Biomedical subjects

C J Bagley

Publications and source records attributed to C J Bagley.

At least 37 records · Page 2Linked to original sources

PubMed1996

Interacting residues in the extracellular region of the common beta subunit of the human granulocyte-macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 receptors involved in constitutive activation.

A previous study using random mutagenesis identified an activating mutation in the common beta subunit (hbetac) of the human granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5 receptors in which an isoleucine residue (Ile374) in the extracellular region of hbetac is replaced by asparagine (Jenkins, B. J., D'Andrea, R., and Gonda, T. J. (1995) EMBO J 14, 4276-4287). To investigate the mechanism by which this mutation (I374N) acts, we employed site-directed mutagenesis to explore predictions based on a structural model of hbetac. We focused on possible interactions between Ile374 and other hydrophobic residues in its vicinity and found that replacement of two such residues, Leu356 and Trp358, with asparagine resulted in constitutive activation of hbetac. Hydrophilic substitutions at both of these positions and at position 374 resulted in the greatest degree of activation, as measured by the growth rate of factor-independent cells, while hydrophobic substitutions had lesser or no effects. Moreover, these "weak" substitutions appeared to synergize, since factor-independent cells expressing the double mutants I374F/W358F and I374F/L356A showed substantially higher growth rates than the single mutants. Taken together, these results suggest that Ile374 normally interacts with Leu356 and Trp358, and that disruption of these interactions results in a conformational change in hbetac that leads to constitutive activity. A model relating this notion to the predicted structure and to ligand- and alpha subunit-dependent activation of hbetac is proposed.

Amino Acid Sequence↗

PubMed1996

A single tyrosine residue in the membrane-proximal domain of the granulocyte-macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 receptor common beta-chain is necessary and sufficient for high affinity binding and signaling by all three ligands.

The beta-chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) receptors functions as a communal receptor subunit and is often referred to as beta common (betac). Analogous to other shared receptor subunits including gp130 and the IL-2R gamma chain, betac mediates high affinity binding and signal transduction of all of its ligands. It is not clear, however, how these common receptor subunits can recognize several ligands and indeed whether they exhibit a common binding pocket to accomplish this. We have performed molecular modeling of betac based on the known structures of the growth hormone and prolactin receptors and targeted the putative F'-G' loop for mutagenesis. Substitution of this whole predicted loop region with alanines completely abrogated high affinity binding of GM-CSF, IL-3, and IL-5. Individual alanine substitutions across the loop revealed that a single residue, Tyr421, is critical for high affinity binding of GM-CSF, IL-3, and IL-5, whereas alanine substitution of adjacent residues has little or no effect on high affinity binding. Significantly, reintroducing Tyr421 into the polyalanine-substituted mutant restored high affinity ligand binding of GM-CSF, IL-3, and IL-5, indicating that within this region the tyrosine residue alone is sufficient for high affinity ligand interaction. Functional studies measuring STAT5 activation revealed that alanine substitution of Tyr421 severely impaired the ability of betac to signal. These results show for the first time that a single residue in a shared receptor subunit acts as a binding determinant for different ligands and may have implications for other receptor systems where communal receptor subunits exhibit hydrophobic residues in their putative F'-G' loops. These results also raise the possibility that a single compound targeted to this region may simultaneously inhibit the binding and function of multiple cytokines.

Alanine↗

PubMed1996

Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist.

The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3-dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R alpha-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R alpha-chain expressed by transfected cells and bound to primary cells expressing IL-3R alpha. MoAb 7G3 bound the IL-3R alpha-chain with a kd of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low-affinity IL-3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R alpha-chain MoAbs failed to inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R alpha-chain mutants and IL-3R alpha/GM-CSFR alpha chimeras revealed that 31 amino acids in the N-terminus of IL-3R alpha were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3R alpha in IL-3 binding. Since this domain is unique to the IL-3/GM-CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.

Animals↗

PubMed1996

Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding.

The human interleukin-3 receptor (IL-3R) is a heterodimer that comprises an IL-3 specific alpha chain (IL-3R alpha) and a common beta chain (beta C) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. These receptors belong to the cytokine receptor superfamily, but they are structurally and functionally more related to each other and thus make up a distinct subfamily. Although activation of the normal receptor occurs only in the presence of ligand, the underlying mechanisms are not known. We show here that human IL-3 induces heterodimerization of IL-3R alpha and beta c and that disulfide linkage of these chains is involved in receptor activation but not high-affinity binding. Monoclonal antibodies (MAb) to IL-3R alpha and beta c were developed which immunoprecipitated, in the absence of IL-3, the respective chains from cells labelled with 125I on the cell surface. However, in the presence of IL-3, each MAb immunoprecipitated both IL-3R alpha and beta c. IL-3-induced receptor dimers were disulfide and nondisulfide linked and were dependent on IL-3 interacting with both IL-3R alpha and beta c. In the presence of IL-3 and under nonreducing conditions, MAb to either IL-3R alpha or beta c immunoprecipitated complexes with apparent molecular weights of 215,000 and 245,000 and IL-3R alpha and beta c monomers. Preincubation with iodoacetamide prevented the formation of the two high-molecular-weight complexes without affecting noncovalent dimer formation or high-affinity IL-3 binding. Two-dimensional gel electrophoresis and Western blotting (immunoblotting) demonstrated the presence of both IL-3R alpha and beta c in the disulfide-linked complexes. IL-3 could also be coimmunoprecipitated with anti-IL-3R alpha or anti-beta c MAB, but it was not covalently attached to the receptor. Following IL-3 stimulation, only the disulfide-linked heterodimers exhibited reactivity with antiphosphotyrosine antibodies, with beta c but not IL-3R alpha being the phosphorylated species. A model of IL-3R activation is proposed which may be also applicable to the related GM-CSF and IL-5 receptors.

Animals↗

PubMed1995

Interaction of GM-CSF and IL-3 with the common beta-chain of their receptors.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL-3) are cytokines active in both normal and abnormal hemopoiesis, inflammation, and immunity. Their biological activity is mediated via receptors that comprise a ligand-specific alpha chain and a beta chain that is common to the GM-CSF, IL-3, and IL-5 receptors. To understand the mechanism of action of GM-CSF and IL-3 in both normal and pathological conditions, we are seeking to define the structural elements required for ligand/receptor and receptor/receptor contact and their role in cellular activation. To this end we have identified a conserved motif in the first helix of GM-CSF, Glu21 that is critical for high affinity binding and biological activity. Charge-reversal mutagenesis of this residue generates a GM-CSF analogue that is devoid of biological activity and can antagonize the activity of wild-type GM-CSF. This probably results from the selective deficiency in interaction with the beta chain of the receptor and suggests that similar antagonists for IL-3 and IL-5 are also feasible. Complementary mutagenesis studies on the receptor beta chain have identified the putative B'-C' loop in the membrane-proximal domain as being critical for the high affinity binding of GM-CSF but not IL-3. Characterization of the specificity of sites of interaction between the ligands and receptors may permit the design of specific or genetic antagonists that may have important therapeutic implications.

Amino Acid Sequence↗

PubMed1994

Three residues in the common beta chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.

Amino Acid Sequence↗

PubMed1994

Specific human granulocyte-macrophage colony-stimulating factor antagonists.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activities. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor alpha chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor alpha-chain-common beta-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor alpha-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.

Amino Acid Sequence↗

PubMed1994

Identification of residues in the first and fourth helices of human granulocyte-macrophage colony-stimulating factor involved in biologic activity and in binding to the alpha- and beta-chains of its receptor.

Residues within the first and fourth helices of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) were analyzed for their role in biologic activity and interaction with the alpha- and beta-chains of the hGM-CSF receptor. Within the first helix substitution of the surface residues Glu14, Asn17, Gln20, Arg23, Arg24, and Asn27 or the buried residues Ala18, Leu25, and Leu28 did not significantly impair bioactivity or receptor binding. Substitutions at the buried residues Ala22 and Leu26 had intermediate bioactivity. However, substitutions of the surface residue Glu21 or the buried residue Ile19 reduced the relative bioactivity of the analogues to as little as 0.45% and 0.3%, respectively. Substitution of the charged surface residues of the fourth helix showed that substitution at Glu104, Lys107, and Lys111 had no significant effect on bioactivity, but substitution at Glu108 and Asp112 reduced the potency of the analogues to 34% and 7%, respectively. Receptor binding studies showed that, whereas Glu21 is the critical residue for binding to the hGM-CSF-receptor beta-chain, Asp112 is likely to be involved in binding to the GM-CSF-receptor alpha-chain. These results establish the relative contribution of residues in the first and fourth helices for GM-CSF bioactivity and receptor binding, and support a model where the fourth helix of GM-CSF interacts with the alpha-chain, and the first helix with the beta-chain of the GM-CSF receptor.

Amino Acid Sequence↗

PubMed1994

Two contiguous residues in human interleukin-3, Asp21 and Glu22, selectively interact with the alpha- and beta-chains of its receptor and participate in function.

We have previously reported that the predicted first helix of human interleukin (IL)-3 contains a hydrophilic region encompassing residues Asp21, Glu22, and Thr25 that is crucial for biological activity and IL-3 receptor binding. Using single amino acid substitution mutagenesis, we have now determined that Asp21 and Glu22, but not Thr25, were crucial for full IL-3 activity. Mutant D21R was 30-fold less potent than wild type IL-3 in the stimulation of biological activity. It also exhibited a similar reduction in its ability to bind to the cloned high affinity IL-3 receptor complex (alpha- and beta-chains) or to the receptor alpha-chain alone, indicating that residue 21 is involved in contacts with the alpha-chain. Mutant E22R was approximately 20,000-fold less potent than wild type IL-3 in the stimulation of biological activity and in binding to the IL-3 receptor high affinity complex. However, the binding of E22R to the IL-3 receptor alpha-chain alone was similar to that of wild type IL-3, suggesting that this mutant was defective in interactions with the receptor beta-chain. These results show that two contiguous residues in the N-terminal region of IL-3 mediate binding to the two different chains of the IL-3 receptor and emphasize the functional significance of the conserved Glu in the first helix of the IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 cytokine subfamily.

Amino Acid Sequence↗

PubMed1993

On the biosynthesis of bovine pancreatic trypsin inhibitor (BPTI). Structure, processing, folding and disulphide bond formation of the precursor in vitro and in microsomes.

The natural gene for bovine pancreatic trypsin inhibitor (BPTI) was expressed by in vitro transcription/translation systems as the 100-residue pre-proBPTI, with a signal peptide for translocation into the endoplasmic reticulum. Expression in the presence of microsomes defined the site of co-translational cleavage of the signal peptide. The resulting proBPTI in the microsomes consists of the 58 residues of mature BPTI, plus an additional 13 residues at the N terminus, including a cysteine residue at position -10, and seven residues at the C terminus. ProBPTI remained in the unfolded, reduced form within microsomes when synthesized under reducing conditions, but folded and formed disulphide bonds rapidly when the disulphide form of glutathione was added. Complete folding could occur within about one minute, even when residue Cys10 was replaced by Ser. The structure of proBPTI was determined by circular dichroism and two-dimensional NMR and found to be that of mature BPTI with flexible extensions on both termini. Its inhibition of the activity of alpha-chymotrypsin was indistinguishable from that of the mature protein. The extensions of the precursor appeared to play only very minor roles in refolding in vitro under conditions where folding and disulphide bond formation are coupled. Under pH and redox conditions thought to reflect those in vivo, complete folding and disulphide bond formation required several hours. Addition of protein disulphide isomerase to in vitro folding experiments caused substantial and similar increases in the rate of formation of the fully folded state for both mature BPTI and proBPTI; the half time for folding to the native state was reduced to approximately two minutes, which is comparable to that occurring in microsomes. The absence of substantial effects of the N and C-terminal extensions on the protein structure, inhibitor activity and refolding leaves their functional roles to be discovered.

Amino Acid Sequence↗

PubMed1993

A model for the interaction of the GM-CSF, IL-3 and IL-5 receptors with their ligands.

The high affinity receptors for GM-CSF, IL-3 and IL-5 are heterodimers consisting of a ligand-specific alpha chain and a common beta chain. These proteins are members of a family of proteins known as the "cytokine receptor family" which is characterized by the presence of a 200-residue ligand-binding module. The GM-CSF, IL-3 and IL-5 receptor alpha chains constitute a distinct subgroup and share features not found in other members of the cytokine receptor family, features which we propose to be important for their interaction with the common beta chain and for their binding of the structurally-related ligands. The growth hormone receptor is a well-characterized member of the cytokine receptor family. Based on the structure of the complex between growth hormone and its receptor, we have proposed sites of contact between the GM-CSF, IL-3 and IL-5 receptors and their cognate ligands.

Amino Acid Sequence↗

PubMed1992

Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3, following their expression in Escherichia coli as fusion proteins.

The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.

Amino Acid Sequence↗

PubMed1990

Synthesis of insulin-like growth factor I using N-methyl pyrrolidinone as the coupling solvent and trifluoromethane sulphonic acid cleavage from the resin.

Insulin-like growth factor I (IGF-I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N-methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double-coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S-acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF-I peptides were full agonists of natural IGF-I in a radioimmunoassay, in an IGF-I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF-binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF-I.

Animals↗

PubMed1990

Enhanced potency of truncated insulin-like growth factor-I (des(1-3)IGF-I) relative to IGF-I in lit/lit mice.

The relative potencies of insulin-like growth factor (IGF-I) and the N-terminal truncated derivative, des(1-3)IGF-I, have been compared in lit/lit mice. Injection of 30 micrograms IGF-I, 30 micrograms des(1-3)IGF-I or 3 micrograms des(1-3)IGF-I daily for 3 weeks increased total length and nose-rump length of the animals substantially more than in controls or animals treated with 3 micrograms IGF-I daily. Body weight changes were not statistically significant. The lower dose of des(1-3)IGF-I, but not that of IGF-I, led to increases in kidney and heart weights relative to controls, while the higher dose of either IGF-I or des(1-3)IGF-I also increased the weights of liver, lungs and stomach. These results indicate that the higher potency of des(1-3)IGF-I demonstrated in cultured cells also applies in vivo to at least one strain of GH-deficient animals.

Animals↗

PubMed1989

A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide.

In order to elucidate the role of the N-terminus of insulin-like growth factor 1 (IGF-1) with respect to its biological properties, we chemically synthesized analogues of IGF-1 truncated by one to five amino acid residues from the N-terminus. In a bioassay that measured the stimulation of protein synthesis in rat L6 myoblasts, the concentrations required to produce a half-maximal response were: IGF-1, 13 ng/ml; des-(1)-IGF-1, 10 ng/ml; des-(1-2)-IGF-1, 13 ng/ml; des-(1-3)-IGF-1, 1.5 ng/ml; des-(1-4)-IGF-1, 5.1 ng/ml; des-(1-5)-IGF-1, 1200 ng/ml. When tested for their abilities to compete with 125I-IGF-1 binding to L6 myoblasts at 3 degrees C, the concentrations required for 50% competition were: IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1, 20 ng/ml; des-(1-3)-IGF-1, 14 ng/ml; des-(1-4)-IGF-1, 40 ng/ml; des-(1-5)-IGF-1, greater than 1000 ng/ml. Receptor-binding experiments at 25 degrees C, however, gave results suggesting that the myoblasts were secreting a binding protein selective for the three longest peptides. This interpretation was confirmed by binding studies with medium conditioned by the L6 myoblasts as well as binding protein purified from MDBK-cell-conditioned medium. In both cases IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1 competed for tracer IGF-1 binding at least 60-fold better than did the three shorter peptides. The results obtained account for the increased potency of des-(1-3)-IGF-1 and des-(1-4)-IGF-1, since their activities are not attenuated by the binding protein, and the relatively lower potency of des-(1-4)-IGF-1 is a consequence of this peptide binding less well to the L6-myoblast receptor.

Animals↗

PubMed1989

Effects of insulin-like growth factors on protein metabolism: why are some molecular variants more potent?

The insulin-like growth factors (IGFs) produce a dual anabolic effect on protein metabolism in cultured cells via a stimulus of the synthetic pathway and an inhibition of the degradative pathway. Accordingly, they offer promise as agents that may retard the extensive and life-threatening negative nitrogen balance that accompanies human polytrauma. In this report we describe the discovery of a novel, more potent, form of IGF-1, des-(1-3)-IGF-1, and explain its increased action as resulting from higher concentrations of the free peptide produced because des-(1-3)-IGF-1 binds very poorly to IGF carrier proteins released from most cell types. The truncated growth factor retains a long biological half-life because it is attached to blood IGF-binding proteins. Further understanding of the biological significance of IGF, coupled with synthetic approaches directed at the production of mutant IGF-1 molecules, can be expected to yield significant advances in the treatment of polytrauma.

Animals↗

PubMed1987

Natural and synthetic forms of insulin-like growth factor-1 (IGF-1) and the potent derivative, destripeptide IGF-1: biological activities and receptor binding.

Insulin-like growth factor-1 (IGF-1), whether recombinant, chemically-synthesised or purified from bovine colostrum, was equipotent in radioreceptor assays with IGF-1 or insulin-like growth factor-2 (IGF-2) as radioligand as well as in its ability to stimulate protein synthesis in L6 myoblasts. The N-terminal truncated, destripeptide derivative of IGF-1 was approximately 7 times more potent than IGF-1 in the protein synthesis bioassay. This increased activity occurred equally with the peptide purified from bovine colostrum as with chemically-synthesised material. The higher potency of the truncated form was not associated with an increased ability to compete for IGF-1 binding to L6 myoblasts.

Cross Reactions↗

PubMed1986

Purification and partial sequence analysis of insulin-like growth factor-1 from bovine colostrum.

Growth-promoting activity in bovine colostrum has been detected as the capacity to stimulate protein synthesis in L6 myoblasts. By using this assay as a measure of bioactivity, a growth factor has been purified to near homogeneity from centrifuged colostrum by a series of steps including acid extraction, chromatography on sulphopropyl-Sephadex, followed by adsorption to, and elution from, C18 columns using acetonitrile and propan-1-ol gradients. The purified growth factor has a low solubility at neutral and alkaline pH and has an Mr of 7800 by gel-permeation chromatography. Sequence analysis of the first 30 amino acids from the N-terminus indicated complete identity in this region with human insulin-like growth factor-1. Accordingly we conclude that the purified growth factor is bovine insulin-like growth factor-1.

Amino Acid Sequence↗