Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [3H]flunitrazepam and [3H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [3H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [3H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [3H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [3H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [3H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.     

Photoaffinity labelling of Ca2+ channels with [3H]azidopine

A 1,4-dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a Kd of 350 pM to guinea-pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited steroselectively by 1,4-dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of Mr approximately 145 000. The covalent labelling of the Mr approximately 145 000 band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.     

Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone

We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000.     

Photoaffinity labeling by [3H]-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity

N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive [3H]MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein. Specific binding was optimal at pH 7.5 and inhibited in the presence of Na+ and Li+. Inhibition by amiloride exhibited biphasic kinetics. After resolution of solubilized membranes by high-pressure liquid chromatography, MIA binding activity cofractionated together with Na+/H+ antiport activity, measured after reconstitution in asolectin vesicles, into a major and a minor peak. When fractions containing the major peak of Na+/H+ antiport activity were incubated with [3H]MIA and then photolyzed with a mercury arc lamp, covalent incorporation of label into polypeptides of apparent molecular mass 81 and 107 kDa was observed. These photolabeled bands were also observed in intact brush border membranes in addition to labeled polypeptides of apparent molecular mass 60 and 46 kDa, respectively. Labeling was inhibited by amiloride, reduced in the presence of Na+, and not observed in the absence of photolysis. These data point to the 81- and 107-kDa polypeptides as candidates for identification as components of a Na+/H+ antiport system in renal brush border membranes.     

Photoaffinity labeling with [3H]RU 28362: a powerful tool for the study of rat brain glucocorticoid receptors

In order to study the receptor system for adrenocortical steroids in rat brain the synthetic glucocorticoid RU 28362 (11 beta, 17 beta-dihydroxy-6-methyl-17 alpha-(1-propynyl) androsta-1,4,6-trien-3-one) has been used for photoaffinity labeling. Competition and dissociation studies revealed a single class of binding sites for RU 28362 in rat brain cytosol. Photoaffinity labeling was performed by u.v.-irradiation for 2 min with a coupling efficiency of about 25%. The high efficiency permitted investigation of crude cytosolic preparations under denaturating conditions. Sodium dodecyl sulfate (SDS) and high resolution two-dimensional gel electrophoresis confirmed the high specificity of the photoaffinity labeling. The molecular weight (93 kD) as well as the isoelectric point (5.6) evaluated by these methods corresponded well to data reported for the classical glucocorticoid receptor in rat liver.     

Pharmacological characterization of [3H]-JTH-601, a novel alpha1-adrenoceptor antagonist: binding to recombinant human alpha1-adrenoceptors and human prostates

Several alpha1-adrenoceptor (AR) selective antagonists are now widely used to improve lower urinary tract symptoms in benign prostatic hyperplasia patients. However, these drugs often result in orthostatic hypotension, because of their poor uroselectivity; the blockade of alpha1-AR not only in prostate but also in vasculature. Here we have investigated uroselectivity of JTH-601, a newly developed antagonist, in radioligand binding experiment using recombinant human alpha1-AR subtypes and human prostate. In saturation experiments, [3H]-JTH-601 showed subtype selectivity: high affinity to alpha1a-AR (pKd; 9.88+/-0.09), lower affinity to alpha1b-AR (pKd; 8.96+/-0.17) and no specific binding at concentrations up to 3000 pM to alpha1d-AR. In competition experiments, JTH-601 and its metabolic compound (JTH-601-G1) also showed alpha1a-AR selectivity, exhibiting approximately 5 times higher affinity for alpha1a-AR than for alpha1b-AR, 10 to 20 times higher affinity than for alpha1d-AR, respectively. [3H]-JTH-601 also bound to human prostate membranes in monophasic manner with high affinity constant (pKd; 9.89+/-0.12, Bmax=123.6+/-16 fmol/mg protein). JTH-601 is a unique alpha1-AR antagonist that shows high affinity and selectivity for human recombinant alpha1a- and human prostate. This new compound is useful for understanding alpha1-AR pharmacology and may have a therapeutic value.     

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.     

Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate]

It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [3H]25-OH-D3 or [3H]25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [3H]25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of [3H]25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These data provide strong evidence that [3H]25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein.     

Photoaffinity labeling of D-2 dopamine binding subunits from rat striatum, anterior pituitary and olfactory bulb with a new probe, [3H]azidosulpride

A novel photoaffinity probe [3H] azidosulpride has been developed for biochemical studies of D-2 dopamine receptors. This ligand binds to the receptors with high affinity (Kd = 3.1 +/- 0.2 nM) and, upon photoactivation, about 20% of the radioactivity bound to membranes becomes covalently incorporated. More than 90% of this irreversible binding is protectable by dopaminergic agents including D-2 selective compounds, whereas D-1 selective and non-dopaminergic compounds are ineffective. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals a single band at Mr = 85 kDa for labeled receptors in striatum, anterior pituitary or olfactory bulb, where pharmacologically distinct binding sites have been previously detected.     

O-Methylated and sulfoconjugated catecholamines: differential activities at human platelet alpha 2-adrenoceptors.

The physiological effects of the sulfoconjugates of epinephrine, norepinephrine, and the 3-O-methylated catecholamines, metanephrine, normetanephrine, and methoxytyramine were examined with regard to their alpha 2-adrenoceptor binding properties and aggregation activity in human platelets. Sulfoconjugation of catecholamines resulted in the loss of both their competitive potency for [3H]yohimbine binding and their influence on platelet aggregation. O-Methyl substituted catecholamines showed attenuation of their alpha 2-adrenoceptor binding affinities when compared with those of the corresponding non-esterified amines. Unlike the free amine epinephrine, which stimulated platelet aggregation, the O-methylated catecholamine derivatives inhibited aggregation. Inhibition was dose-dependent and restricted to the alpha 2-adrenoceptor mediated aggregation response stimulated by epinephrine (1 microM) or potentiated by subthreshold concentrations of epinephrine (30-300 nM) in the presence of subaggregatory doses of vasopressin (10-30 nM). Collagen- and ADP-induced platelet aggregation was not affected. The hydrophilic beta-antagonist CGP 12177 displayed no effects. However, high concentrations (0.1 mM) of both isomers of the strongly lipophilic beta-adrenoceptor antagonist propranolol inhibited the actions of all aggregators by stabilizing the membrane. Such a nonspecific membrane interaction of the methylated catecholamines could be excluded because of their low lipid solubility calculated in a n-octanol-phosphate buffer system at pH 7.4. We suggest therefore that methylated catecholamines are biological alpha 2-adrenoceptor antagonists acting on alpha 2-adrenoceptor stimulated reactions of human platelets. Whether this receptor antagonism is relevant to other human tissues needs clarification. Sulfated catecholamines, however, are wholly ineffective at this receptor site and may constitute a pathway to control the concentration of the active free catecholamines.     

Photoaffinity labeling of the purified skeletal muscle calcium antagonist receptor by a novel benzothiazepine, [3H]azidobutyryl diltiazem

Previous photoaffinity-labeling studies with [3H]azidopine, (+) [3H]PN200-110, and [3H]LU 49888 have demonstrated that 1,4-dihydropyridines (nifedipine-like drugs) and phenylalkylamines (verapamil-like drugs) bind exclusively to the 165-kDa alpha 1 subunit of skeletal muscle calcium channels. However, it has not been conclusively determined whether benzothiazepines (diltiazem-like drugs), which represent the third group of calcium antagonists, also bind to the alpha 1 subunit. Here we report data obtained with a newly developed benzothiazepine photoaffinity probe, [3H]azidobutyryl diltiazem. This drug competes with diltiazem for the benzothiazepine-binding site and, in purified calcium channel preparations, specifically labels the 165-kDa polypeptide which does not change its electrophoretic mobility upon disulfide reduction. These data show that benzothiazepines, just like 1,4-dihydropyridines and phenylalkylamines, bind to the alpha 1 subunit of the skeletal muscle calcium channels.     

Photoaffinity labeling of human IRBP with all-trans-retinoic acid

Interphotoreceptor retinoid-binding protein (IRBP), found only in photosensitive tissues, is a large approximately 135-kDa glycoprotein that contains a fourfold repeat structure. IRBP may function as a buffer and prevent retinoid toxicity and retinoid degeneration. Here we asked (i) whether each repeat of IRBP possesses the capability of photo-crosslinking all-trans-retinoic acid (RA), (ii) within Repeat 1 whether a single retinoic acid-binding domain exists, and (iii) whether protease and CNBr digestion of Repeat 1 bound RA indicate the exact location of the binding site. 3H-RA cross-linked to all four repeats, consistent with the current model of multiple binding sites in IRBP. Acetone precipitation was effective in removing unbound 3H-RA. LysC and tryptic digestion of the RA-Repeat 1 detected 18- and 5-kDa bands, respectively. CNBr digestion showed two bands about 9 and 11 kDa in size. Our data suggests a single binding site near positions 151-160 in the center of Repeat 1.     

Photoaffinity labeling of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) on intact platelets with 8-azido-[gamma-32P]ATP.

The fibrinogen receptor GPIIb-IIIa plays a crucial role in platelet aggregation. Here we show that the adenine nucleotide, 8-azido-ATP, inhibits ADP-induced conformational change of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3). Photoaffinity labeling of intact platelets with 8-azido-[gamma-32P]ATP exclusively modifies two plasma-membrane glycoproteins which are identical with both subunits of GPIIb-IIIa. The presence of adenine-nucleotide-binding sites on GPIIb-IIIa implies that the platelet fibrinogen receptor is directly regulated by extracellular adenine nucleotides.     

Photoaffinity labelling of androgen receptors from human foreskin and cultured genital fibroblasts with tritiated methyltrienolone [3H]R1881

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from either normal foreskins or normal cultured fibroblasts. Partially purified receptors were covalently labelled with [3H]R1881 (tritiated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from both sources showed two peaks of specific binding, Mr approximately 40 K and approximately 85 K, under denaturing conditions. Photoaffinity labelling of the human androgen receptor may thus provide a useful tool in further studies on disorders of androgenization.     

Photoaffinity labeling of mitochondrial adenosinetriphosphatase by 2-azidoadenosine 5'-[alpha-32P]diphosphate

2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623] contained an average of 2.9 mol of tightly bound ADP plus ATP per mole of enzyme. Short-term incubation of F1 with micromolar concentrations of [alpha-32P]-2-azido-ADP in the dark in a Mg2+-supplemented medium resulted in the rapid supplementary binding of 3 mol of label/mol of F1, consistent with the presence of six nucleotide binding sites per F1. The Kd relative to the reversible binding of [alpha-32P]-2-azido-ADP to mitochondrial F1 in the dark was 5 microM in the presence of MgCl2 and 30 microM in the presence of ethylenediaminetetraacetic acid. A linear relationship between the percentage of inactivation of F1 and the extent of covalent photolabeling by [alpha-32P]-2-azido-ADP was observed for percentages of inactivation up to 90%, extrapolating to 2 mol of covalently bound [alpha-32P]-2-azido-ADP/mol of F1. Under these conditions, only the beta subunit was photolabeled. Covalent binding of one photolabel per beta subunit was ascertained by electrophoretic separation of labeled and unlabeled beta subunits based on charge differences and by mapping studies showing one major radioactive peptide segment per photolabeled beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin

A radioiodinated, photoactive cardiac glycoside derivative, 4'-(3-iodo-4-azidobenzene sulfonyl)cymarin (IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC inhibited the activity of (Na+K+)-ATPase in electroplax microsomes from Electrophorus electricus with the same I50 as cymarin. [125I]IAC binding, in the presence of Mg2+ and Pi, was specific, of high affinity (KD = 0.4 microM), and reversible (k-1 = 0.11 min-1) at 30 degrees C. At 0 degree C, the complex was stable for at least 3 h, thus permitting washing before photolysis. Analysis of [125]IAC photolabeled electroplax microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7-14%) showed that most of the incorporated radioactivity was associated with the alpha (Mr = 98,000) and beta (Mr = 44,000) subunits of the (Na+K+)-ATPase (ratio of alpha to beta labeling = 2.5). A higher molecular weight peptide (100,000), similar in molecular weight to the brain alpha(+) subunit, and two lower molecular weight peptides (12,000-15,000), which may be proteolipid, were also labeled. Two-dimensional gel electrophoresis (isoelectric focusing then SDS-PAGE, 10%) resolved the beta subunit into 12 labeled peptides ranging in pI from 4.3 to 5.5. When (Na+K+)-ATPase in synaptosomes from monkey brain cortex was photolabeled and analyzed by SDS-PAGE (7-14%), specific labeling of the alpha(+), alpha, and beta subunits could be detected (ratio of alpha(+) plus alpha to beta labeling = 35). The results show that [125I]IAC is a sensitive probe of the cardiac glycoside binding site of (Na+K+)-ATPase and can be used to detect the presence of the alpha(+) subunit in crude membrane fractions from various sources.