Low background scintillation counting

Counting radioactive samples with Beckman Instrument's Ready Caps, using a restricted energy window, LL-UL = 400-1000, resulted in machine backgrounds of under 2 cpm and efficiencies of counting relative to liquid scintillation cocktails (LSC) of 51%, 65%, 57%, 62%, and 1% for 32P, 125I, 14C, 35S and 3H, respectively. Signal-to-noise ratios from a quantitative molecular hybridization technique were increased 8-10 fold. There may be a general application for this product in experiments yielding low amounts of radioactivity in liquid samples.     

Comparative Study of 125I- and [3H]Acetate-Labeled Antibodies in Detecting Iridescent Viruses

Radioimmunoassays for detecting cell-associated or released virus are described using either (125)I- or [(3)H]acetate-labeled antibodies. In the first assay system, antigen-antibody complexes were separated from free antibody by centrifugation. Sensitivities of 0.1 mug of iridescent virus could be achieved with either (125)I- or [(3)H]acetate-labeled antibody. In the second assay, the antigen was fixed to cover-slip cell cultures, and then reacted with labeled antibody, unbound radioactivity being removed by repeated washing. Nonspecific binding with this method was 0.5 to 1% of the total radioactivity added and sensitivities of 0.1 or 10 mug were achieved with (125)I and [(3)H]acetate, respectively. Immunoglobulins were labeled at the rate of 1 in 300 for (125)I and 1 in 200 with [(3)H]acetate although there was a 400-fold greater isotopic abundance of (125)I relative to (3)H. The possibility of preparing labeled protein of high specific activity using carrier-free [2-(3)H]iodoacetic acid is discussed.     

Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether

Intralipid was sonicated with [3H]cholesteryl linoleyl ether (a nonhydrolyzable analog of cholesteryl linoleate) and incubated with rat HDL and d greater than 1.21 fraction of rabbit serum at a ratio of 0.012 mg triacylglycerol to 1 mg HDL protein. 25% of [3H]cholesteryl linoleyl ether was transferred to HDL. The labeled HDL was injected into donor rats and was screened for 4 h. [125I]HDL was subjected to the same protocol as the 3H-labeled HDL, including screening. The screened, labeled sera were injected into acceptor rats and the disappearance of radioactivity from the circulation was compared. The t1/2 in the circulation of [125I]HDL was about 10.5 h, while that of [3H]cholesteryl linoleyl ether-HDL was about 8 h. The liver and carcass were the major sites of uptake of [3H]cholesteryl linoleyl ether-HDL and accounted for 29-41% (liver) and 30% (carcass) of the injected label. Maximal recovery of [3H]cholesteryl linoleyl ether in the liver was seen 48 h after injection, and thereafter there was a progressive decline of radioactivity, which reached 7.8% after 28 days. The maximal recovery of [125I]HDL in the liver was about 9%. Pretreatment of the acceptor rats with estradiol for 5 days resulted in a 20% increase in the hepatic uptake of [3H]cholesteryl linoleyl ether-HDL and a 5-fold increase in adrenal uptake. The present findings indicate that in the rat the liver is the major site of uptake of HDL cholesteryl ester and that part of the HDL cholesteryl ester may be cleared from the circulation separately from the protein moiety. On the basis of our previous findings (Stein, Y., Kleinman Y, Halperin, G., and Stein, O. (1983) Biochim. Biophys. Acta 750, 300-305) the loss of the [3H]cholesteryl linoleyl ether from the liver after 14-28 days was interpreted to indicate that the labeled [3H]cholesteryl linoleyl ether had been taken up by hepatocytes.     

On the enterohepatic cycle of triiodothyronine in rats; importance of the intestinal microflora

Until 70 h after a single iv injection of 10 uCi [125I]triiodothyronine (T3), normal rats excreted 15.8 +/- 2.8% of the radioactivity with the feces and 17.5 +/- 2.7% with the urine, while in intestine-decontaminated rats fecal and urinary excretion over this period amounted to 25.1 +/- 7.2% and 23.6 +/- 4.0% of administered radioactivity, respectively (mean +/- SD, n = 4). In fecal extracts of decontaminated rats 11.5 +/- 6.8% of the excreted radioactivity consisted of T3 glucuronide (T3G) and 10.9 +/- 2.8% of T3 sulfate (T3S), whereas no conjugates were detected in feces from normal rats. Until 26 h after ig administration of 10 uCi [125I]T3, integrated radioactivity in blood of decontaminated rats was 1.5 times higher than that in normal rats. However, after ig administration of 10 uCi [125I]T3G or [125I]T3S, radioactivity in blood of decontaminated rats was 4.9- and 2.8-fold lower, respectively, than in normal rats. The radioactivity in the serum of control animals was composed of T3 and iodide in proportions independent of the tracer injected, while T3 conjugates represented less than 10% of serum radioactivity. These results suggest an important role of the intestinal microflora in the enterohepatic circulation of T3 in rats.     

Preparation of 6-125I-labeled amiloride derivatives

Amiloride and certain of its derivatives are effective inhibitors of Na/H antiporters and of epithelial Na channels. We describe a simple method for the preparation of a variety of pharmacologically active 6-iodoamiloride derivatives that are labeled with 125I at high specific radioactivity. 6-Dechloroamiloride derivatives (bearing a hydrogen atom instead of the chlorine at the 6 position of the amiloride molecule) are reacted with 125ICl, prepared by the oxidation of the iodide in Na125I preparations. The 125I-labeled derivatives are separated from free 125I by anion exchange chromatography, or purified by thin layer chromatography. Both 6-dechloroamiloride and 5-(N-alkyl)-6-dechloroamiloride derivatives can be labeled by this method, with yields varying between 10 and 70%, depending on the ICl concentration and the structure of the 5-N-alkyl group. Efficient radiolabeling at high specific radioactivity also depends on the use of freshly prepared batches of 125I. Using carrier-free 125I, [125I]6-iodoamiloride and [125I]6-iodo-5-(N-tert-butyl)amiloride were prepared with yields of 27 and 22%, respectively. Potential applications of the 125I-labeled amiloride derivatives include ligand binding and affinity labeling experiments.     

Comparative labelling of α1-acid glycoprotein by (3H)-borohydride or (125I)-iodide for use in a radioimmunoassay

The labelling of α₁-acid glycoprotein (AGP) with (³H)-sodium borohydride was compared to the labelling with (¹²⁵I)-sodium iodide by the chloramine T method in view to its use in a radioimmunoassay. The tritium labelling allowed to reach a high specific radioactivity similar to that obtained with iodide ((³H)-AGP: 29.8 mCi/mg; (¹²⁵I)-AGP: 30.5 mCi/mg). Each mole of sialic acid residue of AGP contains one atom of tritium. The stability of (³H)-AGP was better than that of (¹²⁵I)-AGP as indicated by its immunoreactivity as a function of time. Immunoreactivities and standard curves were similar for the two tracers but affinity of antiserum was higher for (¹²⁵I)-AGP than for (³H)-AGP. Tritium labelling by (³H)-borohydride will be very useful for glycoprotein antigens which cannot be labelled with (¹²⁵I)-iodide.     

Specific photoaffinity labelling of inhibitory adenosine receptors

N6(L-phenylisopropyl)adenosine (L-PIA) and N6(3-iodo-4-azido benzyl)-adenosine (IAzBA) inhibit the adenylate cyclase activity in synaptic membranes of chick cerebellum via Ri adenosine receptors. [3H]L-PIA and [125I]AzBA bind to these membranes with Kd values of approximately 1 nM and Bmax values of approximately 1000 fmol/mg protein. Photolysis of [125I]AzBA bound to synaptic membranes results in the specific incorporation of radioactivity into a protein with Mr = 36,000. This photoincorporation is blocked by simultaneous exposure to L-PIA, theophylline, an adenosine receptor antagonist, or Gpp(NH)p, but not by cytosine, suggesting that the 36,000 dalton protein is the Ri adenosine receptor or a subunit of the receptor that contains the adenosine binding site.     

Evidence for different forms of human chorionic gonadotropin receptor complex in pseudopregnant rat ovaries.

After in vivo application of [125 I]-iodo-hCG to pseudopregnant rats, radioactivity extracted from ovarian particulate fractions can be eluted from Sephadex G--200 columns in three various fractions (Vo, Kav = 0.15, Kav = 0.27) differing from hCG (Kav = 0.35). By lowering the pH 50% of the radioactivity is precipitated and the resuspended material shows a conversion to the smaller complexes. The supernatant material is dissociated to [125I]-iodo-hCG. In alkaline milieu [125I]-iodo-HCG is dissociated completely from the complexes.     

Multiplicity of molecules carrying blood-group-I antigen on erythrocyte membranes.

A human serum containing a monoclonal anti-(blood-group I) antibody was used to investigate the distribution of blood-group-I antigen on erythrocyte membrane components. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis profiles of immuneprecipitates by using 3H-labelled (by the galactose oxidase/NaB3H4 method) and 125I-labelled solubilized stroma were compared. Different radioactive profiles were revealed by the two radiolabelling methods. In the immunoprecipitates the predominant 125I radioactivity within the gel had the electrophoretic mobility of Band-3 protein (apparent mol.wt. 90 000--100 000), whereas the 3H radioactivity revealed a diffusely migrating component(s) (apparent mol.wt. range 40 000--70 000) in addition to radioactivity compatible with glycolipids at the dye front. The diffusely migrating 3H-labelled component was shown to have a similar electrophoretic mobility to a subpopulation of erythrocyte poly(glycosyl)ceramides with blood-group-I activity.     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.     

Antibody accumulation in small tissue samples: Assessment by quantitative autoradiography

Uptake of radiolabeled ¹²⁵I monoclonal antibodies in small metastases can only be characterized by autoradiographic techniques. To obtain quantitative data out of autoradiographic images, a transformation of the essentially two-dimensional signal into the Bq per unit volume information is needed. Part of the calibration problem could be solved by using tissue-equivalent standard preparations. However, when aiming at a quantification of radioactivity in small areas (⩽- 2 mm diameter), special criteria had to be expanded upon for the reconstruction of the area in the dose matrix and for the correct integration of the radioactivity content.     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.     

Simultaneous identification of estrogen and progesterone receptors by HPLC using a double isotope assay.

Polymorphism of estrogen (ER) and progestin receptors (PR) was analyzed simultaneously using high performance hydrophobic interaction chromatography (HPHIC). HPHIC was used previously to characterize four ER isoforms [Hyder et al., J. Chromat. 397 (1987) 251] based on retention times on Synchropak propyl (100 x 6 mm) HPLC columns (Synchrom, Inc.). ER and PR were prepared from human breast cancer. ER was labeled with 3 nM of either [3H]estradiol-17 beta ([3H]E) or [125I]iodoestradiol-17 beta ([125I]E) while PR was associated with 5 nM of either [3H]R5020 ([3H]R) or [125I]iodovinylnortestosterone ([125I]V). ER was resolved by HPHIC into isoforms MI (Rt = 11 min), I(Rt = 16 min), and II (Rt = 24 min). Isoforms I and II each accounted for ca 45% of specific binding. PR separated into isoforms MI (Rt = 14 min) and I (Rt = 21 min, 80% of specific binding) when eluted with the same gradient used for ER chromatography. Upon inclusion of 10 mM molybdate ER resolved into isoforms MI and MII (Rt = 16 min) and PR into isoforms MI and I (here however isoform MI represented 80-95% of specific binding). Elution patterns were preserved with different batches of stationary phase suggesting the integrity of the isoform distribution. HPLC profiles of ER isoforms labeled with earlier [125I]E or [3H]E were identical as were PR isoform profiles labeled with either [3H]R or [125I]V. Pairs of 125I- and 3H-labeled ligands were used in either combination to monitor ER and PR profiles simultaneously. Isoforms analyzed in 50 biopsies gave reproducible retention times, however the ratio between I and II for ER and MI and I for PR varied. This method allows rapid, simultaneous monitoring of the chromatographic behavior of ER and PR isoforms or other associating proteins or nucleotides. One may now better elucidate their interrelationship as it relates to the hormone-response mechanism.     

Protein transfer between A-I-containing lipoprotein subpopulations: evidence of non-transferable A-I in particles with A-II.

Transfer of apolipoproteins (apo) between the two subpopulations of apo A-I-containing lipoproteins in human plasma: those with A-II [Lp(AI w AII)] and those without [Lp(AI w/o AII)], were studied by observing the transfer of 125I-apo from a radiolabeled subpopulation to an unlabeled subpopulation in vitro. When Lp(AI w AII) was directly radioiodinated, 50.3 +/- 7.4 and 19.5 +/- 7.7% (n = 6) of the total radioactivity was associated with A-I and A-II, respectively. In radioiodinated Lp(AI w/o AII), 71.5 +/- 6.8% (n = 6) of the total radioactivity was A-I-associated. Time-course studies showed that, while some radiolabeled proteins transferred from one population of HDL particles to another within minutes, at least several hours were necessary for transfer to approach equilibrium. Incubation of the subpopulations at equal A-I mass resulted in the transfer of 51.8 +/- 5.0% (n = 4) of total radioactivity from [125I]Lp(AI w/o AII) to Lp(AI w AII) at 37 degrees C in 24 h. The specific activity (S.A.) of A-I in the two subpopulations after incubation was nearly identical. Under similar incubation conditions, only 13.4 +/- 4.6% (n = 4) of total radioactivity was transferred from [125I]Lp(AI w AII) to Lp(AI w/o AII). The S.A. of A-I after incubation was 2-fold higher in particles with A-II than in particles without A-II. These phenomena were also observed with iodinated high-density lipoproteins (HDL) isolated by ultracentrifugation and subsequently subfractionated by immunoaffinity chromatography. However, when Lp(AI w AII) radiolabeled by in vitro exchange with free [125I]A-I was incubated with unlabeled Lp(AI w/o AII), the S.A. of A-I in particles with and without A-II differed by only 18% after incubation. These data are consistent with the following: (1) in both populations of HDL particles, some radiolabeled proteins transferred rapidly (minutes or less), while others transferred slowly (hours); (2) when Lp(AI w AII) and Lp(AI w/o AII) were directly iodinated, all labeled A-I in particles without A-II were transferable, but some labeled AI in particles with A-II were not; (3) when Lp(AI w AII) were labeled by in vitro exchange with [125I]A-I, considerably more labeled A-I were transferable. These observations suggest the presence of non-transferable A-I in Lp(AI w AII).     

The ND5 subunit was labeled by a photoaffinity analogue of fenpyroximate in bovine mitochondrial complex I

Fenpyroximate is a potent inhibitor of the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). We synthesized its photoaffinity analogue [(3)H](trifluoromethyl)phenyldiazirinylfenpyroximate ([(3)H]TDF). When bovine heart submitochondrial particles (SMP) were illuminated with UV light in the presence of [(3)H]TDF, radioactivity was mostly incorporated into a 50 kDa band. There was a good correlation between radioactivity labeling of the 50 kDa band and inhibition of the NADH oxidase activity, indicating that a 50 kDa protein is responsible for the inactivation of complex I. Blue native gel electrophoresis of the [(3)H]TDF-labeled SMP revealed that the majority of radioactivity was found in complex I. Analysis of the complex I band on an SDS gel showed a major peak of radioactivity at approximately 50 kDa. There are three subunits in complex I that migrate in this region: FP51K, IP49K, and ND5. Further analysis using the 2D gel electrophoresis implied that the labeled protein was the ND5 subunit. Labeling of the ND5 subunit was stimulated by NADH/NADPH but was prevented by various complex I inhibitors. Amiloride derivatives that are known to be inhibitors of Na(+)/H(+) antiporters also diminished the labeling. In agreement with the protective effect, we observed that the amiloride derivatives inhibited NADH-ubiquinone-1 reductase activity but not NADH-K(3)Fe(CN)(6) reductase activity in bovine SMP. These results suggest that the ND5 subunit is involved in construction of the inhibitor- and quinone-binding site(s). Furthermore, it seems likely that the ND5 subunit may participate in H(+)(Na(+)) translocation in coupling site 1.     

Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine.

Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the plasma membrane.     

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.     

Binding Properties of 3-[125I]Iodophencyclidine, a New Radioligand for N-Methyl-D-Aspartate-Gated Ionic Channels

The binding properties of the 125I-labeled phencyclidine derivative N-[1-(3-[125I]iodophenyl)cyclohexyl]piperidine (3-[125I]iodo-PCP), a new ligand of the N-methyl-D-aspartate (NMDA)-gated ionic channel, were investigated. Association and dissociation kinetic curves of 3-[125I]iodo-PCP with rat brain homogenates were well described by two components. About 32% of the binding was of fast association and fast dissociation, and the remaining binding was of slow association and slow dissociation. Saturation curves of 3-[125I]iodo-PCP also were well described using two binding sites: one of a high affinity (KDH = 15.8 +/- 2.3 nM) and the other of a low affinity (KDL = 250 +/- 40 nM). 3-Iodo-PCP inhibited the binding of 3-[125I]iodo-PCP with inhibition curves that were well fitted by a two-site model. The binding constants (KiH, BmaxH; KiL, BmaxL) so obtained were close to those obtained in saturation experiments. Ligands of NMDA-gated ionic channels also inhibited the binding of 3-[125I]iodo-PCP with two constants, KiH and KiL. There was a very good correlation (r = 0.987) between the affinities of these ligands to bind to NMDA-gated ionic channels and their potencies to inhibit the binding of 3-[125I]iodo-PCP with a high affinity. Moreover, the regional distribution of the high-affinity binding of 3-[125I]-iodo-PCP paralleled that of tritiated N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). In contrast to that of [3H] TCP, the binding of 3-[125I]iodo-PCP to well-washed rat brain membranes was fast and insensitive to glutamate and glycine.(ABSTRACT TRUNCATED AT 250 WORDS)