Sustained delivery of growth factors with high loading efficiency in a layer by layer assembly.

Layer by layer (LBL) assembly has garnered considerable interest due to its ability to generate multifunctional films with high tunability and versatility in terms of substrates and polyelectrolytes, allowing the option to use complex devices and drugs. Polyelectrolytes, such as growth factors (GFs), are essential, but costly, delicate, biological molecules that have been used in various tissue regeneration applications. For this reason, the controlled drug delivery of efficiently loaded GFs via LBL assembly (GF-LBL) can contribute to the establishment of cost-effective biologically triggered biomedical applications. We have developed an LBL method to load GFs (specifically, transforming growth factor beta 1, platelet-derived growth factor ßß, and insulin growth factor 1), with up to 90% efficiency approximately, by gas plasma surface activation and tuning the pH to increase the ionic strength of polyelectrolytes. Poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI) have been used to provide the initial necessary charge for multilayer build-up. Heparin and dextran sulphate have been investigated as counter polyelectrolytes to enhance the activity of GFs by protecting their ligands, where heparin resulted in the highest achievable loading efficiency for all GFs. Oxygen gas plasma and acidic pH levels also resulted in a significant increase in GF loading efficiency. The three GFs were released by diffusion and erosion in a controlled manner over lengthy time scales and the bioactivity was maintained for up to 14 days. When tested as implants in vitro, GF-LBL constructs increased fibroblast proliferation, influenced cell morphology and migration, and enhanced myofibroblast differentiation, indicating that the biological functionalities of the GFs were preserved. In conclusion, this developed LBL assembly method can provide a simple drug delivery system, which may yield more effective applications for tissue regeneration as well as biomedical sciences at large.

Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities.

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications.

Biological activity of the e domain of the IGF-1Ec as addressed by synthetic peptides.

Insulin-like growth factor-1 (IGF-1) is a multipotent growth factor involved in the growth, development and regulation of homeostasis in a tissue-specific manner. Alternative splicing, multiple transcription initiation sites and different polyadelynation signals give rise to diverse mRNA isoforms, such as IGF-1Ea, IGF-1Eb and IGF-1Ec transcripts. There is increasing interest in the expression of the IGF-1 isoforms and their potential distinct biological role. IGF-1Ec results from alternative splicing of exons 4-5-6 and its expression is upregulated in various conditions and pathologies. Recent studies have shown that IGF-1Ec is preferentially increased after injury in skeletal muscle during post-infarctal myocardium remodelling and in cancer tissues and cell lines. A synthetic analogue corresponding to the last 24 aa of the E domain of the IGF-1Ec isoform has been used to elucidate its potential biological role. The aim of the present review is to describe and discuss the putative bioactivity of the E domain of the IGF-1Ec isoform.

Synthesis, characterization, and biocompatibility of a novel biomimetic material based on MGF-Ct24E modified poly(D, L-lactic acid).

Mechano-growth factor (MGF) is an alternative splicing variant of Insulin-like growth factor I. MGF and its 24 amino acid peptide analog corresponding to the unique C-terminal E-domain (MGF-Ct24E) are the positive regulator for tissue regenesis in bone. A novel biomimetic poly(D, L-lactic acid) (PDLLA) modification was designed and synthesized based on MGF-Ct24E grafted maleic anhydride modified PDLLA (MPLA). MGF-Ct24Es were grafted into the side chain of MPLA via a stable covalent amide bond using 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as the condensing agent to produce biomimetic MPLA materials (MGF-Ct24E-MPLA). Fourier transform infrared spectrometry, amino acid analyzer, and elementary analysis were used to characterize the MGF-Ct24E-MPLA. The hydrophilicity of MGF-Ct24E-MPLA was evaluated by means of the water-uptake ratios and static water contact angle. Data revealed that the grafting efficiency of MGF-Ct24E was about 29.9%. MGF-Ct24E-MPLA had better hydrophilicity than PDLLA and MPLA. The osteoblasts behavior of proliferation, differentiation, and mineralization on PDLLA, MPLA, and MGF-Ct24E-MPLA films was investigated and the results indicated that the introduction of MGF-Ct24E could improve osteoblasts proliferation, mineralization, and delay differentiation. The MGF-Ct24E modified MPLA with higher bioactivity may have potential application for bone tissue engineering.

Contribution of residue B5 to the folding and function of insulin and IGF-I: constraints and fine-tuning in the evolution of a protein family.

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6-A11, A7-B7, and A20-B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7-A11, A6-B7, and A20-B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His(B5) --> Thr markedly destabilizes the hormone (DeltaDeltaG(u) 2.0 +/- 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr(B5) --> His (residue 4) specifies a unique structure with native (1)H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His(B5)-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His(B5) does not significantly enhance thermodynamic stability (DeltaDeltaG(u) 0.2 +/- 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr(B5)-insulin is decreased 5-fold, His(B5)-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr(B5) in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His(B5) in insulin highlights its critical role in insulin biosynthesis.

IGF-1 derived small neuropeptides and analogues: a novel strategy for the development of pharmaceuticals for neurological conditions.

Insulin-like growth factor-1 (IGF-1) is neuroprotective and improves long-term function after brain injury. However, its clinical application to neurological disorders is limited by its large molecular size, poor central uptake and mitogenic potential. Glycine-proline-glutamate (GPE) is naturally cleaved from the IGF-1 N-terminal and it is also neuroprotective after ischemic injury, which provided a novel strategy of drug discovery for neurological disorders. GPE is not enzymatically stable, thus intravenous infusion of GPE becomes necessary for stable and potent neuroprotection. The broad effective dose range and treatment window of 3-7 h after the lesion suggest its potential for treating acute brain injuries. G-2meth-PE, a GPE analogue designed to be more enzymatic resistant, has a prolonged plasma half-life and is more potent in neuroprotection. Neuroprotection by GPE and its analogue may involve modulation of inflammation, promotion of astrocytosis, inhibition of apoptosis and vascular remodelling. Acute administration of GPE also prevents 6-OHDA-induced nigrostrial dopamine depletion. Delayed treatment with GPE does not prevent dopamine loss, but improves long-term function. Cyclo-glycyl-proline (cyclic Gly-Pro) is an endogenous DKP that may be derived from GPE. Cyclic Gly-Pro and its analogue cyclo-L-glycyl-L-2-allylproline (NNZ 2591) are both neuroprotective after ischaemic injury. NNZ2591 is highly enzymatic resistant and centrally accessible. Its peripheral administration improves somatosensory-motor function and long-term histological outcome after brain injury. Our research suggests that small neuropeptides have advantages over growth factors in the treatment of brain injury, and that modified neuropeptides designed to overcome the limitations of their endogenous counterparts represent a novel strategy of pharmaceutical discovery for neurological disorders.

Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulfide-bridged D-peptide analogue of insulin-like growth factor 1.

IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGF1 receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen-sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS-1 may serve as an alternative target to overexpressed IGF1R in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRS1 siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.

Synthesis and characterization of biotinylated forms of insulin-like growth factor-1: topographical evaluation of the IGF-1/IGFBP-2 AND IGFBP-3 interface.

Activation of the insulin-like growth factor-1 (IGF)-1 receptor signaling pathways by IGF-1 and IGF-2 results in mitogenic and anabolic effects. The bioavailability of the IGFs is regulated by six soluble binding proteins, the insulin-like growth factor binding proteins (IGFBPs), which bind with approximately 0.1 nM affinity to the IGFs and often serve as endogenous antagonists of IGF action. To identify key domains of IGF-1 involved in the interaction with IGFBP-2 and IGFBP-3, we employed IGF-1 selectively biotinylated on residues Gly 1, Lys 27, Lys 65, and Lys 68. All monobiotinylated species of IGF-1 exhibited high affinity ( approximately 0.1-0.2 nM) for IGFBP-2 and IGFBP-3 in solid-phase-binding assays. However, different labeling intensities were observed in ligand blot analysis of IGFBP-2 and IGFBP-3. The N(epsilon)(Lys65/68)(biotin)-IGF-1 (N(epsilon)(Lys65/68b)-IGF-1) probe exhibited the highest signal intensity, while N(alpha)(Gly1b)-IGF-1 and N(epsilon)(Lys27b)-IGF-1 demonstrated significantly lower signals. When taken together, these results suggest that, once bound to IGFBP-2 or IGFBP-3, the biotin moieties of N(alpha)(Gly1b)-IGF-1 and N(epsilon)(Lys27b)-IGF-1 are inaccessible to NeutrAvidin-peroxidase, the secondary binding component. Ligand blots using IGF-1 derivatized with a long chain form of the N-hydroxysuccinimide biotin (NHS-biotin) to yield N(alpha)(Gly1)(LC-biotin)-IGF-1 and N(epsilon)(Lys27)(LC-biotin)-IGF-1 demonstrated increased signal intensity compared with their NHS-biotin counterparts. In BIAcore analysis, IGFBP-2 and IGFBP-3 bound only to the N(epsilon)(Lys65/68b)-IGF-1-coated flowcell of a biosensor chip, confirming the inaccessibility of Gly 1 and Lys 27 when IGF-1 is bound to IGFBP-2 and IGFBP-3. These data confirm the involvement of the IGFBP-binding domain on IGF-1 in binding to IGFBP-2 and IGFBP-3 and support involvement of the IGF-1R-binding domain in IGFBP binding.

Synthesis and characterization of insulin-like growth factor (IGF)-1 photoprobes selective for the IGF-binding proteins (IGFBPS). photoaffinity labeling of the IGF-binding domain on IGFBP-2.

Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes, N(alphaGly1)-(4-azidobenzoyl)-IGF-1 (abG(1)IGF-1) and N(alphaGly1)-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1 (bedG(1)IGF-1), selective for the IGFBPs were synthesized by derivatization of the alpha-amino group of Gly(1), known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG(1)IGF-1.recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG(1)IGF-1.rhIGFBP-2 complex revealed photoincorporation within residues 212-227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2.

Critical evaluation of a theory of molecular recognition using human insulin-like-growth-factor-I fragment 21-40 and its complementary peptide.

Using solid-phase methods we have synthesized human insulin-like-growth-factor-I (IGF-I) fragment 21-40 (IGF-I 21-40) and the peptide derived from the 5'----3' translation of the complementary nucleic acid of this peptide, 'I-FGI 20-40' (the complementary peptide). According to a recently proposed theory of molecular recognition, these two peptides should bind specifically to each other. We have tested this theory by using both solid- and solution-phase direct-binding assays for this complementary-peptide pair. We have also investigated the ability of I-FGI 20-40 to interfere with native IGF-I binding during radioimmunoassay (r.i.a.), radio-receptor (r.r.a.) assay and ligand-blot analysis of IGF-binding proteins. We have obtained no evidence of any interaction between IGF-I 21-40 and I-FGI 20-40 in either solid- or solution-phase assays. In addition, I-FGI 20-40 does not interfere in the assays used to detect IGF-I binding antibodies (r.i.a.), receptors (r.r.a.) or binding proteins (ligand blots). Our data therefore question the universality of this particular theory of molecular recognition.

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.

An insulin-like hybrid consisting of a modified A-domain of human insulin-like growth factor I and the B-chain of insulin.

We have synthesized an insulin-like compound, consisting of the B-chain of bovine insulin and an A-chain corresponding to the A-domain of human insulin-like growth factor-I (IGF-I), in which the isoleucine residue normally present in position 2 of the A-domain of IGF-I has been replaced with glycine. Biological evaluation of the compound indicated that its insulin-like activity (insulin receptor-binding and stimulation of lipogenesis) was 0.2%, and its growth-factor activity (stimulation of thymidine incorporation) was less than 1%, both relative to natural insulin. We conclude that interactions between IleA2 and TyrA19, which are crucial to high biological activity in insulin, are also present in IGF-I, and are equally critical for its biological activity.

Direct identification of disulfide bond linkages in human insulin-like growth factor I (IGF-I) by chemical synthesis.

The primary structure of human IGF-I, except for the disulfide bond system, has been reported by Rinderknecht and Humbel. IGF-I afforded the corresponding characteristic peptide fragment on V8 protease digestion, which contained Cys6, Cys47, Cys48, and Cys52. Two possible fragments, Type I with Cys6-Cys47 and Cys48-Cys52, and Type II with Cys6-Cys48 and Cys47-Cys52, were synthesized. The disulfide bond system of IGF-I was unequivocally determined to be the Type II form along with Cys18-Cys61. Interestingly, the Type I system was included in the disulfide bond isomer produced as the main by-product in the refolding step on IGF-I synthesis by the recombinant DNA method.

Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology.

Natural human insulin-like growth factor (IGF) I is a relatively large single chain peptide (mol. wt 7649) with a known sequence of 70 amino acids. C6----C48, C47----C52 and C18----C61 assignments have been previously proposed for the three disulphide bonds linking six cysteine residues (C6, C18, C47, C48, C52 and C61), on the basis of analogy (and homology) with proinsulin. In this work, IGF I synthesized by recombinant DNA technology (r-IGF I, with identical biological activity and chromatographic behaviour) was subjected to a three-step mass spectrometric analysis in combination with degradation methods for structural verification. Firstly, the correct molecular weight of the intact peptide was determined by high-mass fast atom bombardment (FAB) analysis. Secondly, twofold enzymatic degradation (chymotrypsin followed by V8 protease, 'FAB mapping' of the cleavage products) was employed in order that fragments with 'isolated' S-S bonds would be produced which allow an unambiguous assignment. This immediately established the C18----C61 linkage as it was contained in a singly bridged two-chain peptide. The two other S-S bonds, which cross-link C6 and the 'tight' C47 to C52 segment, remained 'unresolved' within a more complex, doubly bridged triple-chain peptide. Thirdly, further degradation of this structural block, in which cleavage of the C47-C48 bond was required to discern these bonds, was carried out by using FAB tandem mass spectrometry and (for additional corroboration) manual Edman degradation. Both procedures confirmed the original C6----C48/C47----C52 prediction.(ABSTRACT TRUNCATED AT 250 WORDS)

Determinants of growth-promoting activity reside in the A-domain of insulin-like growth factor I.

A two-chain, disulfide linked, insulin-like compound embodying the A-domain of insulin-like growth factor I (IGF-I) and the B-chain of insulin has been synthesized and characterized with respect to insulin-like biological activity and growth-promoting potency. The compound displays a potency of ca. 41% relative to insulin in assays for insulin-like activity (e.g., lipogenesis) but significantly higher activity than insulin, ca. 730% relative to insulin, in growth factor assays (e.g., thymidine incorporation). The compound is, however, a less potent growth factor than IGF-I itself, ca. 26.5% relative to IGF-I, and is not recognized by IGF carrier proteins. We conclude that structural features contained in the A-domain of IGF-I are primarily responsible for the growth-promoting ability displayed by IGF-I, while features in the B-domain are responsible for recognition by IGF carrier proteins.

Assay of somatomedin C by cartridge extraction prior to radioimmunoassay with antiserum developed against synthetic somatomedin C.

The whole molecule of human somatomedin C (SM-C) prepared by the total synthesis method was used as an antigen to produce an antiserum for a radioimmunoassay. Since plasma proteins that bind SM-C interfere with the assay, a method was developed that uses acid dissociation followed by C-2 cartridge extraction to strip SM-C from its binding proteins before assay. This assay has no cross-reactivity with human proinsulin or insulin-like growth factor II (IGF-II). The SM-C values in 339 normal subjects showed age-dependence, increasing from childhood to a peak at age 14 to 16 years and decreasing sharply before adulthood. In adults, the SM-C values decreased gradually with age. All 13 patients with acromegaly who were tested had an increased SM-C value, with no overlap with the normal range. The 12 patients with prolactinoma but non-growth-hormone-producing pituitary tumor had no increase in SM-C. Two children with pituitary deficiency had low SM-C values; one of these children received growth hormone therapy, and his SM-C value increased from undetectable to normal. By three weeks after discontinuation of the therapy, his SM-C value was again undetectable. Of 20 children with short stature and constitutional delay of growth and development, SM-C was below normal in 70 percent and normal in 30 percent. Two patients with malnutrition had below-normal SM-C values.

Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-II receptor: comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I.

Although insulin-like growth factors (IGF) I and II bind with high affinity to structurally discrete receptors, they bind with a lesser affinity to each other's receptor. We have evaluated the affinity of five different IGF-I preparations (three natural IGF-I preparations, one synthetic preparation, and one recombinant DNA-derived) for the IGF-II receptor in rat placental membranes, 18-54,SF cells and BRL-3A cells. In all tissues tested, the natural IGF-I preparations demonstrated an affinity for the IGF-II receptor which was 10-20% that of IGF-II. However, the recombinant and synthetic IGF-I preparations exhibited substantially lower affinities than natural IGF-I for this receptor, with only 10-25% reduction in (125-I)iodo IGF-II binding at peptide concentrations up to 400 ng/ml. Radioimmunoassay of the natural IGF-I preparations with an antibody directed against the unique C-peptide region of IGF-II demonstrated that contamination of IGF-I preparations with immunoreactive IGF-II could not exceed 5%. These results demonstrate that IGF-I purified from human plasma has a different affinity for the IGF-II receptor than does synthetic or recombinant IGF-I. Furthermore, these data are consistent with the hypothesis that IGF-I, itself, may be heterogeneous, and that subforms may vary in their affinities for the IGF receptors. Alternatively, IGF-I preparations which have been considered to be pure may be contaminated with small amounts of IGF-II, resulting in overestimation of the affinity of IGF-I for the type II IGF receptor.

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.