Characterization of proteins released from legume seeds in hot water.

When immersed in water at 50-60 degrees, mature soybean seeds release a large amount of protein. The major protein released was basic 7S globulin (Bg), which is present in the cotyledons of soybean seeds. The released Bg consisted of the 27,000 and 16,000 subunits which were linked by disulphide bonding and glycosylated. The released Bg exhibited an identical structure with the mature Bg which was synthesized in the normal developing seeds. Proteins like Bg were also found to be released into hot water from the seeds of legume species such as azuki-bean, cowpea, mung-bean and winged-bean. Besides Bg and Bg-like proteins, a few proteins including the 9,000 hydrophobic protein in soybean, ubiquitin in cowpea and mung-bean, and Kunitz trypsin inhibitor in winged-bean, were released from the seeds in hot water.     

(Thr-59)-insulin-like growth factor I stimulates 2-deoxyglucose transport in BC3H1 myocytes through the insulin-like growth factor receptor, not the insulin receptor

The murine non-fusing muscle cell line contains distinct receptors for insulin and insulin-like growth factors. Pretreatment of myocytes with insulin for 20 h at 37 degrees C inhibits the binding of [125I]iodoinsulin by 60% without affecting the binding of [125I]iodoinsulin-like growth factor I. The ED50 values for down-regulation of the insulin and insulin-like growth factor receptor by their respective ligands are 1 nM and 3 nM, respectively. Insulin, (Thr-59)-insulin-like growth factor I and multiplication-stimulating activity stimulate 2-[3H]deoxyglucose transport in myocytes with ED50 values of 5 nM, 5.6 nM and 33 nM, respectively. In order to determine whether (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes via its own receptor or the insulin receptor, we determined the activity of these peptides after down-regulation of the insulin receptor. The rate of 2-[3H]deoxyglucose transport in myocytes pretreated with insulin (5 nM) is elevated but returns to control levels by 1 h after the washout of insulin. The dose-response curve for insulin-stimulated 2-[3H]deoxyglucose transport is shifted to the right (ED50 greater than 100 nM) immediately after insulin washout but is normal by 1 h after insulin washout. In contrast, the dose-response curve for (Thr-59)-insulin-like growth factor I is unchanged in insulin-pretreated cells immediately after insulin washout. These data show that (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes by acting through an insulin-like growth factor receptor and not through the insulin receptor. Since multiplication-stimulating activity is 6-fold less active than (Thr-59)-insulin-like growth factor, they both may be acting through a type 1 insulin-like growth factor receptor.     

Receptors for insulin and insulin-like growth factor-I in the human adrenal gland

Human adrenal glands contain high-affinity receptors for insulin and insulin-like growth factor I (IGF-I). Comparative studies with rat, hamster and human adrenal membranes confirmed that IGF-I receptors are most abundant in rat and hamster adrenals, whereas insulin and IGF-I receptors are present in equivalent numbers in human adrenal glands. Covalent crosslinking studies revealed that the human adrenal gland IGF-I receptor binding subunit migrated on dodecyl sulfate polyacrylamide gels with Mr = 135,000, which is identical to the migration of IGF-I receptor binding subunits isolated from other tissues. Autoradiography of frozen human adrenal slices incubated with [125I]insulin showed prominent, displaceable binding of this radioligand to the zona reticularis, zona glomerulosa, vasculature and medulla; in contrast, [125I]IGF-I binding to human adrenal tissue was most prominent in the zona reticularis and negligible in the medullary region.     

Isolation and characteristics of insulin-binding proteins from soybeans

Two soybean insulin-binding proteins were isolated using affinity chromatography on insulin-Sepharose. Both proteins have molecular mass about 39 kDa and consist of two subunits linked by disulphide bonds. According to the amino acid composition and N-terminal sequences of the subunits, these proteins, characterized by the absence of free thiol groups and sugar residues, are variants of the previously described soybean basic 7S globulin. The blotted proteins as well as their subunits were shown to bind 125I-labelled bovine insulin. For one of the proteins and insulin, dissociation constant of 4.10(-9) M was measured. The existence of plant insulin-binding proteins suggests the insulin-like regulation in the plant metabolism.     

Synthesis of an insulin-like compound consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I

An insulin-like hybrid molecule consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I (growth factor I sequence 1-30) has been synthesized essentially by the procedures developed in this laboratory for the synthesis of insulin and analogues. The hybrid competed with 125I-insulin for insulin receptors in rat liver plasma membranes and was a full agonist in stimulating incorporation of [3(-3)H]glucose into lipids in rat adipocytes. In both assays, the compound displayed ca. 2% of the potency of insulin. The compound was recognized by anti-insulin antibodies but was only ca. 0.25% as potent as insulin in this activity. The hybrid exhibited growth-promoting activity in fibroblasts, displaying 3-8% of the activity of insulin. In contrast, the compound was recognized by insulin-like growth factor carrier proteins, a property not associated with insulin. Two points of nonhomology between the B chain of insulin and the B domain of insulin-like growth factor I are considered in connection with these observations.     

Difference in the number of insulin binding sites between cortisol-sensitive and cortisol-resistant lymphoma P1798 cells.

Cortisol-sensitive and cortisol-resistant lymphoma P1798 cells specifically bind [25I]insulin. Resistant lymphocytes bind 40% less insulin than sensitive cells. These results suggest that insulin (or insulin-like substances) may play a role in growth regulation and/or response of this tumor to glucocorticoid therapy.     

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.     

Characterization of insulin-like growth factor I receptors on Madin-Darby canine kidney (MDCK) cell line

Specific insulin-like growth factor I (IGF-I) receptors on the Madin-Darby canine kidney (MDCK) cell line were identified and characterized. [125I]IGF-I specifically bound to the cells, but [125I]insulin bindings to the cells was minimal. Unlabeled IGF-I displaced both the IGF-I and insulin bindings with potencies that were 100 and 10 times as great as insulin. By an affinity labeling technique, IGF type I receptors were present in the MDCK cells. IGF-I stimulated DNA synthesis and cell proliferation at physiological concentrations. On the other hand, insulin had a little effect on DNA synthesis. These data suggest that IGF type I receptors as demonstrated in MDCK cells are involved in DNA synthesis and cell proliferation.     

Effect of an anti-insulin-like growth factor I receptor antibody on insulin-like growth factor II stimulation of DNA synthesis in human fibroblasts

To investigate the role of insulin-like growth factor II in the control of DNA synthesis in human fibroblasts, dose-response curves for insulin-like growth factor I and II stimulation of [3H]thymidine incorporation were compared in the absence and presence of alpha IR-3, a highly specific monoclonal antibody directed against the type I insulin-like growth factor receptor. Specific binding of [125I]insulin-like growth factor I to human fibroblast monolayer cultures was inhibited 60-70% in the presence of alpha IR-3. alpha IR-3 had no effect on [125I]insulin-like growth factor II binding to human fibroblasts. However, alpha IR-3 inhibited both insulin-like growth factor I and II stimulated [3H]thymidine incorporation. These data indicate that the type II insulin-like growth factor receptor does not function as a transducer of insulin-like growth factor II's mitogenic effect in human fibroblasts.     

Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines

Conditioned serum-free media (CM) from small-cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth-factor-binding proteins (IGF-BP). 6/9 SCLC cell lines secreted binding proteins with high affinity for IGFs. When [125I]IGF-I or [125I]IGF-II was incubated with the CMs, complexes of tracer with proteins could be demonstrated by gel filtration, by precipitation with polyethylenglycol, and after adsorption of unbound tracer with activated charcoal. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF-I and IGF-II. The dissociation constants were determined to be 0.106 nM for IGF-I and 0.209 nM for IGF-II binding. Cross-linking of [125I]IGF-I or [125I]IGF-II to the CMs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions revealed the presence of IGF-BPs with molecular masses in the range 24-32 kDa. The binding was competitively inhibited by addition of cold IGF-I and IGF-II but not by insulin. Northern blot hybridization with an IGF-BP cDNA probe encoding a low-molecular-weight IGF-BP from a human placenta cDNA library and Western blot analysis with a corresponding polyclonal antibody showed no expression of this gene. These data demonstrate that SCLC cell lines release IGF-BPs in culture supernatants, which differ from IGF-BPs detected in liver and placenta. These IGF-BPs might be important mediators in the autocrine/paracrine growth regulation of IGFs in SCLC.     

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37–40, 32, 30–31, 28, and 24 kDa. The 30–31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM-conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [¹²⁵I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [¹²⁵I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [¹²⁵I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an ∼400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the α2β2 form of the IGF-I receptor, and two β subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two β subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two β subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system. © 1996 Wiley-Liss, Inc.     

IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes.

Recombinant human insulin-like growth factors (rhIGF-I and rhIGF-II) and human insulin promoted the differentiation of spermatogonia into primary spermatocytes in newt testes fragments cultured in a chemically defined medium. The biological potency for promoting differentiation was dose-dependent for all the ligands with the highest potency displayed by IGF-I, followed by IGF-II, and the least by insulin. The difference in potency was larger between IGF-II and insulin than that between IGF-I and IGF-II. This order of biological potency was in good accordance with the order of affinity in binding specificity of [125I]IGF-I to the testicular membrane fractions: IGF-II and insulin competed the binding of [125I]IGF-I only at concentrations 20-fold and 100-fold higher, respectively, than IGF-I. Specific binding was observed in both somatic cells (mostly Sertoli cells) and germ cells (spermatogonia and primary spermatocytes), though the binding to somatic cells was about 2.7 times higher than that to germ cells. These results indicate that (1) specific binding sites for IGF-I are present in the newt testes, (2) IGF-II and insulin also bind to these receptors but to a lesser degree, and (3) IGF-II and insulin as well as IGF-I promote spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor.     

Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.

To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.     

Receptor autoradiographic localization of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands

We report here the first evidence of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands, obtained at autopsy. Sections of tissue were incubated with 0.1 nM [125I]IGF-I and analyzed using [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry. Specific binding sites of [125I]IGF-I were found to be localized in the definitive zone, fetal zone, and fetal medulla of the fetal adrenal glands. In the adult adrenal glands, the entire cortex and medulla were specifically labeled with [125I]IGF-I. Specific binding obtained at a concentration of 0.1 nM [125I]IGF-I to areas in the fetal and adult human adrenal glands was competitively displaced by unlabeled IGF-I, with an IC50 value of 0.34-2.54 nM, and 0.38-0.73 nM, respectively, whereas insulin was much less potent in displacing the binding. Acquisition of this knowledge will aid in studies on cell growth and steroid-catecholamines biosynthesis of the human adrenal gland.     

Tissue distribution of insulin-like growth factor receptors in the turkey.

1. The distribution and relative insulin-like growth factor (IGF) binding capacities of membranes derived from 14 tissues of the turkey were examined. 2. Affinity cross-linking analyses using [125I]IGF-I and [125I]IGF-II with membranes derived from the liver, pectoralis major muscle, gizzard, heart and brain indicated that both IGFs interact with only type-I IGF receptors on these tissues. 3. There was no evidence for the existence of a type-II IGF receptor in any tissue. 4. Although considerable variation was detected in the molecular weights of the IGF receptor alpha subunits between tissues (112.2-132.9 kDa), these differences did not appear to influence receptor-ligand affinities.     

Decrease in IGF-I binding sites on human promyelocytic leukemia cell line (HL-60) with differentiation

Specific insulin-like growth factor I (IGF-I) receptors on human promyelocytic leukemia cell line (HL-60) were identified and characterized. [125I]IGF-I specifically bound to the cells, and [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. [125I]IGF-I binding to the cells were displaced by multiplication stimulating activity (MSA) and porcine insulin, with potencies that were 10 and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present on the HL-60 cells. After the cells were differentiated to the macrophage-like cells by 12-o-tetra-decanoyl-phorbol-13-acetate (TPA) and 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), [125I]IGF-I binding to the cells decreased significantly. By Scatchard analysis, it was found to be due to a decrease in the number of IGF-I receptors. Thus, the differentiation of HL-60 cells to the macrophage-like cells was accompanied by a decrease in IGF-I receptors.     

Ultrastructural Localization of the Basic 7S Globulin in Soybean (Glycine max) Cotyledons

The basic 7S globulin (Bg) is a cysteine-rich glycoprotein insoybean seeds. Mature Bg is composed of subunits of high andlow molecular mass that are linked by disulfide bonds. Bg andBg-like proteins from seeds of other legumes and from carrotcells in suspension culture are capable of binding to insulinand to insulin-like growth factors. The ultrastructural localizationof Bg in immature soybean cotyledons was investigated immunocytochemicallyusing polyclonal antibodies and immunogold labeling. Bg waslocalized mainly in the middle lamella of the cell wall on theside facing the intercellular space. A few gold particles werealso detected near the plasma membrane, in Golgi vesicles thatwere fusing with the plasma membrane and in the rough endoplasmicreticulum (rER). These observations suggest that Bg is a novelcell-wall protein in the middle lamella of soybean seeds andthat is synthesized in the rER and secreted into the cell wallvia Golgi vesicles. (Received January 6, 1994; Accepted August 12, 1994)     

Insulin induces a selective heterologous desensitization of the mitogenic response of Swiss 3T3 cells to vasopressin

Prior incubation of confluent, quiescent cultures of Swiss 3T3 cells with insulin leads to a selective loss of mitogenic stimulation on re-addition of the combination of vasopressin and insulin in serum-free medium. The desensitization is specific for the action of vasopressin as insulin is fully active in the refractory cells when added in combination with other mitogens, whereas vasopressin is not. A prolonged treatment with insulin is required for induction of the refractoriness, half-maximal loss of response occurs after about 7 h and desensitization is complete after 12 h treatment. The refractory cells recover their response to vasopressin after more than 24 h incubation in the absence of insulin. A rapid response of the cells to vasopressin, inhibition of 125I-epidermal growth factor (125I-EGF) binding, is also desensitized by insulin. Desensitization is induced by insulin-like growth factor I (IGF-I), and partially by desoctapeptide insulin, but not by insulin B chain. Although the characteristics of insulin-induced desensitization are very similar to those of the homologous desensitization induced by vasopressin treatment, insulin does not bind to vasopressin antiserum or the [3H]vasopressin receptors of Swiss 3T3 cells. Insulin treatment also does not lead to any down-regulation of [3H]vasopressin receptors, and the refractoriness of the cells must therefore lie at a post-receptor step. Both insulin- and vasopressin-induced refractoriness to the mitogenic action of vasopressin can be blocked by a low level of cycloheximide. Both these agents therefore seem to induce the synthesis of specific protein(s) which selectively inhibit the mitogenic response of the cell to vasopressin.     

Degradation of somatomedins by the thioredoxin system

The insulin disulfide reducing thioredoxin system from E. coli was used to investigate a possible mechanism of degradation for the two somatomedins, insulin-like growth factor I and II (IGF-I and -II). The amounts of IGF-I and -II remaining after degradation were measured by use of human placenta radioreceptor assay. The results show that both IGF-I and -II were as sensitive to disulfide reduction as insulin.     

Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster

An insulin-degrading enzyme (IDE) from the cytoplasm of Drosophila Kc cells has been purified and characterized. The purified enzyme is a monomer with an s value of 7.2 S, an apparent Km for porcine insulin of 3 microM, and a specific activity of 3.3 nmol of porcine insulin degraded/(min.mg). N-Terminal sequence analysis of the gel-purified enzyme gave a single, serine-rich sequence. The Drosophila IDE shares a number of properties in common with its mammalian counterpart. The enzyme could be specifically affinity-labeled with [125I]insulin, has a molecular weight of 110K, and has a pI of 5.3. Although Drosophila Kc cells grow at room temperature, the optimal enzyme activity assay conditions parallel those of the mammalian IDE: 37 degrees C and a pH range of 7-8. The Drosophila IDE activity, like the mammalian enzymes, is inhibited by bacitracin and sulfhydryl-specific reagents. Similarly, the Drosophila IDE activity is insensitive to glutathione as well as protease inhibitors such as aprotinin and leupeptin. Insulin-like growth factor II, equine insulin, and porcine insulin compete for degradation of [125I]insulin at comparable concentrations (approximately 10(-6) M), whereas insulin-like growth factor I and the individual A and B chains of insulin are less effective. The high degree of evolutionary conservation between the Drosophila and mammalian IDE suggests an important role for this enzyme in the metabolism of insulin and also provides further evidence for the existence of a complete insulin-like system in invertebrate organisms such as Drosophila.