Specific, saturable, and high affinity binding of 125I-low density lipoprotein to glass beads.

¹²⁵I-Low density lipoprotein (¹²⁵I-LDL)¹ binds tightly to glass beads at physiologic pH and ionic strength. This binding shows saturability, high affinity (half maximal binding achieved at 10–15 μg protein/ml), and specificity (unlabeled LDL but not HDL or albumin competes with ¹²⁵I-LDL for binding to the glass beads). In contrast to the binding of ¹²⁵I-LDL to the physiologic LDL receptor on the surface of human fibroblasts and lymphocytes, the binding of ¹²⁵I-LDL to bind to inert substances such as glass must be considered in the interpretation of studies in which ¹²⁵I-LDL binding to membrane receptors is measured. The data emphasize the importance of correlating observed ¹²⁵I-LDL binding with a physiologic action of the lipoprotein.     

Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor

Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride.     

Inhibition of beta nerve growth factor binding to PC12 cells by alpha nerve growth factor and gamma nerve growth factor

Pheochromocytoma (PC12) cells have been found to differ from dorsal root ganglionic cells with respect to the modulation of the beta nerve growth factor (beta NGF) binding properties elicited by alpha NGF and gamma NGF. In contrast to our previous results with intact dorsal root ganglionic cells in which only high-affinity binding was blocked, alpha NGF and gamma NGF were found to block competitively all steady-state binding of iodinated beta NGF to PC12 cells at both 37 and 0.5 degrees C. The EC50 that was found for the alpha NGF displacement was 9-10 microM, and the gamma NGF effect had an EC50 of 200 nM, in the predicted range based upon the apparent Kd for dissociation of the alpha beta or the beta gamma complex in solution. The concurrence of the binding EC50 and the Kd for each complex indicates that the formation of alpha beta or beta gamma complexes in solution competes with the process of PC12 receptor binding with 125I-beta NGF. Experiments were carried out examining the dissociation kinetics following the addition of excess unlabeled beta NGF or alpha NGF at both 37 and 0.5 degrees C. Three dissociation components were observed with alpha NGF, in contrast to the two normally found with beta NGF. Lowering the chase temperature to 0.5 degrees C changed the relative contributions made by each component without dramatically changing any of the rate constants. The "slow" receptor was further examined by the dependence on 125I-beta NGF concentration of the slowest component with a chase of either excess alpha NGF or excess gamma NGF at 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity binding of [H]neurotensin to rat uterus

[³H]Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that [³H]NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited [³H]NT binding with the following potencies (IC₅₀): NT 8–13 (0.4 nM), NT 1–13 (4 nM), NT 9–13 (130 nM), NT 1–11, NT 1–8 (>100 μM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.     

Immunoassay of indomethacin: The use of [125I] protein A to detect specific serologic binding

The sera of rabbits that were injected with indomethacin covalently linked to human albumin bound increasing amounts of [¹⁴C]indomethacin during the course of immunization. The serum-binding components chromatographed with the γ-globulin fraction and were precipitated with goat-anti-rabbit-γ-globulin. When measured by radioimmunoassay with [¹⁴C]indomethacin under optimal conditions, 34 ng (95 pmoles) of unlabeled indomethacin inhibited [¹⁴C]indomethacin binding 50%. The antibodies reacted most effectively with indomethacin and the desmethylated analogue, 1-(p-chlorobenzoyl)-2-methyl-5-hydroxyindole-3-acetic acid, but not 2-methyl-5-methoxy-indole-3-acetic acid.An immunoassay based on the use of [¹²⁵I]Protein A as tracer for IgG antibody was developed for quantitative determination of indomethacin at the picogram level. Indomethacin, immobilized by covalent linkage to a solid sup port, bound the rabbit anti-indomethacin. Protein A, labeled with [¹²⁵I], measured the levels of the bound IgG antibody. Fluid phase indomethacin competed with solid-phase indomethacin for the anti-indomethacin which resulted in decreased anti-indomethacin and consequently decreased [MI]Protein A on the immobilized indomethacin-anti-indomethacin complex. The serologic specificity with the immobilized ligand immunoassay was the same as that found with the [¹⁴C]indomethacin radioimmunoassay, but the sensitivity for detection of indomethacin was increased over 300-fold.     

Naloxazone irreversibly inhibits the high affinity binding of [125I]D-ala2-D-leu5-enkephalin

Scatchard analyses of [¹²⁵I]D-ala²-D-leu⁵-enkephalin binding in rat brain membranes are curvilinear, suggesting low and high affinity sites. Treating the membranes with naloxazone abolishes the high affinity binding with slight effect on low affinity binding. Displacement of [¹²⁵I]-D-ala²-D-leu⁵-enkephalin binding by morphine in untreated membranes is biphasic. Displacement by morphine in naloxazone-treated tissue is monophasic, with no inhibition by low concentrations of morphine. Naloxazone treatment has little effect on displacements by unlabeled D-ala²-D-leu⁵-enkephalin. Binding in N4TG1 neuroblastoma cells, which demonstrates a linear Scatchard plot with single affinity constant similar to that of the low affinity binding in brain, is less sensitive to naloxazone's actions. Naloxazone treatment $\text{in vivo}$ inhibits D-ala²-D-leu⁵-enkephalin analgesia.     

High affinity binding of [3H] cocaine to rat liver microsomes

[3H]Cocaine bound reversibly, with high affinity (KD 2.3 +/- 1.1 nM) and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow (T1/2 for association, 6 min and for dissociation 17 min), and the kinetically calculated KD was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in [3H]cocaine binding. On the other hand, chronic administration of cocaine reduced [3H]cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of [3H]cocaine to rat liver microsomes was insensitive to monovalent cations and greater than 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced [3H]cocaine binding to liver with a different rank order of potency than their displacement of [3H]cocaine binding to striatum. This high affinity [3H]cocaine binding protein in liver is not likely to be a monooxygenase, but may have a role in cocaine-induced hepatotoxicity.     

High affinity binding of [125I-Tyr11]somatostatin-14 to human small cell lung carcinoma (NCI-H69)

The in vitro binding of [125I-Tyr11]somatostatin-14 (SRIF-14) to membranes prepared from cultured human small cell lung carcinoma (SCLC) cells (NCI-H69) has been characterized. Binding to SCLC was monophasic and of high affinity (Kd = 0.59 +/- 0.02 nM, n = 3). The estimated Bmax was 173 +/- 2.4 fmol/mg protein. Receptors were also present on solid NCI-H69 tumors grown in vivo in the athymic nude mouse. However, the concentration was only about 10% of that observed in cell culture. Biologically-active SRIF analogues were potent inhibitors of [125I-Tyr11]SRIF-14 binding, and an analysis of the pharmacological specificity indicated that the SCLC receptor was of the peripheral (e.g., non-neural) subtype. The presence of SRIF receptors on SCLC membranes may indicate that SRIF has a role in regulation of SCLC function.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

Characterization of picomolar affinity binding sites for [125I]-human calcitonin gene-related peptide in rat brain and heart

We have characterized picomolar affinity binding sites for human calcitonin gene-related peptide (CGRP) in rat brain and heart (atria and ventricle) membranes. By saturation analysis, apparent dissociation constant (KD) values of high affinity sites for [125I]-human CGRP are 9 approximately 15 pM (brain), 34 pM (ventricle) and 85 pM (atria). Low affinity sites with KD values of about 50 nM are found in rat brain and ventricle, but not in atria. Human and rat CGRP potently inhibited [125I]-human CGRP binding to these high affinity sites with apparent inhibition constant (Ki) values comparable to their KD values. Salmon calcitonin marginally inhibited these binding with Ki values between 0.1 microM and 1 microM. Extremely potent cardiovascular and gastrointestinal actions of CGRP might be mediated through CGRP binding sites with picomolar affinity which are similar to those we characterized in this study.     

Comparative affinities of adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) for [125I] AM and [125I] CGRP specific binding sites in porcine tissues.

We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM > or = human AM > or = human AM(22-52) > h alpha CGRP > or = h alpha CGRP(8-37) > sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20-300 fold more potent than hAM (22-52). When the same experiments were performed using [125I] h alpha CGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > h alpha CGRP > h alpha CGRP(8-37) in most of the tissues except in spleen and liver where h alpha CGRP was the most potent ligand. In lung, h alpha CGRP was almost as potent as rAM and hAM in displacing [125I] h alpha CGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

[125I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes

[¹²⁵I]LSD (labeled at the 2 position) has been introduced as the first ¹²⁵I-labeled ligand for serotonin 5-HT₂ (S2) receptors. In the present study we examined the binding of [¹²⁵I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [¹²⁵I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal K_d = 1.1 nM, B_max = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC₅₀ values for a variety of compounds show a clear 5-HT₂ (S2) serotonergic pattern at this [¹²⁵I]LSD site. Blockage of this primary 5-HT₂ (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [¹²⁵I]LSD binding site with a K_d = 9.1 nM, B_max = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [¹²⁵I]LSD is a sensitive and selective label for 5-HT₂ (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [¹²⁵I]LSD binding site with mianserin allows the high sensitivity of [¹²⁵I]LSD to be applied to D2 dopamine receptor studies as well.     

Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography

We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.     

The structural basis for insulin-like growth factor I receptor high affinity binding

We have recently identified high and low affinity insulin-like growth factor I (IGF I) binding sites in solubilized human placental membranes and purified the high affinity IGF I receptor by IGF I affinity chromatography (Tollefsen, S. E., Thompson, K., and Petersen, D. J. (1987) J. Biol. Chem. 262, 16461-16469). To define the structural basis for high affinity IGF I binding, we have examined the effect of disulfide bond reduction on the binding parameters of the high affinity IGF I receptor. We find that the disulfide bonds linking the two alpha beta dimers of the IGF I receptor heterotetramer are reduced by incubation at pH 8.75 with 2 mM dithiothreitol (DTT) for 5 min at room temperature. Gel filtration chromatography on a Superose 12 fast protein liquid chromatography column indicates that the alpha beta dimers do not remain associated by noncovalent interactions after reduction. Scatchard plots of IGF I binding to the IGF I receptor incubated at pH 8.75 with or without DTT indicate that the IGF I receptor alpha beta dimers have a 6.1 +/- 1.6 (mean +/- S.D.) times lower affinity than the heterotetramer for IGF I. The total binding capacity of the IGF I receptor treated with DTT is 1.6 +/- 0.3 (mean +/- S.D.) times higher than that of an equal amount of receptor treated without DTT. These results are consistent with a model in which the heterotetramer binds a single IGF I molecule with high affinity, whereas each of the two alpha beta dimers binds an IGF I molecule with lower affinity after dissociation. We conclude that association of two alpha beta dimers is required for formation of an IGF I receptor with high affinity for its ligand.     

High affinity binding of beta 2-glycoprotein I to human endothelial cells is mediated by annexin II

Beta(2)-glycoprotein I (beta(2)GPI) is an abundant plasma phospholipid-binding protein and an autoantigen in the antiphospholipid antibody syndrome. Binding of beta(2)GPI to endothelial cells targets them for activation by anti-beta(2)GPI antibodies, which circulate and are associated with thrombosis in patients with the antiphospholipid antibody syndrome. However, the binding of beta(2)GPI to endothelial cells has not been characterized and is assumed to result from association of beta(2)GPI with membrane phospholipid. Here, we characterize the binding of beta(2)GPI to endothelial cells and identify the beta(2)GPI binding site. (125)I-beta(2)GPI bound with high affinity (K(d) approximately 18 nm) to human umbilical vein endothelial cells (HUVECs). Using affinity purification, we isolated beta(2)GPI-binding proteins of approximately 78 and approximately 36 kDa from HUVECs and EAHY.926 cells. Amino acid sequences of tryptic peptides from each of these were identical to sequences within annexin II. A role for annexin II in binding of beta(2)GPI to cells was confirmed by the observations that annexin II-transfected HEK 293 cells bound approximately 10-fold more (125)I-beta(2)GPI than control cells and that anti-annexin II antibodies inhibited the binding of (125)I-beta(2)GPI to HUVECs by approximately 90%. Finally, surface plasmon resonance studies revealed high affinity binding between annexin II and beta(2)GPI. These results demonstrate that annexin II mediates the binding of beta(2)GPI to endothelial cells.     

Calcium-induced phosphorylations and [125I]calmodulin binding in renal membrane preparations

Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from th renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [¹²⁵I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of $\text{20 μ}\text{M La}{}^{\mathrm{2+}}$ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.     

2[125I]iodomelatonin binding sites in spleens of guinea pigs.

2-[125I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8 +/- 4.12 pmol/l and binding site density (Bmax) of 0.69 +/- 0.082 fmol/mg protein at mid-light (n = 10). There was no significant change in the Kd (41.8 +/- 3.16 pmol/l) or the Bmax (0.58 +/- 0.070 fmol/mg protein) at mid-dark (n = 10). Kinetic analysis showed a Kd of 23.13 +/- 4.81 pmol/l (mean +/- SE, n = 4), in agreement to that derived from the saturation studies. The 2-[125I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than N-acetylserotonin, 6-hydroxymelatonin greater than 5-methoxytryptamine, 5 methoxytryptophol greater than serotonin, 5-methoxyindole-3-acetic acid greater than 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan greater than tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction (65.5%), the rest are distributed in the microsomal fraction (17.4%), mitochondrial fraction (14.7%) and cytosolic fraction (0.3%). The demonstration of 2-[125I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.     

N-chloroacetyl-[125I]iodotyramine: an alkylating agent with high specific activity

The preparation of iodinated N-chloroacetyltyramine and its evaluation as a specific sulfhydryl reagent are described. N-Chloroacetyltyramine was synthesized by a carbodiimide-mediated condensation of chloro- or iodoacetic acid and tyramine·HCL, and the crystalline product was iodinated in a reaction with chloramine T to yield either a 3,5-[¹²⁵I]diiodotyramine derivative, or a trace-iodinated product when carrier-free ¹²⁵I was employed. These iodinated derivatives react specifically with sulfhydryl groups, as judged by their ability to label reduced but not unreduced ribonuclease A and immunoglobulin E. Specific activities of 1 Ci/mmole in ¹²⁵I or ¹³¹I can be readily achieved with both the diiodinated and trace-iodinated (carrier-free) derivatives, and the specific activity of the former can be used directly to quantitate sulfhydryl groups in subnanomolar quantities of protein. N-Chloroacetyl ¹²⁵I-labeled tyramine prepared by trace iodination with carrier-free ¹²⁵I is more useful when very high specific activities (100–1000 mCi/μmol) are required. The utility of these reagents is discussed.