Steven Jacobs

Quantitative aspects of hormone-receptor interactions of high affinity: Effect of receptor concentration and measurement of dissociation constants of labeled and unlabeled hormones

It is demonstrated that because of limitations in the magnitude of the specific activity of radiolabeled hormone derivatives, direct binding studies of hormone-receptor interactions of high affinity (10(-9) -10(-11) M, depending on whether 3H- or 123I-labeled hormones are used) will be subject to artifactual distortions due to the need to utilize high concentrations of the receptor. If the concentration of the receptor is not ten times lower than the true affinity constant, the apparent dissociation constant obtained from direct concentration binding curves will vary as a linear function of the receptor concentration. In addition, at high receptor concentrations saturability becomes difficult to demonstrate experimentally and the binding data yield apparently non-hyperbolic, sigmoidal curves which can be mistakenly interpreted to depict cooperative interactions. Similar artifacts related to receptor concentration are predicted for measurements of the hormone concentration dependence of biological proce-ses (e.g. activation of adenylate cyclase, transport processes, etc.). Methods for detecting these effects, and correctly measuring affinities for labeled and unlabeled hormones under these conditions, are described. The implications for measuring the binding properties of hormone-receptor interactions are discussed, especially in reference to studies of the comparative analysis of receptor function in altered metabolic states and to studies relating the biological and binding properties of hormones.

Estimation of hormone receptor affinity by competitive displacement of labeled ligand: Effect of concentration of receptor and of labeled ligand

Abstract The concentration of ligand (e.g., hormone) at which a given fraction of bound labeled ligand is competitively displaced from its binding site (e.g., receptor) depends upon the concentration of labeled ligand and of binding sites as well as on the affinity of the ligand. The ligand concentration at which 50% of the bound, labeled ligand is displaced [K′d(app)] is often taken (mistakenly) to be equal to the dissociation constant of the ligand. For an ideal bimolecular reactions, it is shown that K′d(app) actually exceeds the dissociation constant by an amount equal to the labeled ligand concentration plus the binding site concentration minus three halves the concentration of labeled ligand that would be bound in the absence of unlabeled ligand. For both epidermal growth factor and insulin, the concentration of unlabeled hormone at which a given fraction of bound labeled hormone is displaced from placenta membranes is increased by increasing the labeled hormone concentration or the placenta membrane concentration. K′d(app) will give spuriously high estimates of the dissociation constants for these hormones if measured at high labeled hormone or binding site concentrations. These considerations also have important implications for comparative studies of receptors in different species or metabolic states, and for the feasibility of “radioreceptor” assays.

Irreversible stimulation of adenylate cyclase activity of fat cell membranes by phosphoramidate and phosphonate analogs of GTP

SummaryThe ability of 5′-guanylylimidodiphosphate (Gpp(NH)p)1 to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated.The organomercurial,p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NH)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade.GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme.The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30°C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.

Cell Receptors in Disease

Intercellular transfer of information is frequently accomplished through receptors that recognize specific chemical signals such as hormones or neurotransmitters and respond to their presence by in...

[31] Insulin receptor antibodies

Publisher Summary This chapter describes the different aspects of insulin receptor antibodies. Antibodies against insulin receptors occur spontaneously in some human and mouse autoimmune diseases, and have been produced in rabbits that have been immunized with purified rat insulin receptor. These antibodies have been useful in characterizing the structure, function, and regulation of insulin receptors and have also been used to purify the insulin receptors. An assay that measured inhibition of insulin binding was first used to detect the auto-antibodies to the insulin receptor in patients with acanthosis nigricans and insulin resistance. Two types of immunoprecipitation assays are found to be most useful. These utilize solubilized receptor labeled either by incubating it with 125 I-insulin or by directly iodinating the purified receptor using chloramine-T. The amount of 125 I-insulin, that is nonspecifically precipitated, is determined in three sets of controls. In the first set, 2μg of native insulin is added prior to the 125 I-insulin. This saturates the insulin receptor and blocks the binding of 125 I-insulin. It is found that the maximum amount of radioactivity that is precipitated even when a large excess of antibody is used is only about 30%-60% of the total, depending on the batch of iodinated receptor.

Human Insulin Receptor Gene: Data Supporting Assignment to Chromosome 19

Somatic cell hybrid clones constructed by crossing human skin fibroblasts with mouse L cells have been examined for expression of human insulin receptors, using a monoclonal antibody directed against the human insulin receptor. Data obtained in this study support the assignment of the human gene for the insulin receptor to Chromosome 19.

[35] Purification of insulin receptor from human placenta

Publisher Summary The schemes for successful purification of insulin receptors have major purification step affinity chromatography, using either antibodies to insulin receptor or insulin itself as the immobilized ligand. While both types of procedures produce similar results, the limited availability of antireceptor antibodies severely restricts their general usefulness. The chapter presents the description of the schemes that are used for the purification of insulin receptors from human placenta. It is a modification of several published methods. Placenta membranes are solubilized with Triton X-100 and purified by sequential chromatography on concanavalin A-Sepharose, insulin-Sepharose, and wheat germ agglutinin-Sepharose. Approximately 40μg of insulin receptor can be purified from two placentas with an overall yield of approximately 10% determined by the recovery of insulin binding activity.

Affinity Chromatography for Membrane Receptor Purification

Membrane receptors possess no unique gross physical or chemical properties that distinguish them from other membrane proteins of which they constitute only a small fraction of a percent. Their most remarkable characteristic is their ability to bind specific ligands with high affinity and to translate this binding into a relevant biological response. If not for this property, receptors would remain obscure in the sea of the other more numerous proteins that surround them. Therefore, it is not surprising that conventional methods of protein purification, which separate molecules on the basis of physical—chemical properties such as size, shape, charge and solubility, have only limited usefulness for purifying receptors, while affinity chromatography (Cuatrecasas, 1970; Cuatrecasas and Anfinsen, 1971), which depends upon biospecific recognition, is an extremely powerful tool. Table 3.1 lists some of the membrane receptors that have been purified using affinity chromatography.

Activation of adenylate cyclase by Guanosine 5′ α, β methylene triphosphate

Abstract Guanosine 5′ α, β methylene triphosphate activates adenylate cyclase of pigeon erythrocyte ghosts. Activation is inhibited by GTP, persists after free guanosine 5′ α, β methylene triphosphate is removed, and proceeds more rapidly in the presence of isoproterenol. Guanosine 5′ methylene diphosphate neither activates adenylate cyclase nor inhibits activation of adenylate cyclase by guanosine 5′ α, β methylene triphosphate. These results exclude pyrophosphorylation as a mechanism by which GTP normally activates adenylate cyclase.

Monoclonal Antibodies as Probes for Insulin and Insulin-like Growth Factor-I Receptors

Receptors for polypeptide hormones are present in cell membranes in extremely small quantities and comprise only a small fraction of a percent of the total membrane protein. Therefore, any method that can be used for their detection and characterization must have exquisite sensitivity and specificity. Antibodies provide reagents with these essential features. In this chapter we described a series of monoclonal antibodies to receptors for insulin and insulin-like growth factor-I and their use in characterizing these receptors.