume regulatory mechanism. In as much as SGF is very similar (if not identical) to IGF-II, it seems likely that these proposed regulatory functions of SGF in bone are attributable to IGF-II.     

Proteolytic conversion of insulin-like growth factors to an acidic form(s).

The relative amounts of the various forms of bioassayable insulin-like growth factors (IGF) isolated from human serum or serum fraction Cohn IV-1 depend on the purification procedure. With acid gel filtration or acid/ethanol extraction as the initial step, IGF-II (pI approximately 6.5) was the most abundant (40-70%) followed by somatomedin A (pI approximately 7.4; 15-23%), an acidic form of insulin-like activity (ILA pI 4.8) (13-21%) and IGF-I (pI approximately 8.5; 5-27%). If, however, pH 5.5 ion-exchange chromatography on SP-Sephadex was used prior to acid gel filtration, the acidic pI 4.8 form was the major (greater than 90%) species recovered and was accompanied by a quantitative loss of the other IGF species. This suggested a possible conversion of IGF-I, somatomedin A and/or IGF-II to the acidic ILA pI 4.8 form(s) during the SP-Sephadex procedure. Further experiments indicated that differences in the yields of ILA pI 4.8 were not due simply to differences in the initial pH conditions of the various methods (i.e. acid versus neutral), although exposure to pH 9.7 (a pH experienced during elution of IGF activity from the SP-Sephadex) did appear to play a role. The involvement of the carrier protein in the conversion process was tested by subjecting carrier-free IGF-I and IGF-II to the SP-Sephadex procedure. No conversion of the free forms to ILA pI 4.8 occurred. To examine the possible role of proteinase in the conversion of IGFs to ILA pI 4.8, SP-Sephadex chromatography was performed in the presence of a broad spectrum proteinase inhibitor. The IGF distribution pattern obtained closely resembled the 'normal' pattern seen with acid gel filtration, indicating that proteinase inactivation had prevented conversion to ILA pI 4.8. These data suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to more acidic ILA pI 4.8 form(s) (i) occurs during SP-Sephadex chromatography, (ii) is not prevented simply by prior acid exposure, and (iii) takes place only when IGF-I and -II are in their high-Mr carrier-bound forms. Since IGF-I and IGF-II, although homologous, have unique amino acid sequences, the conversion of both IGFs implies that at least two acidic ILA forms exist. Nevertheless, because ILA pI 4.8 retains the full spectrum of IGF bioactivities in vitro, and significant quantities are present in normal human serum (21%), it would suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to ILA pI 4.8 in vivo may be a physiologically significant event.     

Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm,Bombyx mori

A juvenile hormone binding protein (JHBP) has been isolated from Bombyx mori hemolymph by gel filtration, ion-exchange chromatography, chromatofocusing and hydroxyapatite column chromatography. Gel electrophoresis indicates that the isolated protein is homogeneous in the presence or absence of a denaturing agent. The JHBP in question has a relative molecular mass of 32 kDa, determined by denaturing gel electrophoresis. Chromatofocusing analysis indicated that the JHBP is an acidic protein with pI 4.9. The protein exhibits a dissociation constant of 9.0 × 10⁻⁸ M for JH I, 1.14 × 10⁻⁷ M for JH II and 3.9 × 10⁻⁷ M for JH III, and thus its affinity for JH analogues is in the order of JHI >JHII >JHIII. Its amino acid composition indicates that the protein consists of 297 residues of 18 kinds of amino acids. The sequence of the N-terminus of the polypeptide chain was determined for 34 of the first 36 residues: Asp-Gln-Asp-Ala-Leu-Leu-Lys-Pro-?-Lys-Leu-Gly-Asp-Met-Gln-Ser-Leu-Ser-Ser-Ala-Thr-Gln-Gln-Phe-Leu-Glu- Lys-Thr-Ser-Lys-Gly-Ile-Pro-?-Tyr-His-.     

The identification of O-glycosylated precursors of insulin-like growth factor II.

A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.     

Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli

A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.     

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.