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PubMed1978

Photoaffinity labeling of the angiotensin II receptor; pharmacology of the labeling peptides in the dark.

The biological activities of photoaffinity labeling analogs of angiotensin II (ATII) and their precursors were measured in rabbit aorta strips in the dark. Most of the analogs behave as reversible, specific agonists, one as a competitive inhibitor. The activities are discussed in line with the current view of structural requirements. The modifications consisted of substitutions on the aromatic nuclei of Tyr4 and Phe8 in [Sar1]ATII with (4'-NO2) Phe, (4'-NH2)Phe, (4'N3)Phe, (4'-N2 +)Phe, and (4'-NH2-3', 5'-I2)Phe. It is shown that the affinity of the ATII analogs modified in position 4 depends on the electronegativity and not on space-filling properties of the aromatic residue; rising electronegativity lowers the affinity, i.e. [sar1, (4'-NO2)Phe4]ATII has no more measurable activity. Substituting the aromatic side chain in position 8 of [Sar1]ATII gives well-binding analogs with intrinsic activities from 0 to 100% and activity seems to depend only on stereochemical requirements. Agonists and partial agonists bear rather small groups like -NH2, -N3, -NO2, and -N2 +. The only antagonist [Sar1, (4'-NH2-3',5'-I2)Phe8]ATII resembles the antagonist E1Sar1, Leu8]ATII in competitivity and binding.

Angiotensin II↗
PubMed1992

Insulin receptors are bivalent as demonstrated by photoaffinity labeling.

Insulin receptors in human placental membranes were photoaffinity-labeled with a radioactive human insulin-like growth factor I (hIGF-I) photoprobe N epsilon B28-monoazidobenzoyl 125I-hIGF-I either alone or together with a non-radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl insulin. Precipitation of the solubilized receptors with anti-insulin antibody showed that receptors labeled with the radioactive hIGF-I photoprobe were detected in the immunoprecipitate only when photolabeling was carried out in the presence of the non-radioactive insulin photoprobe. Comparable results were obtained in converse experiments using a radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl 125I-insulin, a non-radioactive hIGF-I photoprobe N epsilon B28-monoazidobenzoyl hIGF-I, and an antibody to hIGF-I. The amount of radioactive receptors precipitated by either the anti-insulin antibody or the anti-hIGHF-I antibody was close to the expected amount. These observations demonstrate that the insulin receptor is bivalent being capable of binding two molecules of ligand.

Affinity Labels↗
PubMed1997

Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain.

Photoaffinity labelling of the human poly(ADP-ribose) polymerase (PARP) catalytic domain (40 kDa) with the NAD+ photoaffinity analogue 2-azido-[alpha-32P]NAD+ has been used to identify NAD+-binding residues. In the presence of UV, photo-insertion of the analogue was observed with a stoichiometry of 0.73 mol of 2-azido-[alpha-32P]NAD+ per mol of catalytic domain. Competition experiments indicated that 3-aminobenzamide strongly protected the insertion site. Residues binding the adenine ring of NAD+ were identified by trypsin digestion and boronate affinity chromatography in combination with reverse-phase HPLC. Two major NAD+-binding residues, Trp1014 of peptide Thr1011-Trp1014 and Lys893 of peptide Ile979-Lys893, were identified. The site-directed mutagenesis of these two residues revealed that Lys893, but not Trp1014, is critical for activity. The close positioning of Lys893 near the adenine ring of NAD+ has been confirmed by the recently solved crystallographic structure of the chicken PARP catalytic domain [Ruf, Menissier-de Murcia, de Murcia and Schulz (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7481-7485].

Affinity Labels↗
PubMed1988

Identification and characterization of melanotropin binding proteins from M2R melanoma cells by covalent photoaffinity labeling.

In this study, two melanotropin binding proteins from M2R melanoma cells have been identified based on the photochemical cross-linking of [125I]iodinated porcine beta-MSH ([ 125I]iodo-beta-MSH) to melanoma cell membranes, using N-hydroxysuccinimidyl-azidobenzoate. Autoradiography of photoaffinity-labeled M2R membrane protein, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the specific labeling of two separate bands with an apparent molecular mass of 43 and 46 kilodaltons, respectively. Photoaffinity labeling of both bands was of near-equal intensity and could be inhibited, in a dose-dependent manner, by the addition of unlabeled beta-MSH before photolysis. In addition, agents known to inhibit the binding of beta-MSH to its cellular receptor, such as EGTA, GTP, guanosine 5'-O-(3-thio)triphosphate, and a synthetic analog of the calmodulin-binding domain of myosin light chain kinase-M5, were all found to specifically inhibit the labeling of these two protein bands by the azido derivative of [125I]iodo-beta-MSH. In contrast, addition of a nonrelated peptide, vasoactive intestinal peptide, had no effect upon the labeling of these melanotropin-binding proteins. On the basis of these results we suggest that the two proteins may function as the binding domain(s) of the cellular receptor for melanotropins, or may represent entire receptor moieties themselves.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of the tetracycline binding site of the Escherichia coli ribosome. The uses of a high intensity light source and of radioactive sancycline derivatives.

[3H]Tetracycline (TC) has been shown to photoincorporate into the Escherichia coli ribosome. However, the utility of this process for characterizing the TC binding site on the ribosome is diminished by competing side reactions which also lead to incorporation of radioactivity. In this work we first conducted a detailed study of the labeling processes occurring when ribosomes are irradiated in the presence of [3H]TC with a common, rather low intensity, lamp. On the basis of the results of this study we next explored the usefulness for photoaffinity labeling of the TC site of both irradiation with a high-intensity laser and radioactive, functional TC derivatives having different photochemical properties than TC itself. Labeling patterns determined by polyacrylamide gel electrophoretic analysis of ribosomal proteins extracted from photoaffinity-labeled 30S subunits provided strong evidence that these two approaches offer distinct advantages for characterizing the TC binding site.

Affinity Labels↗
PubMed2001

Identification of the GTP binding site of human glutamate dehydrogenase by cassette mutagenesis and photoaffinity labeling.

It has been reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in glutamate dehydrogenase (GDH) gene that affects enzyme sensitivity to GTP-induced inhibition. To identify the GTP binding site(s) within human GDH, mutant GDHs at Tyr-266 or Lys-450 position were constructed by cassette mutagenesis. More than 90% of the initial activities were remained at the concentration of GTP up to 300 microm for the Lys-450 mutant GDHs regardless of their size, hydrophobicity, and ionization of the side chains, whereas the wild type GDH and the Tyr-266 mutant GDHs were completely inhibited by 30 microm GTP. The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-[gamma-(32)P]azidoguanosine 5'-triphosphate (8-N(3)-GTP). Saturation of photoinsertion with 8-N(3)-GTP occurred apparent K(d) values near 20 microm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N(3)-[gamma-(32)P]GTP was significantly decreased in the presence of 300 microm GTP. Unlike the wild type GDH or the Tyr-266 mutant GDH, less than 10% of photoinsertion was detected in the Lys-450 mutant GDH, and the photoinsertion was not affected by the presence of 300 microm GTP. The results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the GTP binding site and suggest that Lys-450, but not Tyr-266, is required for efficient binding of GTP to GDH. Interestingly, studies of the steady-state velocity showed that both the wild type GDH and the Tyr-266 mutant GDHs were inhibited by ATP at concentrations between 10 and 100 microm, whereas less than 10% of the initial activities of the Lys-450 mutant GDHs were diminished by ATP. These results indicate that Lys-450, but not Tyr-266, may be also responsible for the ATP inhibition; therefore, ATP bound to the GTP site.

Amino Acid Sequence↗
PubMed1988

Characterization of the transport system for beta-lactam antibiotics and dipeptides in rat renal brush-border membrane vesicles by photoaffinity labeling.

The uptake of the alpha-aminocephalosporin cephalexin into brush-border membrane vesicles from rat renal cortex was independent on an inward H+-gradient in contrast to the intestinal transport system. The transport system could be irreversibly inhibited by photoaffinity labeling. Two binding polypeptides for beta-lactam antibiotics and dipeptides with apparent molecular weights 130,000 and 95,000 were identified by photoaffinity labeling with [3H]benzylpenicillin and N-(4-azido[3,5-3H]benzoyl) derivatives of cephalexin and glycyl-L-proline. The uptake of cephalexin and the labeling of the respective binding proteins was inhibited by beta-lactam antibiotics and dipeptides as with intestinal brush-border membranes. These data indicate that the transport systems for beta-lactam antibiotics and dipeptides in the brush-border membrane from rat kidney and small intestine are similar but not identical.

Affinity Labels↗
PubMed1991

A stable diazo photoaffinity label with high absorptivity and effective photoactivation beyond 300 nm.

The sulfosuccinimidyl active ester of 3-(3-carbethoxy-4-diazo-5-oxo-2-pyrrolin-1-yl)propanoic acid (DIAZOPY-SE) has been synthesized for use as a photoaffinity labeling reagent. This compound was obtained from commercial chemicals by a four-step synthesis requiring no complex procedures or special apparatus. The active ester efficiently derivatizes protein amino groups with the chromophore 3-carbethoxy-4-diazo-5-oxo-2-pyrroline (DIAZOPY, epsilon 8800 M-1 cm-1 at lambda max 330 nm), which on irradiation yielded products expected from formation of a reactive carbene intermediate. Brief irradiation of DIAZOPY in 2-propanol using wavelengths greater than 300 nm for photolysis yielded mainly an isopropyl ether resulting from insertion of the carbene into the O-H bond of the alcohol. Formed concurrently and to a somewhat lesser extent was an isopropyl ester, resulting from a ring-contracting Wolff rearrangement of the carbene and subsequent reaction with isopropanol. Analogous products were produced by photolysis in 2-propanol of DIAZOPY-PA (for DIAZOPY propanoic acid), the carboxylic acid precursor of DIAZOPY-SE. Facile protein derivatization by DIAZOPY-SE was demonstrated using actin and sheep IgG. Actin labeled with DIAZOPY-SE and irradiated while in the F-actin (reversibly polymerized) form was crosslinked to yield a covalently-linked dimer, illustrating the potential of the reagent in photoaffinity applications. Advantages of DIAZOPY-SE as a photoaffinity labeling reagent include ease of synthesis, chemical and photostability, efficient photolysis at wavelengths greater than 300 nm, and a capacity for crosslinking by carbene insertion processes.

Actins↗
PubMed1995

Thiomuscimol, a new photoaffinity label for the GABAA receptor.

Thiomuscimol inhibits [3H]muscimol binding to brain GABAA receptors. Exposure of Ag(+)-treated membrane preparations to UV radiation at 254 nm for 40 min in the presence of thiomuscimol (10(-5) M) produced a 20-30% irreversible decrease in high-affinity [3H]muscimol binding sites. The photoaffinity labeling of thiomuscimol was inhibited by GABA (10(-4) M) added prior to exposure to UV light. The data show that thiomuscimol can label the GABAA receptor site and that the ligand can be used as a photoaffinity label for purification and identification of GABA binding sites within the GABAA receptor complex.

Affinity Labels↗
PubMed2002

Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase.

The adenine binding domain of the ADP site within human glutamate dehydrogenase (GDH) was identified by cassette mutagenesis at the Tyr187 position. The wild type GDH was activated 3-fold by ADP at a concentration of 1 mM at pH 8.0, whereas no significant activation by ADP was observed with the Tyr187 mutant GDH regardless of the size, hydrophobicity, and ionization of the side chains. Studies of the steady-state velocity of the mutant enzymes revealed essentially unchanged apparent K(m) values for 2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective apparent V(max) values. The binding of ADP to the wild type or mutant GDH was further examined by photoaffinity labeling with [alpha-(32)P]8-azidoadenosine 5'-diphosphate (8N(3)ADP). 8N(3)ADP, without photolysis, mimicked the stimulatory properties of ADP on GDH activity. Saturation of photoinsertion with 8N(3)ADP occurred with apparent K(d) values near 25 microM for the wild type GDH, and the photoinsertion of [alpha-(32)P]8N(3)ADP was decreased best by ADP in comparison to other nucleotides. Unlike the wild type GDH, essentially no photoinsertion was detected for the Tyr187 mutant GDH in the presence or absence of 1 mM ADP. For the wild type GDH, photolabel-containing peptide generated by tryptic digestion was identified in the region containing the sequence EMSWIADTYASTIG, and the photolabeling of this peptide was prevented >95% by the presence of 1 mM ADP during photolysis, whereas no such a peptide was detected for the Tyr187 mutant GDH in the presence or absence of ADP. These results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide is within the ADP binding domain of the human GDH and that Tyr187 is responsible for the efficient base binding of ADP to human GDH.

Adenine↗
PubMed1982

Photoaffinity labelling of dopamine receptors in molluscan smooth muscle.

Relaxation of catch contraction of the anterior byssus retractor muscle of the sea mussel Mytilus edulis L. by dopamine is mediated through a dopamine receptor but not through adrenoceptors (Takayanagi et al 1981). Photoaffinity labelling is a technique widely used in biochemical in vitro studies to test an interaction between a ligand and its binding site. Therefore, we tried photoaffinity labelling of the dopamine receptor in order to study the dopamine receptor in the anterior byssus retractor muscle of M. edulis. Sea mussels, collected from the east coast of Tokyo Bay were stored in aerated seawater (NaCl 456, KCl 11, CaCl2 2H2O 11, MgCl2 6H2O 48 nM and Tris-HCl 25 mM; pH 7 . 8 to 8 . 0) at 10 degrees C and used within a week of collection. Muscle bundles (about 1 mm in diameter) were dissected from the anterior byssus retractor muscle and suspended in a 10 ml organ bath filled with artificial seawater bubbled with air and kept at 24 to 25 degrees C. Responses to drugs were recorded isotonically under a tension of 0 . 2 g. After the muscle had been exposed to acetylcholine (10(-4)M) for 2 min to induce catch contraction and washed with artificial seawater for 5 min, dopamine was applied. Relaxations following a 10 min exposure to various doses of dopamine were estimated. The response to 3 x 10(-7) M dopamine was considered as the maximum response to obtain dose-response curves (Takayanagi et al 1981). To irradiate the muscle, a Toshiba lamp FL-20E (wavelength: 270 to 350 nm) was used as a light source. The muscle, immersed in artificial seawater containing dopamine (10(-4)M), was irradiated (1 cm from the lamp) for 25 min and then washed with artificial seawater for 60 min (Takayanagi et al 1976). After the muscle was irradiated in the presence of dopamine (10(-4)M) for 25 min and washed for 60 min, the dose-response curve of dopamine was shifted in a parallel way towards doses about 8 times higher (Fig. 1). This inhibition of dopamine-induced responses continued for at least 2 h. The dose-response curve for dopamine was unaffected when the muscle was incubated with both dopamine (10(-4)M) and haloperidol (10(-4)M) for 25 min under the irradiation conditions (Fig. 1). However, the inhibitory action of dopamine was unaffected when the muscle was irradiated in the absence of dopamine and washed for 60 min, suggesting that 20 min irradiation did not influence mechanisms for relaxation of this smooth muscle by dopamine. Furthermore, when the muscle was incubated with dopamine (10(-4)M) or haloperidol (10(-4)M) for 25 min and washed with artificial sea water, the dose-response curve for dopamine was not influenced. When promethazine (10(-4)M), an antihistamine drug found to have no antidopaminergic action in this muscle (Yoshida et al 1981), was used instead of haloperidol (10(-4)M), the dose response curve for dopamine was shifted after irradiation (data not shown). These results indicate the possibility that dopamine is photolysed to a reactive compound which reacts irreversibly with the dopamine receptor.

Affinity Labels↗
PubMed1989

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin.

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.

Affinity Labels↗
PubMed1981

Structure of the lutropin receptor on granulosa cells. Photoaffinity labeling with the alpha subunit in human choriogonadotropin.

Ovarian granulosa cells have specific receptors for lutropin and human choriogonadotropin (hCG). To investigate the nature of the receptor, several different photo-cross-linkable derivatives of radioiodinated hCG were incubated with porcine granulosa cells and hormone derivative-receptor complexes extracted with 0.5% Triton X-100. The extracted complexes appeared in a single peak on gel permeations and in rat zonal sedimentation studies. The estimated molecular weight was found to be 250,000-370,000 with an associated s value of 7.7 +/- 0.3. When Triton X-100 extracts were examined by gel electrophoresis, the autoradiograph revealed four bands. Three were conspicuous, corresponding for molecular weights of 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000, but a fourth band, corresponding to a molecular weight of 120,000 +/- 6,000, was less well defined. Reducing agents, 20 mM dithiothreitol and 2% beta-mercaptoethanol, acid, and 3 M urea did not affect the production of the bands. Furthermore, the bands once formed were resistant to these agents, indicating 1) covalent nondisulfide bond formation in photoaffinity labeling and 2) noncovalent association of photoaffinity-labeled membrane components with the natural hormone receptor.

Animals↗
PubMed1993

GABAA receptor subtypes: ligand binding heterogeneity demonstrated by photoaffinity labeling and autoradiography.

Heterogeneity of binding affinities for a variety of ligands was observed for gamma-aminobutyric acid type A (GABAA) receptors in the rat CNS, at both GABA and benzodiazepine recognition sites. Photoaffinity labeling by [3H]flunitrazepam and [3H]muscimol to affinity column-purified receptor proteins was examined by gel electrophoresis in sodium dodecyl sulfate. Anesthetic barbiturates (pentobarbital) and steroids (alphaxalone) both differentially stimulated the incorporation of [3H]flunitrazepam more so into the 51-kDa alpha 1 subunit than into the 53-kDa alpha 2 polypeptide, and incorporation of [3H]muscimol into the 55-kDa beta 2 subunit more so than the 58-kDa beta 3 polypeptide. Binding to these polypeptides was also affected differentially by other allosteric modulators and competitive inhibitors, including the benzodiazepine "type 1" selective ligand CL218,872. Heterogeneity in affinity of this drug for the single 51-kDa alpha 1 polypeptide strongly suggests that type I receptors, like type II, are heterogeneous. In brain sections, the extent of enhancement of [3H]muscimol binding showed significant regional variation, similar for both steroids and barbiturates, and the GABA analogues THIP and taurine inhibited muscimol binding with regional variations in affinity that were almost opposites of each other. Modulation of [3H]flunitrazepam binding by steroids, barbiturates, and THIP significantly varied with regions. Taken together, ligand binding heterogeneity exhibited by photoaffinity labeling and autoradiography demonstrate the existence of multiple pharmacological-binding subtypes resulting from the combination of multiple polypeptide gene products into several oligomeric isoreceptors. Comparison of the regional distribution of binding subtypes with that of different subunit gene products allows the following conclusions about possible subunit compositions of native pharmacological receptor subtypes present in the brain: Benzodiazepine pharmacology of the oligomeric receptor isoforms is dependent on the nature of alpha and subunits other than alpha, GABA-benzodiazepine coupling is dependent on the nature of the alpha subunits, GABA site pharmacology is dependent on the nature of the beta subunits, and several subunits including alpha and beta contribute to the degree of sensitivity to steroids and barbiturates. Finally, the presence of discrete subunits may be necessary but is not sufficient to postulate a defined pharmacological property.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of whole cells by flashed light: a simple apparatus for high-energy ultraviolet flashes.

A simple apparatus for the photolysis of affinity labels is described. A commercial quartz tube produces high-energy flashes (wavelength from 200 to 1000 nm). A single flash is normally sufficient to activate photoaffinity labels in the presence of cells. Flash photolysis has several advantages over continuous irradiation, e.g. there is no need for cooling and photolabeling may be performed after different preincubation periods. The above apparatus is therefore suitable for investigations on time-dependent uptake of substrates by intact cells. Examples are demonstrated by photoaffinity labeling of rat liver cells by [3H]cyclosporin-diaziridine.

Affinity Labels↗
PubMed1985

Photoaffinity labelling of benzodiazepine receptors: lack of effect on ligand binding to the nucleoside transport system.

This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl-beta-carboline-3-carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (approximately equal to 16-fold) and dipyridamole (approximately equal to sevenfold) for the [3H]Ro 15-1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.

Affinity Labels↗
PubMed1996

Location of two photoaffinity-labeled sites on the ligand-binding domain of retinoic acid receptor alpha.

Retinoic acid receptors (RARs) consist of six domain structures. The C-terminal region (D/E/F-domains) is involved in ligand binding, dimerization, and ligand-dependent transactivation. Structural information about RARs is required for understanding its complex function. A photoreactive retinoid denoted as ADAM-3, which was designed as the result of comparison of two fluorescent retinoids (DAM-3 and DAM-15), was synthesized and used for photoaffinity labeling of recombinant protein MBP-RAR alpha/E. The photoaffinity-labeled site was determined by an endoprotease combination method which utilizes four endoproteinases in a two-phase digestion procedure. Two major labeled fragments were detected in each digestion, and the results of two-phase digestion allowed identification of the labeled residues as being located within residues 492-510 and 585-594, which correspond to 288-306 and 381-390 in human RAR alpha, respectively.

Affinity Labels↗
PubMed1990

Binding of [3H]dihydrotetrabenazine and [125I]azidoiodoketanserin photoaffinity labeling of the monoamine transporter of platelet 5-HT organelles.

The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage organelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodokentanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 +/- 1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values = 1.1 +/- 0.2, 1.2 +/- 0.1 and 0.52 +/- 0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine.

Affinity Labels↗