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PubMed1982

Photoaffinity labeling of the N-formyl peptide receptor of human polymorphonuclear leukocytes.

Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 approximately 0.7 nM). When incubated at 0 degree C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60-70,000-dalton species (as assessed by SDS-PAGE) after exposure to UV light. Addition of 10-100-fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20-40% of the available surface receptor-binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane-rich fractions. The labeled receptor could be completely solubilized with Triton X-100 in a form which eluted as a single species with a Stoke's radius of less than 50 A on Sepharose 4B columns. In addition, the solubilized receptor-ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.

Affinity Labels↗
PubMed1975

The use of aryl azido ATP analogs as photoaffinity labels for myosin ATPase.

The synthesis of ATP analogs containing a photoactive aryl azido grouping coupled to the 3' hydroxyl of ATP is described. The potential effectiveness of these analogs in the investigation of nucleotide-binding regions is outlined and this effectiveness demonstrated by their photodependent inhibition of subfragment 1 ATPase. The use of 14C-labeled azido ATP demonstrates an almost stoichiometric covalent binding of the analog. Because of their potential application to other systems, a number of reactions describing the reactivity of the 3' hydroxyl of the nucleotide ribose are outlined in an Appendix.

Adenosine Triphosphatases↗
PubMed1997

Synthesis of the photoaffinity label [1'-14C]-6C-(azimethyl)octylglucoside and its reaction with isolated renal brush border membranes.

We describe the synthesis of the photolabile analog of n-octylglucoside (OG), 6C-(azimethyl)octylglucoside (diazirino-octylglucoside, DOG). This diazirino derivative (lambda max = 335 nm) can be activated with long-wavelength UV to generate a highly reactive carbene, an intermediate suitable for a covalent ligand-receptor linkage. In inhibitor studies on D-glucose uptake into rabbit renal brush border membrane vesicles (BBMV) DOG showed a competitive inhibition with regard to D-glucose with a Ki of 20 +/- 5 microM, which was not significantly different from that of the template compound OG (Ki = 30 +/- 10 microM). On irradiation (lambda max = 350 nm) of BBMV in the presence of [14C]DOG, proteins of 61, 72 to 78, and 95 kDa were labeled by photoinduction. A band of 73 kDa was also positive in Western blots using an anti-Na+/D-glucose cotransporter antibody. Further investigations revealed that the labeled proteins were presumably mainly glucosidases known to be present in high concentration in renal BBMV. By comparison with authentic material one of the labeled proteins (61 kDa) was identified as trehalase (EC 3.2.1.28). These studies suggest that labels such as DOG carrying the reactive group directly at the D-glucose moiety of the molecule can be used to label proteins with carbohydrate recognition sites.

Affinity Labels↗
PubMed1997

Photoaffinity labeling of yeast farnesyl protein transferase and enzymatic synthesis of a Ras protein incorporating a photoactive isoprenoid.

Farnesyl protein transferase (FPTase) catalyzes the covalent attachment of a farnesyl (C15) group from farnesyl pyrophosphate (FPP) to a specific cysteine residue of Ras and several other proteins. In this report, photoactive farnesyl and geranylgeranyl pyrophosphate analogs 2-diazo-3,3,3-trifluoropropionyloxy-geranyl pyrophosphate (DATFP-GPP) and 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl pyrophosphate (DATFP-FPP) were used to study the active site of Saccharomyces cerevisiae FPTase. Both analogs are substrates for the enzyme, and upon irradiation, DATFP-GPP inhibits FPTase activity in a time-dependent manner. Photoinactivation by DATFP-GPP is prevented by the presence of the natural substrate FPP. Photolysis of radiolabeled DATFP-GPP results in preferential labeling of the beta subunit of FPTase, suggesting that this subunit is involved in recognition of FPP. Of particular importance, DATFP-GPP and DATFP-FPP were used to enzymatically transfer the photoactive isoprenoid moieties to peptides and to Ras; such molecules should be useful for identifying cellular components which specifically recognize farnesylated Ras and other prenylated proteins.

Affinity Labels↗
PubMed1999

Use of DNA photoaffinity labeling to study nucleosome remodeling by SWI/SNF.

The ability of large protein assemblies to reorganize chromatin in an ATP-dependent manner is an important process for regulation of gene expression and potentially for DNA replication and recombination. The manner in which these proteins remodel chromatin to make the DNA accessible to DNA binding proteins such as transcription factors is not well understood. Site-directed DNA-protein cross-linking has been used to understand the architecture of the SWI/SNF-nucleosomal complex and the mechanistic details of how the nucleosome is remodeled. A detailed protocol for such a study is presented along with examples of the extent of this approach.

Base Sequence↗
PubMedDate not supplied

The purified Ca2+ antagonist receptor from skeletal muscle: subunit structure, photoaffinity labeling and endogenous protein kinase activity.

Tritiated analogues of the Ca2+ channel blockers such as [3H] PN200-110, [3H] verapamil and [3H] diltiazem have been used to identify and isolate Ca2+ antagonist receptors. The Ca2+ antagonist binding sites were solubilized from skeletal muscle transverse tubules with the detergent CHAPS and purified by wheat germ lectin column chromatography and sucrose density gradient centrifugation. The isolated proteins retained their ability to bind the various classes of Ca2+ channel blockers. Polypeptides of 170, 150, 108, 56, and 32 kDa were found to be present in the purified receptor fraction when analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions. The apparent molecular weight of the 170 kDa polypeptide changed to 145 kDa in the presence of reducing agents, as where the apparent molecular weight of the 150, 108, 56 and 32 kDa peptides remained unchanged. An endogenous protein-kinase present in the original membranes, co-purified with the receptor and stimulated the phosphorylation of the 150 and 56 kDa polypeptides in the isolated fraction.

Animals↗
PubMed1982

Photoaffinity labelling of the insulin receptor in intact rat hepatocytes, mouse soleus muscle, and cultured human lymphocytes.

Using the photoreactive, biologically active insulin analogue, B2-(2 nitro, 4-azidophenylacetyl)des-PheB1 insulin, which can be covalently bound to receptor molecules upon photolysis, the insulin receptor has been studied in three different types of cells or tissues: isolated rat hepatocytes, intact murine soleus muscle and cultured human lymphocytes. When compared with native insulin, this analogue displayed a slightly reduced binding affinity. Accordingly, the biological potency of the photoreactive analogue was decreased by approximately 30% compared with native insulin when tested for its ability to stimulate amino acid transport in hepatocytes, and deoxyglucose uptake in soleus muscles. It was as effective as insulin, however, at maximally stimulating concentrations and therefore is a full insulin agonist. This photoprobe was used to specifically label the insulin receptor in the three tissues: after ultra-violet irradiation, sodium dodecyl sulphate-polyacrylamide gel analysis of extracts under reducing conditions revealed that most of the radioactivity was associated with a 130,000 dalton band. In isolated hepatocytes, two bands at 125,000 and 23,000 daltons were also specifically labelled. In three different cell types from three different animal species, the 130,000 dalton band appeared to be the major subunit of the insulin receptor.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of the receptor site for alpha-scorpion toxins on purified and reconstituted sodium channels by a new toxin derivative.

1. A methyl-4-azidobenzimidyl (MAB) derivative of the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) specifically labels only the alpha subunit of the rat brain sodium channel in synaptosomes or in purified and reconstituted sodium-channel preparations. 2. Unlike previous photoreactive toxin derivaties, binding and photolabeling by MAB-LqTx are allosterically modulated by tetrodotoxin and batrachotoxin, as observed for native LqTx binding to sodium channels in synaptosomes. 3. Proteolytic cleavage of the alpha subunit photolabeled with MAB-LqTx shows that the label is located within a 60 to 70-kDa protease-resistant core structure in domain I of the sodium-channel alpha subunit. 4. MAB-LqTx will be valuable in further defining the structure-activity relationships at the alpha-scorpion toxin receptor site.

Animals↗
PubMed1985

Detection of antimycin-binding subunits of complex III by photoaffinity-labeling with an azido derivative of antimycin.

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochrome c reductase (Complex III) purified from bovine heart (Ki = ca. 0.5 microM) was considerably less than that of antimycin (Ki less than or equal to 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [3H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m = 13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [3H]DAA, respectively. The labeling of band 7 by [3H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohydride or ascorbate. Based on magnitude of labeling by [3H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m = 16 kDa) showing a lesser but significant amount of specific binding.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of the tetrodotoxin binding component of Electrophorus electricus electroplax.

Radioactive azide derivatives of tetrodotoxin (TTX) were synthesized using 2-nitro-4-azidephenyl-[3H]beta alanine for the purpose of photolabeling of the Na channel. Three azide derivatives, N1, N2 and N3, were separated by ion exchange chromatography on Bio-Rex 70 resin and reversed phase high performance liquid chromatography. N3 was more stable and obtained at a higher yield than the other two derivatives. Bioactivity of N3 was one-twentieth of that of TTX. N3 showed reversible binding to membranes of Electrophorus electricus electroplax in the dark with Kd = 30 nM and B max = 5.2 pmol/mg protein. By photoirradiation, irreversible binding of N3 to the membranes was observed. A N3 binding component was solubilized by lubrol PX and partially purified from the electroplax membranes by Sephadex G25 and Sepharose 6B column chromatography. The component, purified 500 fold from the starting membranes, showed molecular weight of 10,000.

Affinity Labels↗
PubMed1985

An Anolis skin melanophore assay suitable for photoaffinity labeling studies with alpha-MSH.

Visual determination of MSH-induced pigment migration in melanophores of small pieces of Anolis carolinensis skin is standardized by first measuring photoelectrometrically the change in reflection/transmission of the whole dorsal skin in response to different hormone concentrations. This method allows the rapid and precise recording of time-response curves after photo-affinity labeling of MSH receptors or of dose-response curves of large series of synthetic compounds.

Affinity Labels↗