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Results for “Photoaffinity Labels”

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PubMed1980

Identification by photoaffinity labelling of 16S RNA as a component of IF2-binding site of E. coli MRE 600 ribosome.

A photoactivated radioactive derivative of IF2 was obtained by treatment with p-azidobenzaldehyde in the presence of NaBH4, followed by reductive methylation with [14C] formaldehyde. The modified IF2 preserved the functional activity of the native factor in specific binding to 30S ribosomal subparticles to which it specifically cross-linked during mild irradiation. The main affinity-labelled component of the 30S subparticle was found to be 16S RNA.

Affinity Labels↗
PubMedDate not supplied

Biological action and fate of photoaffinity-labelled insulin-receptor complexes.

Covalent linking of two photoactivatable insulin derivatives, B2-(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin and B29-(2-nitro,4-azidophenylacetyl)-insulin to viable rat adipocytes gives a system, which contains a fixed stoichiometry between hormone and receptor. The biological signal of prolonged lipogenesis has been used to study several aspects of insulin binding and action: the role of the site of the crosslink between insulin and receptor, recognition of bound photoinsulin by anti-insulin antibodies, the half-life of the biologically active complex, the pH-dependence of the biological signal, and the possible role of internalization. Furthermore, the effect of trypsin on the insulin receptor, as well as the insulin-receptor complex, has been investigated and a refined model of the receptor is presented.

Adipose Tissue↗
PubMed2003

Solubilization and photoaffinity labeling identification of glucocorticoid binding peptides in endoplasmic reticulum from rat liver.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic effects of steroid hormones. We have previously described that the low-affinity glucocorticoid binding protein (LAGS), present in the endoplasmic reticulum of the male rat liver, has pharmacological and biochemical properties different from those of nuclear receptors. The LAGS is under multihormonal regulation and binds glucocorticoids, progestins, and synthetic steroids but is unable to bind either estradiol, testosterone, or triamcinolone acetonide. In this study, we have solubilized the LAGS and investigated their pharmacological and hydrodynamic properties and their peptide composition. We found that LAGS is an integral protein bound to the endoplasmic reticulum. CHAPS provided its optimal solubilization without changes in its pharmacological properties. Hydrodynamic properties of LAGS showed that it has a molecular mass of at least 135 kDa. SDS-PAGE of covalently-labeled LAGS showed that [3H]dexamethasone binds two peptides of 53 and 37 kDa, respectively. Thus, the LAGS appears as an oligomeric protein under multihormonal regulation. The availability of solubilized LAGS and the fact that it can be induced in vivo represent major steps toward purification and understanding the functional significance of this unique steroid-binding protein.

Animals↗
PubMed2000

Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity.

A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue. As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S-CH2CH2SO3- or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL-BBP enzyme inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3-, or S166C-S-CH2CH2NH3+ and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11-BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs.

Amino Acid Sequence↗
PubMed1999

The binding site for an inhibitor of squalene:hopene cyclase determined using photoaffinity labeling and molecular modeling.

BACKGROUND: The squalene:hopene cyclases (SHCs) are bacterial enzymes that convert squalene into hopanoids, a function analogous to the action of oxidosqualene cyclases (OSCs) in eukaryotic steroid and triterpenoid biosynthesis. We have identified the binding site for a selective, potent, photoactivatable inhibitor of an SHC. RESULTS: SHC from Alicyclobacillus acidocaldarius was specifically labeled by [3H]Ro48-8071, a benzophenone-containing hypocholesteremic drug. Edman degradation of a peptide fragment of covalently modified SHC confirmed that Ala44 was specifically modified. Molecular modeling, using X-ray-derived protein coordinates and a single point constraint for the inhibitor, suggested several geometries by which Ro48-8071 could occupy the active site. CONCLUSIONS: A covalent complex of a potent inhibitor with a squalene cyclase has been characterized. The amino acid modification and molecular modeling suggest that Ro48-8071 binds at the junction between the central cavity and substrate entry channel, therefore inhibiting access of the substrate to the active site.

Affinity Labels↗
PubMedDate not supplied

A laser flash photolysis study of di-, tri- and tetrafluorinated phenylnitrenes; implications for photoaffinity labeling.

Several fluorinated aryl azides (2,6 and 2,4-difluorophenyl azide, 2,4,6-triflurorophenyl azide, and methyl 4-azido-2,3,5,6-tetrafluorobenzoate) were studied by laser flash photolysis techniques. In each case, the azides from singlet nitrenes which can be intercepted with pyridine to form ylides. This allowed determination of the rate constant for ring expansion of the singlet nitrene to the dehydroazepine and measurement of the absolute rate constants for reaction of the singlet nitrenes with typical quenchers.

Affinity Labels↗
PubMed1995

Photoaffinity labeling and photoinactivation of the O2(-)-generating oxidase of neutrophils by an azido derivative of FAD.

A photoactivable derivative of FAD, 4-[N-(4-azido-2-nitrophenyl)amino]butyryl-FAD (NAP4-FAD), was synthesized in a tritiated form with tritium placed in the NAP4 moiety of the photoprobe. [3H]NAP4-FAD was used to photolabel the putative flavin binding site of the O2(-)-generating NADPH oxidase located in the plasma membrane of bovine neutrophils. Effective photolabeling required partial deflavination of membranes, which was achieved by mild treatment with ammonium sulfate added to 50% saturation and 0.05% Triton X-100 for 30 min at 2-4 degrees C. Under these conditions, 40-50% of the oxidase activity was lost, but it could be fully recovered by the addition of nanomolar amounts of FAD (KM = 10-20 nM). Added FAD could be substituted by [3H]NAP4-FAD in photolabeling experiments. In the dark, [3H]NNAP4-FAD bound reversibly with high affinity to deflavinated neutrophil plasma membranes (Kd = 50 nM), did not transport electrons, and efficiently inhibited the FAD-dependent restoration of oxidase activity (Ki = 60 nM). Upon photoirradiation of neutrophil plasma membranes in the presence of [3H]NAP4-FAD, the nitrene derivative formed bound covalently to a 80-120 kDa protein that was identified as the beta-subunit of cytochrome b558 by immunodetection and enzymatic deglycosylation. The amount of [3H]NAP4-FAD covalently incorporated into the beta-subunit of cytochrome b558 was 80-90% of the amount of photoprobe specifically bound to neutrophil plasma membranes. A linear relationship between the extent of specific photolabeling by [3H]NAP4-FAD and the percentage of NADPH oxidase inactivation was observed for percentages of inactivation of up to 70-80%, extrapolating to 0.5 mol of covalently bound [3H]NAP4-FAD per mol of heme b558.

Affinity Labels↗
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↗
PubMed1993

Wheat germ and yeast RNA polymerase II: photoaffinity labeling by 4-thiouracil 5'-monophosphate positioned uniquely at the 3' end of an enzyme-bound [32P]-containing transcript.

A stable ternary transcription complex was formed with either wheat germ or yeast RNA polymerase II using a ribotrinucleotide primer (GpCpG) to initiate transcription on a short synthetic single-strand DNA template. The template was designed to limit the incorporation of a photoprobe S4-UMP (4-thio-UMP) to a unique position at the 3' terminus of the transcript. The resulting stable ternary transcription complex was photolyzed to cross-link the bound transcript ([32P]-labeled by the incorporation of [alpha-32P]CMP) with the protein domain at or near the active site. Separation of the protein components by electrophoresis in polyacrylamide gel containing SDS and analysis by autoradiography and silver staining revealed that for either enzyme only the largest subunit was [32P] labeled.

Affinity Labels↗