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PubMed2002

Biochemistry and pharmacology of epitope-tagged alpha(1)-adrenergic receptor subtypes.

Human alpha(1A)-, alpha(1B)-, and alpha(1D)-adrenergic receptors were tagged at their amino termini with FLAG epitopes and stably expressed in human embryonic kidney (HEK)293 cells. Tagged receptors demonstrated a wild-type pharmacology and mobilization of intracellular Ca(2+). After solubilization and immunoprecipitation, monomers, dimers, and trimers of each subtype were apparent on Western blots. Further denaturation with 6 M urea reduced most oligomers to monomers. Deglycosylation reduced the molecular size of alpha(1A)-, and to a lesser extent alpha(1B)- and alpha(1D)-adrenergic receptors. Radioligand binding site density was highest for alpha(1A)- and much lower for alpha(1B)- and alpha(1D)-adrenergic receptors, but did not correlate with protein expression. Commercial anti-alpha(1)-adrenergic receptor antibodies did not recognize the tagged receptors in Western blots of cell lysates, and substantial cross-reactivity was still observed after solubilization and immunoprecipitation. Surprisingly, only receptor monomers were apparent after photoaffinity labeling with (125)I-arylazidoprazosin, and the intensity of photoaffinity-labeling correlated with the density of radioligand binding sites. We conclude that epitope-tagged alpha(1)-adrenergic receptors exist as both monomers and oligomers in HEK293 cells, but there is substantial discrepancy between protein and binding site expression. Because only monomers are detected by photoaffinity labeling, dimers and trimers observed on Western blots may be pharmacologically inactive.

Blotting, Western↗
PubMed1989

Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes.

The expression of specific membrane receptors for C3a was determined on guinea pig C3a-sensitive (gp R+) platelets and human polymorphonuclear leukocytes (hu PMNL). Binding studies with 125I-labeled C3a from gp or hu sources and Scatchard analysis applied to the binding data revealed the existence of two receptor classes on gp R+ platelets; a high-affinity class with about 200 binding sites/cell and Kd = 1.7 x 10(-9) M, and a relatively low-affinity class with Kd = 10(-8) M and about 500 sites/cell. Hu PMNL express a homogeneous receptor class with Kd = 3 x 10(-8) M and 40,000 sites/cell. Molecular characterization of the C3a receptor on gp R+ platelets was achieved by (a) cross-linking photoaffinity-labeled receptors to bound 125I-labeled C3a; (b) photoaffinity labeling receptors with a 13-amino acid residue C3a analogue 125I-Nap-Ahx-13; and (c) use of chemical cross-linkers like disuccinimidylsuberate to cross-link receptors with 125I-C3a. All three techniques gave rise to very similar labeling patterns. With the photoaffinity labeling methods, a diffuse band pattern was observed with an apparent molecular mass of 95-123 kDa with 125I-C3a as label, and 85-105 kDa with 125I-Nap-Ahx-13 as label. Chemical cross-linking of 125I-C3a revealed three distinct bands with molecular masses of approximately 123, 108 and 95 kDa. Subtracting the contribution of the cross-linked ligands, the C3a receptor on gp R+ platelets appears to be a protein complex, consisting of one to three components with estimated molecular masses between 83-114 kDa.

Adenosine Triphosphate↗
PubMed1997

Evidence that the organic cation/H+ exchanger in the brush border membrane of dog kidney is a 41-kDa protein.

Organic cation (OC+)/H+ exchangers are found in several mammalian tissues and in numerous organisms. In the kidney OC+/H+ exchange activity is localized to the brush border membrane of the proximal tubule cells of the nephron and is believed to be responsible for the efflux of numerous xenobiotics from the tubule cell into the tubular fluid. The objective of the present study was to identify the OC+/H+ exchanger in brush border membrane vesicles isolated from dog kidney by photoaffinity labeling. The results show that [3H]azidopine is an ideal photoaffinity labeling reagent; in the dark it binds reversibly, but irreversibly after photoactivation. The photoaffinity labeling reaction is efficient, specific and sensitive. Our findings are consistent with the conclusions that a 41-kDa protein is the exchanger and that it is present at a concentration of 780 +/- 140 fmol/mg membrane protein (n = 4). A 49-kDa protein is labeled to some extent as well. The relationship between the 41- and 49-kDa proteins has not been resolved.

Acridine Orange↗
PubMed1986

Evidence for a single steroid-binding protein in the rabbit progesterone receptor.

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.

Animals↗
PubMed1989

Subtype-selective immunoprecipitation of the beta 2-adrenergic receptor.

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.

Affinity Labels↗
PubMed1993

Intestinal bile acid absorption. Na(+)-dependent bile acid transport activity in rabbit small intestine correlates with the coexpression of an integral 93-kDa and a peripheral 14-kDa bile acid-binding membrane protein along the duodenum-ileum axis.

The anatomical localization of the Na+/bile acid cotransport system from rabbit small intestine was determined using brush border membrane vesicles prepared from eight different segments of the small intestine. Na(+)-dependent transport activity for bile acids, both for [3H]taurocholate and [3H]cholate, was found in the distal segment 8 only representing the terminal 12% of the small intestine. In contrast, the Na(+)-dependent D-glucose transporter and the H(+)-dependent oligopeptide transporter were found over the whole length of rabbit small intestine in all segments. Photoaffinity labeling with 7,7-azo- and 3,3-azo-derivatives of taurocholate with subsequent fluorographic detection of labeled polypeptides after one- and two-dimensional gel electrophoresis showed that an integral membrane polypeptide of M(r) 87,000 is present in the entire small intestine, whereas an integral membrane protein of M(r) 93,000 together with a peripheral membrane protein of M(r) 14,000 are exclusively expressed in the distal small intestine correlating with Na(+)-dependent bile acid transport activity. Photoaffinity labeling with the cationic bile acid derivative 1-(7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta[3 beta-3H]cholan-24-oyl)-1,2- diaminoethane hydrochloride and 7,7-azo-3 alpha,12 beta-dihydroxy-5 beta[12 alpha-3H]cholan-24-oic acid did not result in a specific labeling of the above mentioned proteins, demonstrating their specificity for physiological bile acids. Photoaffinity labeling of the 93- and 14-kDa bile acid-binding proteins was strongly Na(+)-dependent. Significant labeling of the 93- and 14-kDa proteins occurred only in the presence of Na+ ions with maximal labeling above 100 mM [Na+] showing a parallel [Na+] dependence to transport activity. Inactivation of Na(+)-dependent [3H]taurocholate uptake by treatment of ileal brush border membrane vesicles with 4-nitrobenzo-2-oxa-1,3-diazol chloride led to a parallel decrease in the extent of photoaffinity labeling of both the 93- and 14-kDa protein. Sequence analysis of the membrane-bound 14-kDa bile acid-binding protein surprisingly revealed its identity with gastrotropin, a hydrophobic ligand-binding protein exclusively found in the cytosol from ileocytes and thought to be involved in the intracellular transport of bile acids from the brush border membrane to the basolateral pole of the ileocyte. In conclusion, the present studies suggest that both an integral 93- and a peripheral 14-kDa membrane protein, identified as gastrotropin, and both exclusively expressed in the terminal ileum, are essential components of the Na+/bile acid cotransport system in rabbit terminal ileum.

Affinity Labels↗
PubMed1992

Identification of the template binding polypeptide in the pea chloroplast transcriptional complex.

We have identified the template-binding polypeptide in the pea chloroplast transcriptional complex by photoaffinity labelling. This polypeptide has an apparent molecular weight of about 150 kDa and binds to both, chloroplast ribosomal (16S rRNA) and messenger (psbA) promoters. The 16S rRNA and psbA promoters were amplified from chloroplast DNA by the polymerase chain reaction and labelled with a photoactive analogue of TTP, 5-bromodeoxy UTP, as well as with alpha-32P-dCTP. Using the filter-binding assay, the conditions for binding of the RNA polymerase complex to chloroplast promoters were optimized. The polypeptide directly interacting with the template was photo-crosslinked to it and resolved by denaturing gel electrophoresis. The photoaffinity labelling of the 150 kDa polypeptide was dependent on photoactivation by UV irradiation, and the presence of chloroplast promoters. Competition experiments showed that the protein formed a strong interaction with the plastid promoters which could not be displaced by lambda-phage DNA or synthetic polynucleotides. The photo-crosslinked and nuclease-treated promoter-polypeptide complex was resistant to further digestion with DNase and RNase, but could be hydrolyzed by Proteinase K. Binding of the promoters by the 150 kDa polypeptide could not be surpressed by transcription inhibitors like rifampicin and alpha-amanitin. However, heparin (0.001%) inhibited the formation of the enzyme-promoter complex, and interfered with the photoaffinity labelling of the 150 kDa polypeptide. The extent of photoaffinity labelling of 150 kDa polypeptide exhibits some degree of correlation to total transcriptional activity under various salt concentrations. The results demonstrate that the 150 kDa polypeptide is a functional template binding polypeptide of the pea chloroplast transcription complex.

Base Sequence↗
PubMed1986

Identification of a novel Mr = 76-kDa form of beta-adrenergic receptors.

The structure of the human beta-adrenergic receptor in purified basal membranes of human placental syncytiotrophoblast was probed using photoaffinity labeling. Basal membranes display a high specific activity of receptors (4-5 pmol/mg protein) and possess both beta 1- and beta 2-adrenergic receptors subtypes. Autoradiography of membranes that were incubated with the beta-adrenergic antagonist [125I]iodoazidobenzylpindolol, photolyzed and then subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, identified four radiolabeled peptides, Mr = 65-kDa, 54-kDa, 43-kDa and a novel higher molecular weight 76-kDa form of the receptor. Photoaffinity labeling of each of these four peptides displayed the pharmacological properties expected for true beta-adrenergic receptors. The 76-kDa photoaffinity labeled receptor peptide observed in human placenta basal membranes has not been reported elsewhere. Competition studies with the beta1-selective ligand CGP-20712A demonstrate that the photoaffinity labeled receptor peptides are composed of both beta 1- and beta 2-adrenergic receptor subtypes.

Adrenergic beta-Antagonists↗
PubMed1995

Astroglial cells express large amounts of GABAA receptor proteins in mature brain.

GABAA receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [3H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [3H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [3H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABAA receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABAA receptors. The presence of alpha-subunit(s), as revealed by [3H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mas 51-53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABAA receptors are extrasynaptic and expressed in astroglia.

Aging↗
PubMed1981

Autoradiographic localization of benzodiazepine receptors in immunocytochemically identified gamma-aminobutyrergic synapses.

Benzodiazepine receptors can be visualized in regions of synaptic contact by electron microscopic autoradiography using [3H]flunitrazepam as a photoaffinity label in fresh brain tissue. Perfusion fixation of the tissue prior to photoaffinity labeling left the ligand binding characteristics and the light and electron microscopic distribution of benzodiazepine receptors unaltered. Therefore, the immunocytochemical localization of a neuronal marker in fixed tissue could be combined with photoaffinity labeling in order to identify the types of synapses containing benzodiazepine receptors. By using antiserum to glutamate decarboxylase, a marker of gamma-aminobutyrergic neurons, one-third of the photolabeled benzodiazepine receptors were found to be associated with immunocytochemically stained nerve endings. Thus, these synapses are the site of at lest some benzodiazepine receptors. The enhancement of gamma-aminobutyrergic synaptic transmission by benzodiazepines, shown electrophysiologically, appears to be a primary mechanism of action of this group of drugs.

Affinity Labels↗
PubMed1986

RNA contacts subunits IIo and IIc in HeLa RNA polymerase II transcription complexes.

Subunits of RNA polymerase II that come into contact with nascent RNA transcripts have been determined by photoaffinity labeling. Transcription was carried out in a cell-free extract in the presence of 4-thio-UTP, and utilized the major late promoter of adenovirus-2 DNA. The transcript length was limited by inclusion of the chain terminator 3'-O-methyl-GTP. Transcription complexes were irradiated with near-UV light (lambda greater than 300 nm) to specifically photoactivate 4-thio-uridine. After photocross-linking, radiolabeled proteins were separated by electrophoresis, blotted onto nitrocellulose, and visualized by autoradiography. Specific photoaffinity labeling of enzyme subunits IIo and IIc was observed. In some experiments, radiolabeled IIa was also detected. Based on the level of photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription reaction, the transcriptional activity of RNA polymerase IIO appears to be greater than 10 times that of IIA. RNA attached to subunits IIo and IIc was estimated to be 16-40 nucleotides in length, whereas RNA attached to subunit IIa was approximately 27-40 nucleotides long. Photoaffinity labeling was sensitive to alpha-amanitin, and required DNA containing an RNA polymerase II promoter.

Adenoviridae↗
PubMed1989

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe.

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1988

Transport systems for amphipathic compounds in normal and neoplastic hepatocytes.

Photoaffinity labeling of plasma membrane subfractions from liver and of intact liver tissue with a photolabile bile salt derivative, the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid, revealed that the hepatobiliary transport of bile salts is accomplished by transport systems different for sinusoidal uptake and canalicular secretion. Polypeptides with apparent Mr values 54,000 and 48,000 interact with bile salts at sinusoidal membrane, whereas a polypeptide with an apparent Mr of 100,000 is involved in bile salt secretion through the canalicular membrane. Photoaffinity labeling with photolabile derivatives of uncharged and cationic compounds provided evidence that the sinusoidal membrane polypeptides exhibit a broad binding specificity. Photoaffinity labeling studies and kinetic studies suggest that hepatic uptake of different amphipathic anions, uncharged compounds and even of cations is mediated by the sinusoidal transport systems which are involved in the uptake of bile salts. Relatively little is known about the specificity of the canalicular bile salt transport system. The fluorescent bile salt derivative, the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid, is readily taken up into the hepatocytes of all acinar zones and may be used for the evaluation of the functional state of bile salt transport by fluorescence microscopy. Fluorescent microscopic studies with the fluorescent bile salt derivative showed that ascites hepatoma AS 30D cells do not have the ability to take up bile salts and demonstrated the absence of hepatobiliary bile salt transport in the solid Morris hepatoma 7777. Photoaffinity labeling studies revealed that in both tumor cell models, in hepatoma AS 30D and in Morris hepatoma 7777, the plasma membranes were devoid of the polypeptides having affinities to bile salts and amphipathic cations. A slight labeling of bile salt binding membrane polypeptides in plasma membranes from Morris hepatomas 9618A and TC 5123 opens the possibility to study transport in neoplastic hepatocytes.

Animals↗
PubMed1987

Chemical modification and cross-linking of neurophysin tyrosine-49.

Photoaffinity labeling of the single neurophysin tyrosine, Tyr-49, with Met-Tyr-azido-Phe amide has been reported to inhibit both neurophysin self-association and peptide binding. Accordingly, we investigated the functional consequences of modification, principally by tetranitromethane, of Tyr-49. Tetranitromethane-mediated tyrosine-tyrosine cross-linking permitted synthesis of covalent neurophysin "dimers" and of peptide-protein conjugates, the latter potentially analogous to the photoaffinity-labeled product. The self-association and binding properties of the covalent dimers were found to be similar or enhanced relative to those of the native protein. In contrast to the photoaffinity-labeled product, covalent conjugates of Tyr-49 with the ligand peptides Met-Phe-Tyr amide, Phe-Tyr amide, and Tyr-Phe amide also generally exhibited normal or increased binding affinity for exogenous peptide; a subfraction of the Phe-Tyr amide adducts showed evidence of reduced affinity. Diiodination of Tyr-49 had no significant effect on binding. However, among the products of tetranitromethane treatment in the absence of peptide was a novel inactive non-cross-linked product, representing modification only of Tyr-49 but containing no demonstrable nitrophenol. As evidenced by circular dichroism and nuclear magnetic resonance (NMR), this product was not significantly unfolded and retained the ability to self-associate. These latter results provide the strongest evidence thus far of a role for Tyr-49 in peptide-hormone binding. The disparate effects of different Tyr-49 modifications are collectively interpreted and reconciled with NMR data and the properties of the photoaffinity-labeled protein to suggest potential mechanisms of Tyr-49 participation in binding and the probable orientation of Tyr-49 relative to peptide residue 3 in neurophysin complexes.

Amino Acids↗
PubMed1985

Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486.

A steroid binding protein (Mr = 110,000) has previously been identified in the plasma membrane of Xenopus laevis oocytes by photoaffinity labeling with [3H]R5020. In order to further characterize this steroid receptor, the photoaffinity labeled receptor protein was solubilized with 0.1% Brij 35. The solubilized labeled receptor yielded an approximate mol. wt of 102,000 +/- 2,000 by sucrose density gradient centrifugation, suggesting that the solubilized receptor exists as a monomer. RU 486, a synthetic progestin antagonist for mammalian cytosolic receptor systems, inhibited up to 70% of [3H] R5020 photoaffinity binding to the 110,000-Dalton receptor with an IC50 of 5 microM and induced germinal vesicle breakdown (GVBD) with an EC50 of 9.0 +/- 0.6 microM. GVBD induced by RU 486 was slower than with progesterone, and RU 486 was less powerful than progesterone. Micromolar concentrations of RU 486 also potentiated GVBD induced by sub-optimal concentrations of progesterone or R5020. Furthermore, RU 486 inhibited oocyte plasma membrane adenylate cyclase with an apparent IC50 of 7.5 +/- 2.5 microM. The close correlation of the EC50 value for RU 486 induction of GVBD with the IC50 values for inhibition of [3H]R5020 photoaffinity labeling of the 110,000-Dalton receptor and inhibition of adenylate cyclase activity further supports the physiological significance of the oocyte plasma membrane steroid receptor.

Affinity Labels↗
PubMed1989

Potentiation of vincristine and actinomycin D by a new synthetic imidazole anti-tumor agent YM534 active against human cancer cells and multidrug-resistant cells.

Ethyl 6-p-5-(l-imidazolyl) pentyloxyphenoxy-2, 2-dimethylhexanoate hydrochloride (YM534) is a new synthetic anti-tumor compound. Combinations of YM534 with other anti-cancer agents were examined to ascertain whether YM534 potentiated other anti-cancer agents against the KB cell line and its multidrug-resistant counterpart, VJ-300. YM534 potentiated the cytotoxic action of vincristine and actinomycin D about 2-fold against KB cells, but not those of daunomycin and adriamycin. By contrast, YM534 only slightly reversed drug-resistance to adriamycin and daunomycin in VJ-300 while it reversed 5-fold vincristine resistance and 60-fold actinomycin D resistance in VJ-300. The reversal effect of YM534 on actinomycin D and vincristine-resistance in VJ-300 cells appeared to be due to enhanced accumulation of [3H] actinomycin D and [3H] vincristine in VJ-300 cells by YM534. YM534 inhibited efflux of actinomycin D and vincristine from VJ-300 cells, and it also enhanced cellular uptake of these anti-cancer agents. YM534 enhanced cellular accumulation of both actinomycin D and vincristine in the sensitive KB cells. YM534 is thus a unique anti-cancer agent since combinations of other anti-cancer agents with YM534 are expected to augment anti-tumor activity of them. By contrast, YM212, a carboxy analog of YM534, had much less activity to potentiate vincristine and actinomycin D). YM534 at 100-1000 microM almost completely inhibited the photoaffinity labeling of [3H] azidopine to the 170-kD P-glycoprotein of VJ-300 cell membranes, but YM212 showed much less inhibitory action on the photoaffinity labeling. YM534 could also inhibit the photoaffinity labeling of deglycosylated P-glycoprotein.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Novel tropane-based irreversible ligands for the dopamine transporter.

3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.

Animals↗
PubMed1996

Chemical cross-linking of the substance P (NK-1) receptor to the alpha subunits of the G proteins Gq and G11.

We have previously shown that the high-affinity binding of substance P (SP) to its receptor is dependent on an interaction with a PTX-insensitive G protein. This G protein couples SP receptor activation to stimulation of its effector, phospholipase C. In this study, we combined photoaffinity labeling, chemical cross-linking techniques, and immunological characterization using sequence-specific antibody probes to identify G proteins that couple to the SP receptor. First we covalently labeled the SP receptor present on rat submaxillary gland membranes with a radioiodinated photoreactive derivative of SP, p-benzoyl-L-phenylalanine(8)-substance P (125I-[Bpa8]SP). Photoincorporation of this SP derivative was susceptible to guanine nucleotide inhibition, indicating that the receptor was coupled to its G protein during labeling. We then used a chemical cross-linking agent to covalently link the photoaffinity labeled SP receptor and its associated G protein. Cross-linking generated a 96 kDa product, formation of which was prevented by the addition of a guanine nucleotide, but not an adenine nucleotide, following photolabeling, but prior to cross-linking. Furthermore, the 96 kDa cross-linked complex was absent in membranes which had been depleted of G proteins by treatment with alkaline buffer prior to addition of the cross-linking agent. Reductive cleavage of the cross-link in the isolated 96 kDa complex yields two products: the 53 kDa SP receptor and a 42 kDa protein identified by immunoblot analysis as either G alpha q or G alpha 11. Antisera against a common sequence within G alpha s, G alpha i, and G alpha o showed no immunoreactivity to the complex or its cleavage products. These results provide the first direct evidence of specific interaction between photoaffinity labeled SP receptor and the alpha subunits of Gq and G11, members of a family of G proteins known to be associated with pertussis toxin-insensitive phospholipase C activation.

Affinity Labels↗