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PubMed2002

Structure-based design of cyclooxygenase-2 selectivity into ketoprofen.

We have recently described how to achieve COX-2 selectivity from the non-selective inhibitor indomethacin (1) using a combination of a pharmacophore and computer 3-D models based on the known X-ray crystal structures of cyclooxygenases. In the present study we have focused on the design of COX-2 selective analogues of the NSAID ketoprofen (2). The design is similarly based on the combined use of the previous pharmacophore together with traditional medicinal chemistry techniques motivated by the comparative modeling of the 3-D structures of 2 docked into the COX active sites. The analysis includes use of the program GRID to detect isoenzyme differences near the active site region and is aimed at suggesting modifications of the basic benzophenone frame of the lead compound 2. The resulting series of compounds bearing this central framework is exemplified by the potent and selective COX-2 inhibitor 17 (LM-1669).

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Photoactivatable peptides based on BMS-197525: a potent antagonist of the human thrombin receptor (PAR-1).

Photoactivatable analogs of the human thrombin receptor (PAR-1) antagonist, N-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2 (BMS-197525), were prepared with benzophenone substitutions in the N-terminal, Leu, or Arg position. The analogs retained antagonist activity (with reduced potency); the tritium-labeled isotopomers are potential photoaffinity labels for the receptor. C-Terminal extension of the analogs with ornithine(biotin) did not significantly alter antagonist potency.

Chromatography, High Pressure Liquid↗
PubMed2002

Synthesis of alkenyldiarylmethane (ADAM) non-nucleoside HIV-1 reverse transcriptase inhibitors with non-identical aromatic rings.

The existing methods for the synthesis of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors proceed from symmetrical benzophenones and therefore result in products with identical aromatic rings. New methods have therefore been devised for the preparation of stereochemically defined ADAMs with non-identical aromatic rings. The new routes rely on palladium-catalyzed reactions, including Sonogashira, Suzuki, Stille, and hydroarylation methodology. Several of the new ADAMs inhibited the cytopathic effect of HIV-1 in cell culture and HIV-1 reverse transcriptase at submicromolar concentrations.

Biochemistry↗
PubMed2001

The historical aspects of sunscreens.

While there is little literature on the way in which people protected themselves against the sun, evidence from paintings suggest that clothing covering the body, veils and large brim hats were used by ancient Greeks, and that umbrellas existed in ancient Egypt, Mesopotamia, China and India. Veiel in 1887 (Vierteljahresschr. Derm. Syph. 14, 113-116) was able to protect a patient with eczema solare by the use of a tightly woven red veil. In 1889, Widmark (Uber den Einfluss des Lichtes auf die Haut. Hygiea, Festband #3, Samson and Wallin, Stockholm) used acidified quinine sulfate to absorb UVB, apparently because, since quinine fluoresces when irradiated with UVR, he rightly assumed that it would absorb the short wavelengths. In 1891, Hammer (Uber den Enfluss des Lichtes auf die Haut, Ferdinand Enke, Stuttgart) repeated Widmark's experiments and used quinine prepared in lotion or ointment as the first human sunscreen. Various plant extracts had been used at the turn of the century in folk medicine. One of the most effective was a chestnut extract from which aesculin was derived. Unna (Med. Klinik. 1911;7:454-456) developed several glycosides of aesculin,which were introduced as Zeozon and Ultrazeozon. In 1922, Eder and Freund (Wiener. Klin. Wchnschr. 35, 681-684) introduced 2-naphthol-6,8-disulfonic acid salts (Antilux) which were quite effective in both the UVB and UVA region. Over the next 40 years a number of different chemicals were introduced for sunscreen purposes: tannic acid (1925), benzyl salicylate (1931), para-aminobenzoic acid derivatives and 2-phenylimidazole derivatives (1942), anthranilic acid (1950), various cinnamates (1954), chloroquine (1962), benzophenones (1965) and many more since then. The list of chemical useful for sunscreen formulation is now extensive, the US Food and Drug Administration and the European Community have published compendia of approved chemicals and inorganic filters.

Drug Design↗
PubMed1999

Mediation of sperm-egg fusion: evidence that mouse egg alpha6beta1 integrin is the receptor for sperm fertilinbeta.

BACKGROUND INTRODUCTION: A key step leading to fertilization is the binding of sperm to the egg plasma membrane. When a mammalian sperm reaches the egg plasma membrane, fertilinbeta, an extracellular sperm membrane protein, is believed to bind to an egg plasma membrane receptor triggering fusion. We set out to identify the fertilinbeta binding partner on the egg plasma membrane. RESULTS: We synthesized an 125-labeled peptide with the consensus Asp-Glu-Cys-Asp (DECD) sequence of fertilinbeta's disintegrin domain. This peptide contains a benzophenone photoaffinity probe and inhibits sperm-egg fusion. Upon photoactivation in the presence of whole mouse eggs, a single polypeptide was covalently labeled. This polypeptide has been identified by immunoprecipitation as an alpha6 integrin complexed with beta1 integrin. CONCLUSIONS: Our experiments establish that small peptides containing the consensus DECD sequence of sperm fertilinbeta bind specifically to an alpha6beta1 integrin receptor on the egg membrane. We conclude that fertilinbeta binds directly to the alpha6beta1 integrin on the egg surface and this partnership mediates sperm-egg fusion.

ADAM Proteins↗
PubMed1999

Illumination of human keratinocytes in the presence of the sunscreen ingredient Padimate-O and through an SPF-15 sunscreen reduces direct photodamage to DNA but increases strand breaks.

On illumination with simulated sunlight, the UVB-absorbing sunscreen chemical 2-ethylhexyl-4-dimethylaminobenzoate (Padimate-O) generates excited species which inflict non-ligatable strand breaks on DNA in vitro and it also becomes mutagenic to yeast in vivo. Padimate-O is known to penetrate human skin but its effects on human cells are not clear. Here, we first simulate the sunlight which penetrates human skin and use it to illuminate human keratinocytes. The DNA damage observed in terms of UV-endonuclease-sensitive sites (ESS) and direct strand breaks per kilobase (kb) of DNA per joule per square metre agrees well with that predicted from action spectra based on monochromatic light. Using plasmid DNA in vitro, we find a very similar pattern of results. Next, we simulate the spectrum that results when the incident light is first attenuated by a film of sunscreen (SPF-15; 2 mg/cm(2)) containing benzophenone-3 (a UVA absorber), octyl methoxycinnamate (a UVB absorber), and Padimate-O. If the sunscreen is not in contact with keratinocytes it reduces direct DNA damage from sunlight (ESS). However, any Padimate-O in contact with the cells substantially increases indirect damage (strand breaks) even though the film of sunscreen reduces direct photodamage. We estimate that applying an SPF-15 sunscreen which contains Padimate-O to human skin followed by exposure to only 5 minimum erythemal doses (MED) of sunlight could, while suppressing the formation of ESS, increase strand breaks in cells under the epidermis by at least 75-fold compared to exposure to 1 MED in the absence of sunscreen.

4-Aminobenzoic Acid↗
PubMed2000

Functional tethered lipid bilayers.

Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support. Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support. An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite. And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates. All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques. For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer. Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step. Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e. the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively. The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes. The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies. The results obtained for reconstituted H(+)-ATPase from chloroplasts and E. coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.

Biopolymers↗
PubMed2004

Photoimmobilization of proteins for affinity capture combined with MALDI TOF MS analysis.

Affinity capture surfaces can be prepared in a number of ways. A method of obtaining such surfaces through UV-activated immobilization of binding proteins using a benzophenone derivative is reported. Photoimmobilized protein G was used to selectively capture and preconcentrate bovine IgG from a mixture with BSA, and the affinity of photoattached concanavalin A toward ovalbumin was compared with that of commercially available concanavalin A on agarose beads. The results of the capture after tryptic digestion were analyzed by MALDI TOF MS. Immobilized trypsin was also prepared through photoimmobilization and later used to digest hemoglobin. Immobilized enzyme digestion resulted in more partial cleavages than solution-phase digestion. More methionine and tryptophan oxidation was also observed. Photoimmobilization was shown to be a quick and easy way of immobilizing ligands on surfaces.

Amino Acid Sequence↗
PubMed2004

Surface-directed, graft polymerization within microfluidic channels.

We demonstrate a simple procedure to coat the surfaces of enclosed PDMS microchannels by UV-mediated graft polymerization. In prior applications, only disassembled channels could be coated by this method. This limited the utility of the method to coatings that could easily and tightly seal with themselves. By preadsorbing a photoinitiator onto the surface of PDMS microchannels, the rate of polymer formation at the surface was greatly accelerated compared to that in solution. Thus, a gel did not form in the lumen of enclosed microchannels. We demonstrate that the photoinitiator benzophenone remained on the surface of PDMS even after extensive washing. After addition of a variety of monomer solutions (acrylic acid, poly(ethylene glycol) monomethoxyl acrylate, or poly(ethylene glycol) diacrylate) and illumination with UV light, a stable, covalently attached surface coating formed in the microchannels. The electroosmotic mobility was stable in response to air exposure and to repeated cycles of hydration-dehydration of the coating. These surfaces also supported the electrophoretic separation of two model analytes. Placement of an opaque mask over a portion of the channel permitted photopatterning of the microchannels with a resolution of approximately 100 microm. By using an appropriate mixture of monomers combined with masks, it should be possible to fabricate PDMS microfluidic devices with distinct surface properties in different regions or channels.

Journal Article↗
PubMed2005

Automated on-line column-switching HPLC-MS/MS method with peak focusing for the determination of nine environmental phenols in urine.

We developed a method using isotope dilution on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination in urine of nine environmental phenolic compounds: Bisphenol A; 4-tert-octylphenol; o-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; benzophenone-3 (2-hydroxy-4-metoxybenzophenone); and triclosan (2,4,4'-trichloro-2'-hydroxyphenyl ether). A unique fully automated column-switching system, constructed using 1 autosampler, 2 HPLC pumps, and a 10-port switching valve, was designed to allow for concurrent SPE-HPLC operation with peak focusing. The phenols present in 100 microL of urine were retained and concentrated on a C18 reversed-phase size-exclusion SPE column. Then, the phenols were "back-eluted" from the SPE column and diluted through a mixing Tee before being separated from other urine matrix components using a pair of monolithic HPLC columns. The phenols were detected by negative ion-atmospheric pressure chemical ionization-MS/MS. The efficient preconcentration of the phenols by the SPE column, analyte peak focusing by the dilution, and minimal ion suppression in the LC/MS interface by the buffer-free mobile phases resulted in limits of detection as low as 0.1-0.4 ng/mL for most analytes. The method was validated on spiked pooled urine samples and on urine samples from 30 adults with no known occupational exposure to environmental phenols. The method can be used for quick and accurate analysis of large numbers of samples in epidemiologic studies for assessing the prevalence of human exposure to environmental phenols.

Chromatography, High Pressure Liquid↗
PubMed2006

Site selectively templated and tagged xerogels for chemical sensors.

We report on a new sensor strategy that we have termed site selectively templated and tagged xerogels (SSTTX). The SSTTX platform is completely self-contained, and it achieves analyte recognition without the use of biomolecules. To illustrate the SSTTX scheme's potential, we present results for the selective detection and quantification of a model compound, 9-anthrol. The first-generation SSTTX shows the following: (i) exhibits an apparent dissociation constant of (1.8 +/- 0.3) x 10(-4) M for 9-anthrol; (ii) provides 0.3 muM detection limits for 9-anthrol; (iii) yields a 45-s response time (2-mum-thick film); (iv) is completely reversible (6% relative standard deviation after 25 cycles); (v) yields a selectivity factor for 9-anthrol over several structurally similar analogues/interferences (e.g., anthracene, 9,10-anthracenediol, benzophenone, 2-naphthol, phenol, and pyrene) of between 290 and 520; and (vi) is stable (<2% drift in performance) for at least 10 months when stored under ambient conditions in the dark. The results also suggest SSTTXs as new sensor elements for glycoside, pharmaceutical, prostaglandin, and steroid sensors.

Electrodes↗
PubMedDate not supplied

Photopatterning of antibodies on biosensors.

The immobilization of biomolecules on surfaces in defined micropatterns has become increasingly important for the development of new diagnostic devices and high-throughput genetic and drug screening protocols. We describe the synthesis and testing of thiol-reactive, photoactivatable linkers that will permit laser micropatterning or photolithographic patterning of surfaces. In these linkers, a benzophenone photophore is tethered through a variable-length poly(ethylene glycol) hydrophilic spacer to a maleimide group. Spacers containing one to five ethylene glycol units were examined. Antibodies were photoimmobilized on polystyrene waveguides and the resulting biosensors were used for fluorescence immunoassays. The spacer with five ethylene glycol units optimally decreased the steric interactions among large molecules (antibodies and antigens) and increased binding capacity and response rate of the biosensor. Two different sandwich assay protocols were examined. In the first, the antigen and fluorescently labeled second antibody were added sequentially to the biosensor ("stepwise"). In the second, the antigen and antibody were premixed before injection into the biosensor ("premixed"). The stepwise protocol gave a significantly higher response than that of the premixed protocol. Although the premixed protocol is more convenient, the stepwise protocol provides enhanced sensitivity.

Antibodies↗
PubMedDate not supplied

Solid-phase synthesis of a radiolabeled, biotinylated, and farnesylated Ca(1)a(2)X peptide substrate for Ras- and a-mating factor converting enzyme.

Eukaryotic proteins with carboxyl-terminal Ca(1)a(2) motifs undergo three posttranslational processing reactions--prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding Ca(1)a(2)X endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, localization of Ras2p to the outer membrane is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. A biotinylated, farnesylated Ca(1)a(2)X peptide [(1-N-biotinyl-(13-N-succinimidyl-(S-(E,E-farnesyl)-L-cysteinyl)-L-valinyl-L-isoleucinyl-L-alanine))-4,7,10-trioxatridecanediamine] 1 containing a poly(ethylene glycol) linker was prepared by solid-phase synthesis for use in an assay for Ca(1)a(2)X endoprotease activity that relies on the strong affinity of avidin for biotin. The peptide was radiolabeled in the penultimate step of the synthesis by cleavage of the biotinylated, farnesylated Ca(1)a(2) precursor from Kaiser's oxime resin with [(14)C]-L-alanine methyl ester. [(14)C]1 was a good substrate for yRce1p with K(M) = 1.3 +/- 0.3 microM. Analysis of the carboxyl terminal products by reverse phase HPLC confirmed that VIA was the only radioactive fragment released upon incubation of [(14)C]1 with a yeast membrane preparation of recombinant yRce1p. The solid-phase methodology developed using Kaiser's benzophenone oxime resin to synthesize [(14)C]1 should be generally applicable for peptides containing sensitive side chains. In addition, introduction of the radiolabeled unit at the end of the synthesis mostly circumvents problems associated with handling radioactive materials.

Biotinylation↗
PubMedDate not supplied

Novel carboranyl amino acids and peptides: reagents for antibody modification and subsequent neutron-capture studies.

A new alpha-amino acid derivative incorporating the 1,2-dicarba-closo- dodecarborane(12) cage, namely 5-(2-methyl-1,2-dicarba-closo-dodecarborane(12)-1-yl)- 2-aminopentanoic acid (2), was synthesized by the alkylation of the benzophenone Schiff's base of glycine methyl ester with 3-(2-methyl-1,2-dicarba-closo-dodecaborane(12)-1-yl)pr opyl iodide (8). This amino acid was employed in the synthesis of peptide derivatives such as 19-21 using solid-phase Merrifield methods. Dipeptide 19 was converted to a water-soluble ionic derivative by the pyrrolidine-mediated carborane cage degradation reaction followed by cation exchange to afford sodium salt 22. Dansylation of 22 with dansyl chloride yielded fluorescence-labeled dipeptide 23. Undecapeptide 21 was dansylated while still anchored to the Merrifield resin. Following its cleavage from the resin with hydrogen fluoride, product 25 was acetylated to block the free amino group on the lysine residue and then converted to water-soluble derivative 27. Trial conjugations of dipeptide 23 and undecapeptide 27 to T84.66, an anti-CEA antibody, were carried out by means of carboxyl activation with N-hydroxysulfosuccinimide and N,N-diisopropylcarbodiimide. Studies of the chemical syntheses of these and other peptide derivatives and the conjugation of 23 and 27 to the antibody are described.

Amino Acids↗
PubMedDate not supplied

Synthesis of structurally identical fluorine-18 and iodine isotope labeling compounds for comparative imaging.

The synthesis of a benzophenone-based labeling compound designed for comparative imaging studies with both in vivo positron emission tomograph (PET) and single-photon computed tomography (SPECT) and ex vivo autoradiography is described. The new compound can be labeled with either F-18 or iodine radioisotopes to give two different radioisotopmers: N-[2-fluoro-5-(3-[I-131]iodobenzoyl)benzyl]-2-bromoacetamide (1) and N-[2-[F-18]fluoro-5-(3-iodobenzoyl)benzyl]-2-bromoacetamide (2). Compound 1 and 2 have a 2-bromoacetyl group, which can be used to conjugate with biomolecules through a nucleophilic substitution reaction. Compound 1 was synthesized from the corresponding tributyltin derivatives via an oxidative destannylation reaction, and compound 2 was prepared via a four-step radiosynthesis (nucleophilic aromatic substitution, reduction, oxidation, and alkylation) starting from 4-(N,N,N-trimethylammonio)-3-cyano-3'-iodobenzophenone triflate. A remarkably high radiochemical yield (>90%) was achieved for the F-18 nucleophilic aromatic substitution under mild conditions (room temperature in less than 10 min), indicating the structural advantage of the designed molecule to facilitate the F-18 for trimethylammonium substitution in the presence of two electron-withdrawing groups (nitrile and carbonyl). The overall radiosynthesis time for compound 2 is less than 3 h after end of bombardment (EOB) with an unoptimized radiochemical yield of about 2% (not decay corrected) and specific activity of 0.8 Ci/micromol at EOB. The radiolabeling precursors for compound 1 and 2 were synthesized via a carbon-carbon bond-forming reaction between 2-substituted-5-lithiobenzonitrile and 3-substituted benzaldehyde derivatives. Compounds 1 and 2 should allow us to label biomolecules with F-18 or iodine isotopes and gives structurally identical products, which are expected to have identical biological properties and should be useful for comparative imaging studies.

Acetamides↗
PubMed2000

Mapping the bimolecular interface of the parathyroid hormone (PTH)-PTH1 receptor complex: spatial proximity between Lys(27) (of the hormone principal binding domain) and leu(261) (of the first extracellular loop) of the human PTH1 receptor.

In an effort to characterize the bimolecular interface between parathyroid hormone (PTH) and its human receptor PTH1-Rc (hPTH1-Rc), we previously identified two contact sites in the receptor: one for position 1 and another for position 13 (located at the ends of the principal activation domain) in PTH(1-34). The present study reports a third, novel "contact site" between hPTH1-Rc and Lys(27) of PTH(1-34). Lys(27) is located in the principal binding domain of the hormone (residues 25-34). The photoreactive PTH(1-34) analogue K27 contains a benzophenone (BP) moiety on Lys(27). The analogue binds to stably transfected HEK 293/C-21 cells (which express a high level of recombinant hPTH1-Rc) and stimulates adenylyl cyclase activity with a potency similar to PTH(1-34). In addition, (125)I-K27 cross-links effectively and specifically to the hPTH1-Rc. Enzymatic (Glu-C and Lys-C) and chemical (CNBr and BNPS-skatole) digestions of the photoconjugate between (125)I-K27 and hPTH1-Rc were performed. In addition, photoconjugates involving the bioactive mutants [L261M]- and [R262K]-hPTH1-Rc, transiently expressed in COS-7 cells, were also digested. The data obtained clearly identify L(261) or R(262) of the first extracellular loop of hPTH1-Rc as the contact site for Lys(27) in the hormone. On the basis of (i) the similarity in molecular mass between the CNBr digest of the (125)I-K27-[L261M]hPTH1-Rc conjugate and free (125)I-K27 and (ii) the failure to cross-link (125)I-K27 to a bioactive mutant receptor [L261A]hPTH1-Rc, we conclude that L(261) is the cross-linking site. These results provide the first demonstration of an interaction between the principal binding domain of PTH and the first extracellular loop of hPTH1-Rc. Revealing proximity of Lys(27) (in PTH) to L(261) (in hPTH1-Rc) provides additional insight into the nature of the ligand-receptor bimolecular interface and clearly illustrates that the extracellular loops of the receptor contribute to the specificity of the PTH-PTH1-Rc interaction. Taken together with previous studies, the new findings add important constraints on the possible positioning of the C-terminal helix of PTH (which contains the principal binding domain) relative to the first extracellular loop and the distal C-terminal helix of the large extracellular amino terminal domain of the PTH1-Rc.

Amino Acid Sequence↗
PubMed1995

Probing the bimolecular interactions of parathyroid hormone and the human parathyroid hormone/parathyroid hormone-related protein receptor. 2. Cloning, characterization, and photoaffinity labeling of the recombinant human receptor.

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and cross-linking of a benzophenone-substituted PTH analog, [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of approximately 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of 125I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.

Affinity Labels↗