Metal-binding properties of a calcium-dependent monoclonal antibody.

The calcium-dependent mAb, M1 (also called anti-Flag or 4E11) was studied using a newly developed metal-sensitive enzyme-linked immunosorbent assay (ELISA). This antibody, specific for a calcium complex of the peptide antigen, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, has found widespread use as a mild purification reagent for Flag-epitope tagged recombinant proteins. Although M1 affinity columns release monovalent Flagged proteins in the absence of calcium, the antibody retains substantial affinity for the Flag sequence even in metal-free conditions, so that it has been impossible to use it to develop a metal-sensitive ELISA assay. This is due to the ability of the antibody to remain bound to polyvalent surface-coated antigen, for instance, when Flagged proteins are bound to ELISA plates or blotting filters. The resultant antigen polyvalence raises the avidity of the Flag antibody to a point where the reaction is essentially calcium-independent. However, when the antibody itself was made monovalent, by proteolytic cleavage to the Fab, this situation was reversed and the ELISA reaction became calcium-dependent. This new metal-dependent ELISA assay was used to explore the metal requirements of the antibody in detail. Among divalent metals, binding tapered off with increasing radius above that of calcium, or with decreasing radius below that of calcium. Several smaller metals, such as nickel, acted as inhibitors of the binding reaction. Substantial binding was demonstrated for heavy metals such as cadmium, lanthanum and samarium. Because it is of interest to use this antibody for the co-crystallization of recombinant Flag-fusion proteins, the ability to bind heavy metals was a significant finding.

Efforts toward deriving the CD spectrum of a 3(10) helix in aqueous medium.

There have been two recent reports suggesting that 3(10) helices can be distinguished from alpha helices by circular dichroism. The differentiating feature is stated to be a [theta]222:[theta]208 ratio (R2) distinctly smaller than unity. This has been reported for a C(alpha)alpha'-disubstituted homooctamer [Toniolo et al. (1996), J. Am. Chem. Soc. 118, 2744-2745] and for alanine-rich systems of 16-21 residue length with modest fractional helicity [Millhauser (1995) Biochemistry 34, 3873-3877]. We report here the changes in the CD spectrum produced by inserting aminoisobutyric acid (Aib) residues into the helical domain of human pancreatic amylin. In order to examine this effect at comparable net fractional helicities, CD spectra were measured for each species during the course of a helicity titration by trifluoroethanol addition. The addition of five Aib residues gave results of particular interest. At low net fractional helicity, this Aib-rich system displays a diminished pi-->pi* (circa 208 nm) rotational strength versus the less Aib-rich species. However, NMR data and comparisons of CD difference spectra suggest that fluoroalcohol-induced extension of the short Aib-rich helix is in the form of an alpha helix. Given the diminished intensity of the minimum at 208 nm at low net helicity when 3(10) conformations should contribute, we urge extreme caution in using a [theta]222:[theta]208 ratio smaller than unity as a diagnostic for 3(10) helices.

Selective amylin antagonist suppresses rise in plasma lactate after intravenous glucose in the rat. Evidence for a metabolic role of endogenous amylin.

Data presented here provide the first demonstration that circulating amylin regulates metabolism in vivo, and support an endocrine hormonal role that is distinct from its autocrine action at pancreatic islets. When rats were pre-treated with the potent amylin antagonist AC187 (n = 18), and then administered a 2 mmol glucose load, the rise in plasma lactate was less than in rats administered glucose only (n = 27; P < 0.02). When rats were treated so that plasma glucose and insulin profiles were similar (n = 8), the increase in plasma lactate in the presence of AC187 was only 50.3% as high as the increase when AC187 was absent (P < 0.001). These experimental results fit with the view that some of the lactate appearing in plasma after a glucose load comes from insulin-sensitive tissues. The experiments also support the view that an important fraction of the increase in lactate depends on processes inhibited by a selective amylin antagonist, most likely amylin action in muscle.

A calcium-dependent antibody for identification and purification of recombinant proteins.

We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in a single chromatographic step. The marker peptide could subsequently be removed by proteolysis with the enzyme enterokinase.

Regulation of muscle glycogen metabolism by CGRP and amylin: CGRP receptors not involved.

The aim of the present study was to determine whether amylin and calcitonin gene-related peptide (CGRP) act through shared or distinct receptors to inhibit insulin-stimulated incorporation of [14C]-glucose into glycogen. Rat amylin was 3 fold more potent than either rat alpha CGRP or rat beta CGRP at reducing glycogen synthesis from [14C]-glucose in insulin-treated rat soleus muscle. This action was blocked by peptide antagonists, with the rank order of potency being AC187 > salmon calcitonin8-32 (sCT8-32) > h-alpha CGRP8-37 for antagonism of either amylin or CGRP. The antagonist potency order correlated with affinity for amylin receptors measured in rat nucleus accumbens but not CGRP receptors measured in rat L6 muscle cells. Inhibition of glucose incorporation into glycogen by amylin and CGRP appears to be mediated by shared receptors that have the pharmacological characteristics of amylin receptors, and are distinct from previously described CGRP receptors.

Dose-response for glycaemic and metabolic changes 28 days after single injection of long-acting release exenatide in diabetic fatty Zucker rats.

AIMS/HYPOTHESIS: Exenatide (exendin-4) injected subcutaneously twice daily reduces glycaemic deterioration in diabetic fatty Zucker (ZDF) rats and reduces HbA1c in humans with type 2 diabetes. Because tachyphylaxis may develop with continuous peptide exposure, we examined the activity of a long-acting-release (LAR) formulation of exenatide on HbA1c, insulin sensitivity and beta cell secretion in ZDF rats. METHODS: Single subcutaneous injections of a poly-lactide-glycolide microsphere suspension (3% peptide) containing 0, 1, 10, 100, 1,000, 3,000 or 9,000 mug exenatide were administered to 9-week-old ZDF rats with matched initial HbA1c values (n=7 rats/group). RESULTS: In contrast to the progressive 3.22+/-0.42% increase in HbA1c in control ZDF rats observed over 28 days, single exenatide-LAR injections dose-proportionally prevented such glycaemic deterioration (median effective dose 74 microg+/-0.1 log per rat; median effective concentration 52 pmol/l+/-0.06 log). Hyperinsulinaemic-euglycaemic clamp procedures incorporating an intraclamp glucose challenge performed 28 days after treatment revealed increases in beta cell response to the glucose challenge at lower exenatide-LAR doses, and up to a 2.1-fold increase in insulin sensitivity at higher exenatide-LAR doses. CONCLUSIONS/INTERPRETATION: The finding that a single dose of exenatide-LAR enhanced glucose control for 28 days in the ZDF rat model of type 2 diabetes suggests that tachyphylaxis is unlikely to be a feature of exenatide-LAR preparations, and supports further clinical exploration.

Metabolism of unsaturated derivatives of valproic acid in rat liver microsomes and destruction of cytochrome P-450.

2-n-Propyl-4-pentenoic acid (delta 4-VPA), an unsaturated metabolite of valproic acid (VPA), and the ethyl ester of delta 4-VPA were tested for their ability to cause NADPH- and time-dependent loss of cytochrome P-450 in hepatic microsomal preparations from phenobarbital-pretreated rats. Ethyl delta 4-VPA gave a large amount of destruction (33 +/- 4% over 30 min), whereas delta 4-VPA was a much less effective inhibitor of the enzyme (8 +/- 2% destruction over 30 min). This difference in degree of enzyme loss correlated well with the respective rates at which the substrates underwent oxidative metabolism of the terminal double bond. It is likely, therefore, that the mechanism of action of these compounds is the same as that for allylisopropylacetamide (AIA) and related monosubstituted olefins, which are converted by cytochrome P-450 to chemically reactive species which bind covalently to the prosthetic heme moiety of the cytochrome and thereby destroy the enzyme. In microsomes, both delta 4-VPA and its ethyl ester were metabolized by cytochrome P-450 to a common cyclic end-product, 3-n-propyl-5-hydroxymethyltetrahydro-2-furanone, although stable isotope labeling experiments with oxygen-18 demonstrated that the pathways followed by the two substrates were mechanistically distinct. These findings, together with data from related metabolic studies on AIA, support the view that the efficiency of the initial double bond oxidation reaction determines the extent of cytochrome P-450 destruction during the metabolism of terminal olefins, rather than any subsequent step.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the in vitro metabolism of allylisopropylacetamide to reactive intermediates. Mechanistic studies with oxygen-18.

Metabolism of allylisopropylacetamide (AIA) (1) in microsomal preparations from phenobarbital-pretreated rats is shown to proceed by way of three cytochrome P-450-dependent pathways: (i) aliphatic (C-3') hydroxylation, (ii) allylic (C-3) hydroxylation and (iii) olefin oxidation. The latter represents the major route of biotransformation and leads ultimately to the formation of the gamma-butyrolactone 2. In order to elucidate the mechanism by which AIA is converted to this gamma-lactone, and to gain information on the nature of chemically reactive intermediates in the process, the metabolism of AIA to 2 was investigated in 18O2 or H218O and the pattern of label incorporated into the product was determined by gas chromatography/mass spectrometry (GC/MS). The results support the formation of AIA epoxide as an initial product of olefin oxidation and indicate that this species undergoes rapid intramolecular rearrangement to a protonated iminolactone which, in turn, is hydrolysed to the stable gamma-lactone. On the other hand, the 'dihydrodiol' metabolite of AIA, which would be expected to result from direct hydrolysis of AIA epoxide, was not detected in incubation products and, furthermore, the 18O labeling data specifically exclude the possibility that it served as a precursor of 2. It may be concluded, therefore, that AIA epoxide and the protonated iminolactone to which it gives rise represent reactive intermediates in the oxidation of AIA which may play a key role in the alkylation of certain cellular constituents which accompanies metabolism of AIA by liver enzymes.

Metabolism of valproic acid by hepatic microsomal cytochrome P-450.

Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid omega-1 and omega hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the beta-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes omega-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxy-valproic acid, should not be used to assess in vivo metabolism of valproate via the beta-oxidation pathway.

A sensitive, rapid assay for the detection of human granulocyte-macrophage colony stimulating factor.

Several monoclonal antibodies were developed against recombinant human granulocyte colony stimulating factor (hu-GM-CSF). All were reactive to the protein by enzyme linked immunoabsorbent assay (ELISA), and one, 1G2 was capable of immunoprecipitating significant levels of radiolabeled hu-GM-CSF. When 1G2 and a second GM-CSF reactive monoclonal antibody, 3G11, were used in a double determinant assay, the level of detection of hu-GM-CSF in solution was approximately 500 ng/ml. Additional sensitivity was gained by using affinity purified rabbit polyclonal antibodies, together with the monoclonal 1G2. Using such a configuration in a double determinant assay one could detect 10-90 ng/ml of human-GM-CSF in solution with no reactivity observed to CSF-1 or granulocyte-CSF.

Exendin-4 and glucagon-like-peptide-1: NMR structural comparisons in the solution and micelle-associated states.

Exendin-4, a 39 amino acid peptide originally isolated from the oral secretions of the lizard Heloderma suspectum, has been shown to share certain activities with glucagon-like-peptide-1 (GLP-1), a 30 amino acid peptide. We have determined the structuring preferences of exendin-4 and GLP-1 by NMR in both the solution and dodecylphosphocholine (DPC) micelle-associated states. Based on both chemical shift deviations and the pattern of intermediate range NOEs, both peptides display significant helicity from residue 7 to residue 28 with greater fraying at the N-terminus. Thornton and Gorenstein [(1994) Biochemistry 33, 3532-3539] reported that the presence of a flexible, helix-destabilizing, glycine at residue 16 in GLP-1 was an important feature for membrane and receptor binding. Exendin-4 has a helix-favoring glutamate as residue 16. In the micelle-associated state, NMR data indicate that GLP-1 is less helical than exendin-4 due to the presence of Gly16; chemical shift deviations along the peptide sequence suggest that Gly16 serves as an N-cap for a second, more persistent, helix. In 30 vol-% trifluoroethanol (TFE), a single continuous helix is evident in a significant fraction of the GLP-1 conformers present. Exendin-4 has a more regular and less fluxional helix in both media and displays stable tertiary structure in the solution state. In the micelle-bound state of exendin-4, a single helix (residues 11-27) is observed with residues 31-39 completely disordered and undergoing rapid segmental motion. In aqueous fluoroalcohol or aqueous glycol, the Leu21-Pro38 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure and produces ring current shifts as large as 3 ppm. This tertiary structure is partially populated in water and fully populated in aqueous TFE. The Leu21-Pro38 segment of exendin-4 may be the smallest protein-like folding unit observed to date. When the Trp-cage forms, fraying of the exendin-4 helix occurs exclusively from the N-terminus; backbone NHs for the C-terminal residues of the helix display H/D exchange protection factors as large as 10(5) at 9 degrees C. In contrast, no tertiary structure is evident when exendin-4 binds to DPC micelles. An energetically favorable insertion of the tryptophan ring into the DPC micelle is suggested as the basis for this change. With the exception of exendin-4 in media containing fluoro alcohol cosolvents, NMR structure ensembles generated from the NOE data do not fully reflect the conformational averaging present in these systems. Secondary structure definition from chemical shift deviations may be the most appropriate treatment for peptides that lack tertiary structure.

Development and characterization of two neutralizing monoclonal antibodies to human interleukin-1 alpha.

Mice immunized with homogeneous recombinant interleukin-1 alpha (IL-1 alpha) protein developed specific serum titers to the immunogen. Hybridomas resulting from the fusion of the immune spleen or lymph node cells to myeloma cells were analyzed by an antibody capture assay in which the antigen was present in solution. This assay enabled us to isolate two hybridomas secreting antibodies (designated 2F4 and 4G12) that recognized IL-1 alpha and not interleukin-1 beta as judged by the ability of the antibodies to: (a) precipitate IL-1 alpha, (b) inhibit the binding of 125I-IL-1 alpha to the IL-1 receptor on EL4 cells, (c) inhibit the biological activity of IL-1 alpha as measured in a lectin-induced, IL-1-dependent thymocyte proliferation assay. In a double determinant assay configuration, both antibodies, in conjunction with rabbit polyclonal anti-IL-1 alpha antibodies, could detect nanogram concentrations of IL-1 alpha in solution. Cross-inhibition studies indicated that the 2F4 and 4G12 antibodies bind to the same or spatially related epitopes since each can inhibit the binding of the other to IL-1 alpha.

Beta-structure in human amylin and two designer beta-peptides: CD and NMR spectroscopic comparisons suggest soluble beta-oligomers and the absence of significant populations of beta-strand dimers.

Intensity variation for the positive far UV CD band was observed for three 'beta-sheet' peptides. In 6% HFIP, an amyloidogenic species (human pancreatic amylin) displays, on standing, an extremely intense 192-nm band which diminishes upon physical agitation. A concurrently formed Tyr sidechain band at 274 nm disappears completely with agitation, linking the enhancement of the 192-nm band to the highly ordered stacking of beta-sheets. NMR studies indicate that the beta-states of the three peptides are oligomeric, not beta dimers. A membrane-forming EAK peptide displays NMR peaks due to the low concentration of 'random coil' monomers present in slow equilibrium with beta-oligomers; solutions of a more hydrophobic ELKA peptide, which displays an intense 195-nm band, contain only oligomeric species. NMR studies at 25% HFIP revealed the structural requirements for inhibition of beta-oligomer formation.

Biotransformation and pharmacokinetics in the rhesus monkey of 2-n-propyl-4-pentenoic acid, a toxic metabolite of valproic acid.

2-n-Propyl-4-pentenoic acid (delta 4-VPA), a hepatotoxic metabolite of valproic acid (VPA), was administered by iv bolus injection (14 mg kg-1) to two adult male rhesus monkeys. The plasma concentration vs. time curve for delta 4-VPA in these animals was biexponential and the effective half-life values were 0.53 and 0.67 hr. The pharmacokinetic profile of delta 4-VPA was similar to that of VPA in the monkey, although the unbound fraction of delta 4-VPA in plasma was approximately 2.5-fold greater than the value for the parent drug. The major route of elimination of delta 4-VPA was excretion into urine, and studies with a group of eight animals indicated that delta 4-VPA undergoes extensive biotransformation in this species. A total of 20 metabolites was detected in urine by GC-MS techniques, and 19 of these were identified positively by comparison of their gas-liquid chromatographic and mass spectrometric properties with those of the authentic compounds prepared by synthesis. Many of these metabolites were present largely in the form of glucuronide conjugates, as was delta 4-VPA itself. The major pathways of metabolism of delta 4-VPA were found to be ester glucuronide formation and beta-oxidation, whereas omega- and (omega-1)-oxidation processes were of minor quantitative importance. Excretion of unchanged drug and its metabolites into urine over 24 hr accounted collectively for some 59% of the administered dose, a figure which was appreciably less than the corresponding recovery of metabolites of VPA in the same monkeys. The possibility is raised that beta-oxidation of delta 4-VPA leads to the generation of a chemically reactive intermediate(s) which alkylate(s) cellular macromolecules and thereby forms tissue-bound residues. The significance of such a phenomenon is discussed in relation to the etiology of VPA-induced liver injury.

Localization of human mononuclear cell interleukin 1.

The detection and localization of interleukin (IL) 1 in human monocytes was carried out by flow cytometry using monoclonal antibodies to IL-1 alpha and IL-1 beta proteins. IL-1 alpha was detected on the surface of monocytes and the surface expression increased following lipopolysaccharide activation. No demonstrable IL-1 beta protein could be observed on the cell surface by antibody staining, while both IL-1 alpha and IL-1 beta could be visualized intracellularly by the appropriate monoclonal antibodies following acetone permeabilization of the monocytes. Further experiments with cell associated IL-1 revealed that most of the biological activity of human monocytes could be inhibited by affinity purified polyclonal antibodies to IL-1 alpha protein, whereas no inhibitory activity was observed with IL-1 beta specific antibodies. These data support the hypothesis that a differential localization of IL-1 alpha and IL-1 beta exists within human blood-derived monocytes.

Beta-oxidation of valproate. II. Effects of fasting, glucose, and clofibrate in rats.

The effects of glucose infusion, fasting, and clofibrate pretreatment on valproate (VPA) disposition were investigated in rats to determine the role of endogenous fatty acid beta-oxidation in the metabolic formation of 2-en-VPA. Rats undergoing each treatment received a continuous steady-state infusion of VPA and a single intravenous (i.v.) bolus of 2-en-VPA. Elimination clearance of VPA was significantly higher (median 31%, p = 0.002) with glucose infusion as compared with fasting but was unchanged by clofibrate pretreatment as compared with control. Formation clearance of 2-en-VPA was significantly higher with glucose infusion as compared with fasting (median 147%, p = 0.001) and with clofibrate pretreatment as compared with control (median 73%, p = 0.041). Fractional metabolism of VPA by this route averaged 6% in fasted and control rats and 10% in glucose-infused and clofibrate-pretreated rats. Thus, VPA elimination clearance was not greatly influenced by effects on this route in rats. Elimination clearance of 2-en-VPA was also higher with glucose infusion as compared with fasting (median 149%, p = 0.002), and with clofibrate pretreatment as compared with control (median 167%, p less than 0.001). These observations are consistent with glucose-sparing release of endogenous fatty acids (FAs) to compete with VPA for beta-oxidation, and increased beta-oxidative activity after clofibrate treatment. The results of this study provide strong in vivo evidence for involvement of beta-oxidation in metabolism of VPA.

IL-1 induces rapid phosphorylation of the IL-1 receptor.

The IL-1R on murine T cells is a Mr = 80,000 plasma membrane glycoprotein. cDNA cloning and transfection experiments have shown that this is an integral membrane protein, which binds both IL-1 alpha and IL-1 beta and transduces the IL-1 signal. A mAb, RM-5, which binds an epitope on the receptor which is distinct from the IL-1 binding site has been produced in rats. RM-5 has been used to immunoprecipitate the IL-1R from 32P-orthophosphate labeled CHO cells which express approximately 100,000 functional, murine rIL-1R/cell. Phosphorylation of the receptor was observed as early as 1 min after the addition of IL-1 and continued for periods of up to 30 min. Phosphorylation increases as the concentration of IL-1 increases from 10(-13) to 10(-8) M. Potassium hydroxide hydrolysis of the phosphorylated IL-1R shows that more than 90% of the phosphate is incorporated into serine or threonine. Thus, one of the earliest events after IL-1 binding to the IL-1R is activation of a serine/threonine protein kinase and phosphorylation of the IL-1R itself.

Restricted production of interleukin 4 by activated human T cells.

Interleukin 4 (IL-4) is secreted by activated T cells and pleiotropically modulates both B- and T-lymphocyte function. In murine helper (CD4+) T-cell clones IL-4 production appears to be regulated independently of interferon gamma and interleukin 2. To determine whether production of these lymphokines is also differentially regulated in uncloned human T cells, we studied lymphokine production by normal human peripheral T cells and T-cell subsets after in vitro polyclonal activation. After maximal induction of lymphokine expression, IL-4 mRNA was detectable in less than 5% of CD4+ and 1-2% of unfractionated T cells, whereas approximately 33% and 60% of CD4+ cells expressed detectable mRNA for interferon gamma and interleukin 2, respectively. This finding correlated with dramatically lower production of IL-4 mRNA and protein than of interferon gamma and interleukin 2 by peripheral blood and tonsillar T cells. The helper-inducer (CD4+ CD45R-) T-cell subset, which significantly enhances in vitro immunoglobulin production, accounted for the preponderance of IL-4 mRNA accumulation and protein production by CD4+ T cells; nevertheless, cells with detectable IL-4 mRNA constituted less than 10% of the CD4+ CD45R- subset. Limitation of IL-4 production to a comparatively small population of normal human T cells could selectively regulate the effects of this lymphokine in T-cell-mediated immune responses; such selective regulation may be a fundamental mechanism for restricting the potentially pleiotropic effects of certain lymphokines to appropriate responder cells.

Antimicrobial activity of synthetic bactenecin.

Bactenecin is an antimicrobial peptide isolated from bovine neutrophils. Bactenecin was synthesized by solid-phase peptide synthesis and renatured to a fully disulfide bonded form. The peptide inhibits the growth of Escherichia coli and Staphylococcus aureus at the same concentration reported for the peptide purified from bovine neutrophils. Bactenecin inhibits the growth of other medically important bacteria and yeast, and it kills the fungus Trichophyton rubrum. Acetylation and amidation of the amino- and carboxy-termini of bactenecin do not change its potency, while replacement of its two cysteine residues with serine decreases the potency.

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor.

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.