Human granulosa cells synthesize low molecular weight insulin-like growth factor-binding protein

Human follicular fluid contains insulin-like growth factor I (IGF-I) and its low molecular weight binding protein (IGF-BP). We studied the synthesis of IGF-BP by the granulosa cells obtained after ovarian hyperstimulation for in vitro fertilization. The granulosa cells were cultured for 72 hours in Ham's F-10 medium supplemented with 10% fetal calf serum (FCS). Samples of the culture medium were collected every 24 hours. The IGF-BP concentration in culture medium increased from 1.2 to 2.1 micrograms/l at 48 h and to 3.3 micrograms/l at 72 h. De novo synthesis of IGF-BP was shown by incorporation of labeled methionine into immunoreactive IGF-BP, as detected by SDS polyacrylamide electrophoresis (PAGE) and fluorography. Our results demonstrate synthesis of IGF-BP in the human ovary.     

Insulin regulates the serum levels of low molecular weight insulin-like growth factor-binding protein

The serum levels of 34K insulin-like growth factor (IGF)-binding protein were measured by RIA in 88 type 1 diabetic patients, 9 patients with type 2 diabetes, 7 patients with insulinoma, 19 normal subjects (all after an overnight fast), and 82 normal subjects after a breakfast meal. In addition, the effect of 2- to 3-h euglycemic steady state hyperinsulinemia on serum IGF-binding protein and IGF-1 levels was studied in some subjects in each of the fasted groups. Compared with normal subjects, the mean serum IGF-binding protein levels were 4-fold (P less than 0.001) higher in type I diabetic patients treated with conventional insulin injections, 2.5-fold (P less than 0.001) higher in those treated with continuous sc insulin infusion, and 2-fold (P less than 0.05) higher in patients with type 2 diabetes. In the patients with insulinoma, the mean IGF-binding protein level was 63% below normal (P less than 0.001), and it normalized after removal of the tumor. There was a slight negative correlation between the IGF-binding protein level and insulin dose in the diabetic patients (r = -0.22; P less than 0.05). In normal subjects, serum insulin concentrations were 2-fold higher (P less than 0.001) and the IGF-binding protein level was 29% lower after a meal (P less than 0.05) than in the fasting state. Serum IGF-I concentrations were virtually identical in the type 1 and 2 diabetic patients, insulinoma patients, and normal subjects. During steady state euglycemic hyperinsulinemia, the IGF-binding protein level fell by 40-70% in each group (P less than 0.001), whereas the IGF-I level declined (P less than 0.05) in the type 2 diabetic patients only. The decline of binding protein was closely related to the baseline level (r = 0.94; P less than 0.001). No correlation was found between serum IGF-I and binding protein levels in any group. In conclusion, 1) serum 34K IGF-binding protein levels are elevated in type 1 and 2 diabetic patients and decreased in patients with insulinoma; 2) the serum binding protein, but not IGF-I concentration is decreased by acute hyperinsulinemia; and 3) these data suggest that the serum insulin concentration plays a role in regulation of the serum 34K IGF-binding protein concentration.     

Dose-response characteristics for suppression of low molecular weight plasma insulin-like growth factor-binding protein by insulin

We previously demonstrated that supraphysiological insulin concentrations reduced the plasma 34K insulin-like growth factor-binding protein (IGF-BP) concentrations in humans. In this study we examined whether physiological changes in plasma insulin concentrations regulate IGF-BP and, if so, whether the regulation is influenced by race, glucose tolerance, or rate of glucose metabolism. For these purposes we 1) analyzed the relationship between fasting plasma insulin and IGF-BP concentrations in 2 racial groups (23 caucasians and 35 southwestern American Indians), 2) measured the response of plasma IGF-BP to oral glucose in 20 normal subjects, and 3) determined the dose-response characteristics of plasma IGF-BP to glucose and insulin in 23 normal subjects at 4 different insulin and glucose concentrations. The fasting plasma insulin concentration was inversely related to the plasma IGF-BP concentration in both the caucasian and Indian groups (P less than 0.0001). In the caucasian group the mean plasma IGF-BP concentration was higher [15 +/- 4 (+/- SE) micrograms/L] than in the Indian group (8 +/- 2 micrograms/L; P less than 0.05). This difference was independent of race and glucose tolerance, and it could be explained by lower plasma insulin concentrations in the caucasian (387 +/- 50 pmol/L) than in the Indian group (215 +/- 43 pmol/L; P less than 0.001). After oral glucose administration, the insulin concentration (423 +/- 72 pmol/L) was maximal 30 min after glucose treatment, and significant suppression of the IGF-BP concentration occurred at 90 min. Analysis of the dose-response curves revealed maximal suppression of IGF-BP at about 1150 pmol/L insulin, and half-maximal suppression at about 290 pmol/L. The plasma glucose concentration or the rate of glucose metabolism had no effect on the IGF-BP concentration. These data suggest that insulin is a major regulator of plasma IGF-BP concentrations under physiological conditions.     

Receptors for insulin-like growth factors and growth effects of multiplication-stimulating activity (rat insulin-like growth factor II) in rat embryo fibroblasts

Levels of multiplication-stimulating activity (MSA) in fetal rat serum are high (2-4 micrograms/ml), suggesting that MSA may have a role in fetal growth. We now demonstrate that fibroblasts derived from rat embryos (REFs) have specific MSA receptors and respond to MSA with increased DNA synthesis. Two types of insulin-like growth factor (IGF) receptors were demonstrated by competitive binding of radioiodinated MSA, IGF-I, and IGF-II and by chemical cross-linking of [125I]iodo-MSA and [125I]iodo-IGF-I to REFs. One type of receptor (mol wt, 260,000 under reducing conditions) did not interact with insulin, and another type of receptor (mol wt, 130,000, under reducing conditions) was recognized by insulin. Scatchard analysis of [125I]iodo-MSA binding data was consistent with one class of noninteracting binding sites. A biological response of MSA, increased DNA synthesis, was demonstrated with autoradiography in REFs. During a 16-hr incubation, DNA synthesis was stimulated by normal rat serum, and platelet-poor plasma plus platelet-derived growth factor (PDGF), but not by serum from hypophysectomized (hypox) rats or hypophysectomized (hypox) platelet-poor plasma plus PDGF. However, when MSA was added to hypox serum or to hypox platelet-poor plasma plus PDGF, DNA synthesis was stimulated to the level achieved by normal rat serum. By contrast, during a longer cell multiplication experiment, REFs grew equally well in normal or hypox rat serum, raising the possibility that REFs may produce a MSA-like factor.     

Lack of diurnal rhythm of low molecular weight insulin-like growth factor binding protein in patients with Cushing's disease

A specific radioimmunoassay with antibodies raised against the 25 kD insulin-like growth factor binding protein (25 kD IGFBP) in amniotic fluid was used to measure levels of cross-reacting protein in human serum and plasma. Plasma samples collected continually at 20-min intervals during 24-h in 6 healthy adults revealed a distinct diurnal rhythm in the concentration of 25 kD IGFBP. The lowest levels (9-13 micrograms/l) were found between 13.00 and 24.00 h with a rise after midnight to maximum levels (23-71 micrograms/l) between 03.00 and 09.00 h. There was no relation between the patterns of GH and 25 kD IGFBP. In 3 patients with active Cushing's disease, the levels of 25 kD IGFBP in plasma samples collected during 12 h, 19.00-07.00 h, were generally low and without nocturnal variations. One of the patients studied after extirpation of a pituitary adenoma displayed a nocturnal rhythm with maximum levels of 25 kD IGFBP between 03.00 and 07.00 h. Eight patients treated with stereotactic pituitary irradiation owing to Cushing's disease also showed a distinct nocturnal increase of 25 kD IGFBP. The results indicate the existence of a diurnal rhythm of 25 kD IGFBP in adults. Further, low levels and lack of diurnal rhythm of 25 kD IGFBP are demonstrated in Cushing's disease.     

Transformation-dependent secretion of a low molecular weight protein by murine fibroblasts.

A protocol has been devised to radiolabel proteins secreted by murine fibroblasts in vitro. A radiolabeled polypeptide of molecular weight 35,000 is released into medium in relatively large amounts by transformed cells and in much smaller amounts by nontransformed fibroblasts. This major excreted polypeptide (MEP) is found in the medium of spontaneously transformed mouse cells and in the medium of mouse cells transformed by a DNA tumor virus, RNA tumor viruses, or methylcholanthrene. The appearance of MEP appears to be well correlated with anchorage independence in these transformed cells. MEP can be localized within the cytoplasm of transformed but not untransformed cells by indirect immunofluorescence. The presence of MEP within murine fibroblasts or in their culture medium serves as a novel biochemical marker of transformation. A biological role for this protein has not been assigned.     

Regulation of low molecular weight insulin-like growth factor binding proteins in experimental diabetes mellitus.

Circulating insulin-like growth factor (IGF) bioactivity is reduced in animals and patients with diabetes mellitus. We sought to determine whether the availability and levels of specific IGF binding proteins (BPs) are altered in animals with experimental diabetes, and might contribute to changes in circulating IGF bioactivity in experimental diabetes. Female Sprague-Dawley rats were administered streptozotocin or citrate buffer iv, and then killed either 3 days later, or else after 4-day insulin treatment (7.5 U/kg human NPH twice daily), or 2 days after insulin was discontinued. Serum [125I]IGF-I binding activity was markedly increased in diabetic animals compared to controls when analyzed by Sephacryl S-200 chromatography, dot blot, and affinity labeling techniques, due to increased binding to low mol wt BPs (81 +/- 4% of ligand eluting with low mol wt BPs in diabetic serum vs. 22 +/- 3% in control, P less than 0.001). In contrast, activated charcoal removed ligand from these BPs and underestimated the availability of BPs in diabetes. Serum binding activity fell toward control levels during insulin therapy, then rose again after insulin was withdrawn, corresponding to changes in metabolic status. To distinguish changes in specific BPs, serum proteins were separated by 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to nitrocellulose. Ligand blotting with [125I]IGF-I demonstrated that serum levels of a 32 K mol wt IGF BP are markedly increased in diabetic rats and decline during insulin therapy. Levels of this 32 K IGF BP rose again after insulin was discontinued, demonstrating regulation in accordance with changes in insulin and metabolic status. Western analysis and affinity labeling with immunoprecipitation revealed that this 32 K protein is distinct from the 34 K fetal rat BP, and is immunologically related to the type 1 human IGF BP. We conclude that circulating [125I]IGF-I binding activity is markedly increased in animals with acute streptozotocin-induced diabetes, due to changes in low mol wt proteins, including a 32 K type 1 IGF BP that is regulated by changes in insulin and/or metabolic status. Regulation of low mol wt IGF BPs by insulin, and perhaps other factors, may play an important role in the modulation of tissue growth factor bioactivity in metabolic disease.     

Serum levels of the low molecular weight form of insulin-like growth factor binding protein in healthy subjects and patients with growth hormone deficiency, acromegaly and anorexia nervosa

The low molecular weight form of insulin-like growth factor binding protein (35 kD IGFBP), determined in serum by radioimmunoassay during non-fasting conditions, was high at birth and declined with increasing age during childhood and adolescence (N = 149). Inverse correlation was found between chronological age and 35 kD IGFBP values (r = -0.61, P less than 0.001) during childhood and adolescence, but no age dependency was found in adult subjects aged 20-66 years (N = 73). The mean and 95% confidence limits of immunoreactive 35 kD IGFBP were 34 micrograms/l and 15-79 micrograms/l, respectively, in healthy adults (N = 73) in whom the blood samples were drawn after a one-night fast. The mean level of the 35 kD IGFBP in patients with acromegaly (19 micrograms/l, N = 23) was decreased by 50% in comparison with healthy adults, whereas a 2-fold elevation of the mean levels was found in both anorexia nervosa patients (70 micrograms/l, N = 13) and adult patients with GH deficiency (69 micrograms/l, N = 22). In patients with anorexia nervosa, the 35 kD IGFBP levels were inversely related to the body mass index (r = -0.65, P less than 0.02).     

重型病毒性肝炎患者生长激素-胰岛素样生长因子轴的临床研究

目的研究重型病毒性肝炎患者GH-IGF轴的变化及其临床意义。方法重型病毒性肝炎患者18例,其中急性重型2例,亚急性重型5例,慢性重型11例;正常对照20例。ELISA法测定血清GH、IGF-1、IGFBP1及IGFBP3,全自动生物化学分析仪常规方法测定肝脏生物化学指标。结果重型肝炎患者血清IGF-1、IGFBP3水平明显降低［(5.5±6.2)μg/ml对(17.6±7.0)μg/ml,(2.4±1.3)μg/ml对(9.4±1.7),P＜0.001］。GH、IGFBP1水平增高［(9.1±12.4)ng/ml对(1.6±2.4)ng/ml,P＜0.05;(67.9±50.2)ng/ml对(45.8±33.1)ng/ml,P＜0.01)］。血清IGF-1与IGFBP3呈正相关(r＝0.91,P＜0.001);IGF-1降低与重型肝炎患者预后密切相关(P＜0.001)。IGF-1＜10μg/ml,预测死亡的符合率为90%;IGF-1＞10μg/ml,预测存活的符合率为89.5%。结论重型病毒性肝炎患者GH-IGF轴发生显著异常变化,GH增高与IGF-1水平降低相矛盾,提示重型病毒性肝炎患者存在生长激素抵抗。血清IGF-1水平可作为预测重型肝炎患者预后的指标。     

The insulin-like growth factor-II (IGF II) receptor from rat brain is of lower apparent molecular weight than the IGF II receptor from rat liver

The binding subunits of the insulin and insulin-like growth factor-I (IGF I) receptors from rat brain are of lower molecular weight than the corresponding receptor in rat liver, possibly due to variations in sialic acid content. We have compared the IGF II receptor from rat brain and rat liver. The brain receptor is of smaller apparent mol wt (about 10 K) on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is independent of ligand binding as it persists in iodinated and specifically immunoprecipitated receptors. From studies of wheat germ agglutinin binding and the effect of neuraminidase on receptor mobility, we conclude that this difference is not simply due to variations in sialic acid content. Treatment with endoglycosidase F results in reduction in the molecular size of both liver and brain receptors and after this treatment the aglycoreceptors are of similar size. We conclude that in rat brain tissue the IGF II receptor like the binding subunits of the insulin and IGF I receptors is of lower molecular size than the corresponding receptors in rat liver. This difference is due to differences in N-linked glycosylation.     

Hepatic production of insulin-like growth factors in normal and diseased liver

BACKGROUND/AIMS: IGF-I levels are reduced in cirrhotic patients. However, it is not known whether this decreased level is the result of reduced hepatic production or modified bioavailability secondary to decreased binding proteins. We determined the hepatic production of IGF-I and IGF-II and their receptors in normal and diseased liver. METHODOLOGY: Twenty-five patients included, 11 controls with normal liver and 14 with either chronic hepatitis or cirrhosis. mRNA for IGF-1, IGF-II and their receptors were measured. Immunohistochemical staining was performed to localize the IGF-producing cells. RESULTS: In 11 normal livers, the IGF-I mRNA levels were 4.95 +/- 1.8; in the 14 diseased livers, the levels were 1.22 +/- 0.69 (p < 0.001). IGF-II mRNA levels were 3.78 +/- 1.45 for the control and 5.11 +/- 2.15 in the diseased livers (NS). IGF-I receptor levels were 1.15 +/- 0.83 in the normal and 0.31 +/- 0.22 in the liver disease group (p < 0.05). There was no statistical difference between the two groups for IGF-II receptor. CONCLUSIONS: Patients with chronic liver disease have a significant reduction in their hepatic production of IGF-I, whereas IGF-II tends to be elevated. Treatment with recombinant IGF-I in patients with metabolic or endocrine complications of cirrhosis might prove useful.     

The ontogeny and regulation of a 31,000 molecular weight insulin-like growth factor-binding protein in fetal porcine plasma and sera

Studies were conducted to determine the presence, quantity, and regulation of insulin-like growth factor (IGF)-binding proteins in porcine plasma and sera. The size distribution of IGF-binding protein was determined by affinity cross-linking to [125I]IGF-I, and the binding activity was quantified by a polyethylene glycol precipitation procedure. The major [125I]IGF-I-binding protein complex was of 38,000 mol wt (Mr) in all porcine sera or plasma tested. Binding to this protein was inhibited by excess unlabeled IGF-I, but not by insulin. When plasma samples from fetuses at 45, 70, 90, and 110 days gestation were cross-linked to [125I]IGF-I, there was an increase in the concentration of the 38,000 Mr complex with advancing gestational age, whereas plasma from the mothers of the age-matched fetuses showed little change in the amount of the 38,000 Mr complex. When the binding activity of the fetal plasma was quantified, there was a significant increase in binding activity between 45 and 110 days gestation from 30.5 +/- 3.3% to 77.5 +/- 3.6% (+/- SE) of the assay maximum. This increase was not due to a decrease in binding protein saturation, since the endogenous IGF-I levels (quantified after acid gel filtration chromatography) also increased with advancing gestational age, from 176 to 458 mU/ml. Blood samples were collected from porcine fetuses at 110 days gestation that had either been decapitated or spinal cauterized at 45 days gestation. Decapitation decreased the amount of the 38,000 Mr complex that could be detected by affinity labeling. In contrast, spinal cauterization had no effect on the amount of the 38,000 Mr complex in fetal plasma compared to that in control fetal plasma. These results were verified in the polyethylene glycol assay. These studies show that the amount of the 38,000 Mr IGF-binding protein complex in pig plasma is developmentally regulated and suggest that a pituitary or neural factor may be an important variable in the control of its plasma concentrations.     

Circulating levels and molecular distribution of the acid-labile (alpha) subunit of the high molecular weight insulin-like growth factor-binding protein complex.

A RIA is described for the acid-labile (alpha) subunit of the high mol wt insulin-like growth factor (IGF)-binding protein complex, a glycoprotein of approximately 85,000 daltons (approximately 85K) which combines with the GH-dependent binding protein (BP-53 or IGFBP-3) and IGF-I or IGF-II to form the complex. The assay shows relative specificity for higher primate species. Whereas amniotic fluid, cerebrospinal fluid, and seminal plasma contain virtually no immunoreactive alpha-subunit, the protein is easily detectable in 0.5-microL serum samples. Serum alpha-subunit levels are markedly age dependent, rising over 5-fold from birth to puberty, then remaining relatively constant throughout adulthood. In 170 children, there was a strong association between alpha-subunit and IGFBP-3 levels. Mean alpha-subunit levels (+/- SD) in adults were 24.2 +/- 4.7 mg/L in 93 normal subjects, 54.1 +/- 15.5 mg/L in 12 acromegalics, 6.5 +/- 4.8 mg/L in 10 GH-deficient subjects, and 31.5 +/- 5.7 mg/L in 18 third term pregnant women. In serum fractionated by gel chromatography, alpha-subunit appeared as a broad 100-150K peak. After depleting serum samples of IGFBP-3 by immunoaffinity chromatography, approximately one third of alpha-subunit remained, in a peak of about 100K, suggesting that two thirds of the total alpha-subunit in serum is present in the approximately 150K complex, and one third is uncomplexed. Development of this RIA for alpha-subunit will allow further study of regulation of the GH-dependent complex.     

Elevated activity of low molecular weight insulin-like growth factor-binding proteins in sera of vitamin C-deficient and fasted guinea pigs.

We have previously reported that scorbutic and fasted guinea pig sera contain an insulin-like growth factor-I (IGF-I)-reversible inhibitor of collagen, proteoglycan, and DNA synthesis in cultured cells. Here we report that IGF-binding protein (IGFBP) activity is increased in serum containing the inhibitor [125I]IGF-I or -II bound to these sera was eluted in the 30- to 50-kDa region of an S200 gel column. [125I]IGF-I affinity cross-linking analysis revealed that a 38-kDa cross-linked species increased markedly in fasted and scorbutic sera, with a lesser increase in a 34-kDa species, while scorbutic sera also yielded a 44-kDa species. Gel filtration of unlabeled sera showed a 10-fold increase in the activity of two proteins in the 30- to 50-kDa region from the experimental sera. Their activity correlated with their ability to inhibit binding of [125I]IGF-I to its cellular receptor, suggesting that they have the potential to inhibit IGF-I-dependent functions. Ligand blotting showed that 29 and 35-kDa IGFBPs were almost undetectable in normal serum, but were dramatically induced by scurvy and fasting, so that they accounted for close to 40% of the total circulating BPs. Total IGFBP-3 in the experimental sera was increased about 30%, while there was little effect of scurvy or fasting on the level of BP-3 activity isolated by acid extraction of the high mol wt region of the S200 column. An IGF-I analog with normal affinity for the 30- to 50-kDa BPs from fasted and scorbutic sera, but with reduced affinity for the cell receptor, was equivalent to IGF-I in reversing the inhibition of collagen synthesis by scorbutic guinea pig serum in human fibroblasts. Thus, reversal of inhibition appears to require initial saturation of IGFBPs. The overall results suggest that two circulating IGFBPs with unoccupied binding sites are induced in vitamin C-deficient or fasted guinea pigs and may be responsible for inhibition of IGF-I-dependent functions by sera from these animals.     

Diurnal rhythm of growth hormone-independent binding protein for insulin-like growth factors in human plasma

An antibody raised against the major insulin-like growth factor (IGF)-binding protein in amniotic fluid (BP-28) was used to measure the levels of a cross-reacting protein in human plasma by RIA. Plasma BP-28 immunoreactivity had an apparent mol wt of 35,000 by high performance permeation chromatography. Sampled hourly for 12- or 24-h periods in 15 children with a wide range of GH secretory activity, plasma BP-28 levels showed a marked diurnal cycle, rising 10- to 20-fold between 2400 and 0600 h to a peak of 50-500 micrograms/L, then falling to basal levels (0-40 micrograms/L) by 1200 h. Plasma GH levels were measured at 20-min intervals in the same subjects. Neither peak nor basal BP-28 levels were significantly associated with chronological age, bone age, mean or peak nocturnal GH secretion, relative height, or relative growth velocity in tall, normal, short, or GH-deficient children. Plasma proteins measured in a RIA for 53,000 mol wt GH-dependent IGF-binding protein (BP-53) did not vary diurnally. The IGF-binding activity of plasma BP-28, measured by incubating plasma with IGF tracer and precipitating the BP-28-IGF complex with anti-BP-28 antiserum, closely paralleled the morning rise in BP-28 immunoreactivity. Immunoprecipitable BP-28 bound both IGF-I and IGF-II tracers, with a clear preference for IGF-I, and unlabeled IGF-I was more effective than IGF-II in displacing either tracer IGF. We conclude that plasma BP-28 levels in children have a marked diurnal rhythm which is unrelated to GH secretory activity.     

Purification and immunological characterization of the rat liver insulin-like growth factor-II receptor

Rat liver microsomal insulin-like growth factor-II (IGF-II) receptor has been purified to homogeneity using a single step affinity chromatographic procedure on agarose-IGF-II with elution at pH 4. Determined by either IGF-II binding or a direct RIA for receptor, purification of 2000-fold was obtained. The mean recovery was 28% for five such preparations. Sodium dodecyl sulfate-electrophoresis and autoradiography of purified receptor, radioiodinated receptor, and affinity-labeled receptor all indicated a molecular mass of approximately 250K. Scatchard analysis of IGF-II binding to purified receptor, solubilized microsomal membranes, or plasma membranes showed a single class of binding site with an affinity constant of 6 X 10(10) liter/mol in all cases. Potent antibodies to the purified receptor were raised in rabbits, capable of inhibiting 50% of IGF-II binding at dilutions of 1:170,000 and also of fully precipitating IGF-II-prelabeled receptor at 1:50,000. Both types of antibodies reacted with IGF-II receptors in rat adipose tissue, brain, heart, kidney, lung, and spleen. However, little cross-reactivity was seen with other species. Comparison of the ability of receptor antibodies to inhibit IGF-II binding to microsomal and plasma membranes indicated a specific immunological difference between the IGF-II receptors in the two membrane preparations.     

低出生体重儿童围青春期生长发育和胰岛素样生长因子Ⅰ水平

在我国,低出生体重(LBW) 儿童的发生率为 4.8%～8.5%.大部分LBW儿童在出生后两年内会获得明显的追赶生长,但约有10%的小于胎龄儿出生后生长障碍,最终身高低于其基因型所决定的高度而导致成年身材矮小[1]. 本研究以56例7～14岁LBW儿童作为研究对象,通过与正常出生体重儿童(NBW)比较,探讨LBW儿童青春期生长发育以及生长激素-胰岛素样生长因子Ⅰ(GH-IGF-Ⅰ)轴发挥作用的可能机制.     

Separation of the insulin-like growth factor-binding proteins in plasma and purification of the larger molecular weight species

Methods were developed for purification of the high mol wt (150K) insulin-like growth factor (IGF)-binding protein and its acid stable (70K) component from human plasma. High mol wt IGF-binding protein was highly purified by chromatography of Cohn IV-1 fraction of human plasma on Concanavalin A-Sepharose followed by chromatography on IGF-I-Sepharose. The acid-stable component of the high mol wt IGF-binding protein was purified to near homogeneity by chromatography of Cohn IV-1 fraction of human plasma on Con-A Sepharose, followed by chromatography on Sephadex G-50 at pH 2.5 and subsequent chromatography on IGF-I-Sepharose. Both fractions obtained after IGF-I-Sepharose chromatography were capable of binding [125I]IGF-I and gave a single protein band of 79,000 mol wt, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Coomassie blue staining), suggesting that the large mol wt species may be a dimer of identical or iso-mol wt subunits. Three minor contaminants of less than 5% each were detected upon subsequent silver staining. These methods represent important tools that should aid in furthering our understanding of the role of the IGF carrier proteins in the actions of IGF-I and IGF-II.