An improved assay procedure and a new chemically stable ligand for cytosolic retinoic acid binding protein

A high through-put method to assay for binding of retinoids to their cytosolic binding protein is described. The protein-bound retinoid is quantified following its complete separation from the free ligand by a single gel filtration chromatography step on Sephadex G-25 columns. The method allows a single person to process up to 100 samples per day. A new, substituted benzo[b]thiophene carboxylic acid derivative (tritiated) is proposed as an alternative ligand for cytosolic retinoic acid binding protein. This new analog (CD270), which contains no olefinic double bonds, is characterized by its chemical stability to light and atmospheric oxidation. This molecule shows binding characteristics similar to those of retinoic acid, both in terms of binding affinity and binding specificity (Kd of about 2 nM), and offers the additional advantage that the unbound molecule is absorbed on Sephadex G-25, while the protein-bound ligand is unaffected by the carbohydrate gel matrix. When the affinities of retinoic acid and several analogs were determined by competition binding experiments, similar results were obtained when either tritiated retinoic acid or tritiated CD270 were used as the labeled ligand. The tritiated, heterocyclic retinoic acid analog, CD270, is thus proposed as an alternative ligand for cytosolic retinoic acid binding protein.     

A photoaffinity derivative of colchicine: 6'-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine. Photolabeling and location of the colchicine-binding site on the alpha-subunit of tubulin

A photoaffinity analog of colchicine, 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine, was synthesized by reacting deacetylcolchicine or [3H]deacetylcochicine with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Homogeneity of the photoaffinity analog was established by thin-layer chromatography and high-pressure liquid chromatography. The structure of the photoaffinity analog was determined by 1H and 13C NMR, infrared and ultraviolet-visible spectroscopies, and elemental analysis. Binding of 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine to bovine renal tubulin was measured by competition with [3H]colchicine. The value of the apparent Ki for the photoaffinity analog was 0.28 microM in the concentration range of 0.8-1.2 microM of the analog. A value of 0.50 microM for the apparent Kd was measured by the direct binding of the tritiated photoaffinity analog to tubulin. The analog is slightly more potent an inhibitor of microtubule formation than colchicine. The photoaffinity analog reacted with renal tubulin upon irradiation with a mercury lamp equipped with a 420-nm cutoff filter. Spectral and radiochemical analyses of the tubulin after photolysis and dialysis have demonstrated a stoichiometric incorporation of the photoaffinity analog in the alpha-subunit of the tubulin. Covalent labeling of tubulin with the photoaffinity analog decreases the extent of [3H]colchicine binding by more than 90%.     

Comparison of motilin binding to crude homogenates of human and canine gastrointestinal smooth muscle tissue

Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30 degrees C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu13]motilin or synthetic natural porcine motilin indicated a dissociation constant (Kd) of 3.6 +/- 1.6 nM and a maximal binding capacity (Bmax) of 77 +/- 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent Kd of 2.2 +/- 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 +/- 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.     

Binding characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides

The present paper describes the isolation and characterization of a clone of hybrid myelomas (3-E7) secreting a mouse monoclonal antibody to beta-endorphin. An examination of its specificity against a series of human beta-lipotropin fragments and other opioid peptides revealed that the N-terminus portion of beta-endorphin is the determinant. Complete or almost complete cross-reactivity was obtained to methionine- and leucine-enkephalin, beta-lipotropin 60-65, and BAM 22; partial cross-reactivity was seen to dynorphin1-13 and alpha-neo-endorphin, whereas beta-lipotropin, alpha-N-acetyl-beta-endorphin, Des-Tyr1-beta-endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross-reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) for beta-endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use of beta-endorphin iodinated with its N-terminal tyrosine protected by coupling to an antibody. A solid-phase RIA was developed involving the internally 3H-labeled monoclonal antibody, which resulted in a 10-fold increase in sensitivity as compared with the homogenous RIA. These data indicate that for the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of functional significance for many naturally occurring opioid peptides.     

Partial purification of a receptor for the adipokinetic hormones from Musca autumnalis face flies.

Partial purification of the receptors for the neurohormones, diptera corpora cardiaca factors 1 and 2 (DCC1 and DCC2) was achieved. Receptor proteins were obtained from the abdomens of face fly, Musca autumnalis De Geer. Purification methods included detergent solubilization, affinity chromatography, and polyacrylamide gel electrophoresis. Analysis by gel electrophoresis has identified two proteins from this partial purification with relative molecular weights of 45 and 90 kD. A crude receptor preparation was used to develop a ligand binding assay with radiolabeled (tritiated and iodinated) DCC1. Ligand binding was inhibited by 90% when excess unlabeled DCC1 was added to the assay mixture. Ligand binding was optimum at pH 7.5. Binding saturation occurred at approximately 12 picomole radiolabeled ligand concentration. Because DCC1 and DCC2 have been shown to effect the lipid and trehalose levels in the insect an understanding of the neuropeptide-receptor interaction is important for the development of new methods of control of dairy and poultry muscoid flies.     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-"glyburide", has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values of 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).     

The synthesis of [A 19-3-iodotyrosine] and [A 19-3,5-diiodotyrosine]insulin (porcine)

The chemical synthesis of [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), using the amino-acid derivatives 3-iodotyrosine and 3,5-diiodotyrosine is described. The synthesis of the iodinated A-chains were performed by segment condensation in solution using acid labile protecting groups. The hydroxyl groups of Tyr(I) and Tyr(I2) were unprotected. For the temporary protection of the alpha-amino groups of the A-chain segments containing iodinated tyrosines, the 1-(4-biphenylyl)-1-methylethoxycarbonyl group was selected. After deprotection and sulphitolysis the iodinated A-chain tetra-S-sulphonates were purified by ion exchange chromatography on DEAE cellulose at pH 5.6. Reduction to the sulphhydryl form and the combination with native porcine B-chain yielded [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), respectively. Purification of the first product was achieved by gel filtration and of the later by ion exchange chromatography on CM-cellulose at pH 4.5 and gel filtration. The monoiodinated insulin had a biological activity of 24 +/- 2% and the diiodinated analogue 2.6 +/- 0.2% as determined in an in vitro lipogenesis assay with epididymal adipocytes.     

Solubilization and partial purification of somatostatin-28 preferring receptors from hamster pancreatic beta cells.

Somatostatin-28 (SRIF-28) preferring receptors were solubilized from hamster beta cell insulinoma using the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate. The binding of the iodinated [Leu8-D-TRP22-Tyr25]SRIF-28 analog (referred to as 125I[LWY] SRIF-28) to the solubilized fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the solubilized extract contained two classes of SRIF-28-binding sites: a high affinity site (Kd = 0.3 nM and Bmax = 1 pmol/mg protein) and a low affinity site (Kd = 13 nM and Bmax = 4.7 pmol/mg protein). The binding of 125I[LWY]SRIF-28 to solubilized SRIF-28 receptors was sensitive to the GTP analog guanosine-5'-O-thiotriphosphate, suggesting that receptors are functionally linked to a G-protein. By anion-exchange chromatography of the solubilized extract followed by chromatography on wheat germ agglutinin, a 46-fold purification of SRIF-28 receptors was obtained. At this stage of purification, only high affinity sites were found (Kd = 1 nM) and the GTP effect was not maintained. A specific protein of 37 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling. We suggest that this protein is the putative SRIF-28 receptor or a subunit thereof.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Catalytic hydrogenolysis of poly-iodinated recombinant human insulin-like growth factor II (IGF-II): a potentially useful method for the tritiation of IGF-II.

A method has been developed to prepare, purify, and fully characterize poly-iodinated insulin-like growth factor II (IGF-II) which can then be catalytically deiodinated to produce IGF-II with its native disulfide bonded structure. This method can potentially be adapted to prepare tritiated IGF-II with the use of tritium gas in the hydrogenolysis step. IGF-II was iodinated at all three tyrosines using lactoperoxidase with a three-fold excess of sodium iodide. The iodinated products were purified using reversed-phase HPLC and characterized by peptide mapping. The tyrosine-containing peptides generated by pepsin digestion were characterized by amino acid sequence analysis. Mono- and di-iodinated phenylthiohydantoin tyrosine derivatives were synthesized and used to identify the iodination state of the modified tyrosine residues in the sequence analysis. Purified poly-iodinated IGF-II was deiodinated by hydrogenolysis, over a prereduced palladium (II) oxide catalyst to form IGF-II with its native disulfide bonds intact, as shown by peptide mapping.     

Interaction of local anesthetics with Torpedo californica membrane-bound acetylcholine receptor

The effects of local anesthetics on the rate of the agonist-induced increase in ligand affinity of membrane-bound acetylcholine receptor from Torpedo californica were examined. The rate of the transition in receptor affinity was determined by following the time-dependent increase in inhibition of iodinated alpha-bungarotoxin binding caused by 1 microM carbamylcholine. At concentrations below those that directly inhibited the binding of iodinated alpha-bungarotoxin, dibucaine increased the rate of the transition to a high-affinity state and tetracaine decreased this rate. The measured rate constants were 0.026 +/- 0.008 s-1 in the presence and 0.010 +/- 0.002 s-1 in the absence of dibucaine while tetracaine decreased the rate to 0.006 +/- 0.002 s-1 as compared to a control value of 0.012 +/- 0.003 s-1. A parallel was observed between the effectiveness of a compound in increasing or decreasing the rate of the agonist-induced transition in affinity and the change in its apparent inhibition constant in the presence of carbamylcholine (increase or decrease) measured by the displacement of tritiated perhydrohistrionicotoxin. This parallel could be explained by assuming (a) that local anesthetics bound directly to the specific histrionicotoxin binding site or (b) that they bound to a different site and the observed effects were caused by conformational changes.     

Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.     

Chronic, but not acute, administration of morphine alters antiopiate (Tyr-MIF-1) binding sites in rat brain

Opiate addiction could involve a change in the binding of endogenous antiopiates. A candidate for such a role is Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), a brain peptide that can antagonize exogenous and endogenous opiates and bind to opiate receptors. Its primary action, however, may be through its own binding site in brain, which we now report is altered by chronic administration of morphine. Rats given morphine pellets had reduced binding of both iodinated and tritiated Tyr-MIF-1 on day 5, when substantial tolerance is evident. In contrast, mu and delta opiate receptors were increased. Acute injection of an analgesic dose of morphine did not reduce Tyr-MIF-1 binding, indicating that chronic administration is required for the change. These findings open new approaches to the study of addiction by focusing on antiopiate activity.     

Self-association and phospholipid binding properties of iodinated apolipoprotein A-I

Kinetic turnover studies of apolipoprotein metabolism often utilize radioiodinated tracers. These studies rely on the "tracer assumption" that the modified tracer is physiologically and metabolically identical with the native unmodified tracer. This paper addresses the validity of this assumption on the basis of the examination of the state of self-association and binding properties with egg yolk phosphatidylcholine small unilamellar vesicles of native and iodinated apolipoprotein A-I (apoA-I). Human apoA-I was iodinated to the extent of 1.0 and 3.7 mol of nonradioactive iodine/mol of protein. At concentrations from 0.013 to 0.8 mg/mL, iodinated apoA-I underwent concentration-dependent self-association similar to that of native apoA-I as evidenced by circular dichroism and gel filtration. At all concentrations, however, the iodinated preparations were more highly self-associated as judged by gel filtration in relation to the extent of iodination. Scatchard analysis of fluorometric titrations of apoA-I/vesicle interactions demonstrated that the binding capacity of vesicles for apoA-I increased and apoA-I binding affinity decreased upon iodination. In addition, the kinetics of apoA-I binding to vesicles was enhanced by iodination. The affinity, capacity, and kinetics of apoA-I binding were each altered 2-3-fold dependent on the extent of iodination. Since the dynamic interactions of apoA-I are perturbed by iodination, one may legitimately question whether the "tracer assumption" is valid for 125I-apoA-I under all experimental conditions.     

Interaction of sex steroids with proteins from the soluble fraction of swine and cattle liver (a preliminary analysis)]

The interactions of [3H]estradiol, [3H]testosterone and [3H]progesterone with soluble proteins from porcine and calf liver were studied. The specific binding of [3H]progesterone and [3H]testosterone in calf liver cytosol seems to be due to serum transcortin or its intracellular precursor (analog). Contrariwise, the specific binding of [3H]progesterone observed in porcine liver cytosol was absent in the serum. This binding was characterized by slow association and dissociation dynamics, moderate affinity for the [3H]-ligand and a high binding capacity. The structural determinants of the ligands were studied by competitive inhibition of the [3H]-ligand binding. The delta 4-3-keto group in the steroid A-ring was found to be the most important determinant. An intensive metabolism of [3H]progesterone was observed during its incubation with cytosol (data from thin-layer chromatography). A 3H-metabolite (presumably, 20 beta-dihydroprogesterone) was predominant in the bound ligand fraction. The data obtained suggest that proteins of a steromodulin type are widely distributed in the mammalian liver.     

Prevention of bridge binding in immunoassays: a general estradiol tracer structure

Iodinated estradiol tracers were synthesized with three different bridges connecting the radiolabelled moiety to the steroid core: Hemisuccinate, carboxymethyloxime and amide. Taking these iodinated tracers in combination with ten antibodies raised against estradiol-6-CMO-albumin, titers and slopes of calibration curves have been compared to the corresponding data using a 3H tracer. The data indicate that the tracer with the amide bridge is recognized similarly to the tritiated estradiol by all antibodies tested, whereas the two other iodinated tracers exhibit substantial bridge binding. The results suggest that the amide tracer structure can generally be used to improve the quality of estradiol antibodies suffering from bridge binding effects.     

Receptor binding characterization of the benzodiazepine radioligand 125I-Ro16-0154: potential probe for SPECT brain imaging

The binding of an iodinated benzodiazepine (BZ) radioligand has been characterized, particularly in regard to its potential use as a neuroreceptor brain imaging agent with SPECT (Single Photon Emission Computed Tomography). Ro16-0154 is an iodine-containing BZ antagonist and a close analog of Ro15-1788. In tissue homogenates prepared from human and monkey brain, the binding of 125I-labeled Ro16-0154 was saturable, of high affinity (Kd = 0.5 nM at 37 degrees C), and had high ratios of specific to non-specific binding (approximately 40:1). Physiological concentrations of NaCl (150 mM) enhanced specific binding approximately 15% compared to buffer without this salt. Kinetic studies of association and dissociation demonstrated a temperature dependent decrease in affinity with increasing temperature. Drug displacement studies confirmed that 125I-Ro16-0154 binds to the "central" type BZ receptor: binding is virtually identical to that of 3H-Ro15-1788 except that 125I-Ro16-0154 shows an almost 10 fold higher affinity at 37 degrees C. These in vitro results suggest that 123I-labeled Ro16-0154 shows promise as a selective, high affinity SPECT probe of the brain's BZ receptor.     

Methods for the Determination of the Specific Radioactivity of Radioligands

Abstract

Quantitative analysis of radioligand binding data requires the knowledge of an accurate and actual value for the specific radioactivity of the radiolabeled ligand. Several protocols, based on simple radioligand binding experiments, which allow the determination of the molar assay concentration of the radioligand and of the radioligand/receptor complex, are described. Their appropriateness is demonstrated by the use of tritiated and iodinated beta-adrenergic radioligands.