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At least 649 records · Page 36Linked to original sources
PubMed1987

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide.

N-(4-Azidosalicyl)galactosamine (GalNASA), a photoactivatable, radioiodinatable analogue of N-acetylgalactosamine (GalNAc), has been prepared and characterized. We have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a Ki,app of 800 microM, comparable to that of GalNAc. The Ki,app of GalNASA decreased to 40 microM upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl)ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125I-GalNASA was entirely dependent upon ultraviolet light. A portion of the labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethylenediaminetetraacetic acid. N-Acetylglucosamine, which is not a ligand of discoidin I, was without effect. As a control, no carbohydrate-sensitive labeling was observed upon incubation of 125I-GalNASA with bovine serum albumin. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125I-GalNASA exhibited a Kd of 15-40 microM, in agreement with the Ki,app of prephotolyzed GalNASA observed in the carbohydrate binding assay. Some labeling occurred if 125I-GalNASA was photolyzed prior to incubation with discoidin I, suggesting the involvement of long-lived species in the labeling reaction. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1990

Enthalpic and entropic determinants for the specificity of alkylation of the histidine-12 residue of ribonuclease A by four bromoacetamido nucleoside affinity labels and bromoacetamide.

The binding and alkylation rate constants for the reaction of four bromoacetamido pyrimidine nucleosides and bromoacetamide with bovine pancreatic ribonuclease A (RNase A) have been determined as a function of temperature. The four nucleoside derivatives react exclusively or preferentially with the NE2 atom of histidine-12 and include 2'-bromoacetamido-2'-deoxyuridine, 2'-bromoacetamido-2'deoxyxylofuranosyluracil, 3'-bromoacetamido-3'-deoxythymidine and 3'-bromoacetamido-3'-deoxyarabinofuranosyluracil. Transition-state parameters, delta H++ and delta S++, reveal that nucleosides with "up" OH groups experience relative rate enhancements which have been attributed to contacts between these groups and the enzyme in the transition state (1). Variations in alkylation rates are explained in terms of different degrees of entropic destabilization (2) of the nucleosides in the enzyme.affinity label complex.

Acetamides↗
PubMed1986

Affinity labeling of the DDT1 MF-2 cell alpha 1-adrenergic receptor with [3H]phenoxybenzamine.

In this study, we used phenoxybenzamine to label the alpha 1-adrenergic receptor of a smooth muscle cell line. Our results demonstrate a dose-dependent occupancy of alpha 1-adrenergic receptors by phenoxybenzamine determined by competition for the [3H]prazosin binding site. Following incorporation of [3H]phenoxybenzamine, partially purified membranes were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Despite numerous Coomassie blue-stained bands, only three bands, Mr = 80,000 +/- 500, Mr = 33,000 +/- 2,000, and Mr = 21,000 +/- 400 (N = 4), were labeled with [3H]phenoxybenzamine as determined by autofluorography. Incorporation of [3H]phenoxybenzamine into the Mr = 80,000 band, but not the Mr = 33,000 and Mr = 21,000 bands, was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Labeling of the Mr = 33,000 and Mr = 21,000 bands was partially blocked by phenoxybenzamine. We conclude that [3H]phenoxybenzamine can be used as an affinity probe for the alpha 1-adrenergic receptor and that the ligand binding site of the alpha 1-adrenergic receptor resides in a Mr = 80,000 protein.

Affinity Labels↗
PubMed1990

Affinity labeling of lysine-149 in the anion-binding exosite of human alpha-thrombin with an N alpha-(dinitrofluorobenzyl)hirudin C-terminal peptide.

In order to define structural regions in thrombin that interact with hirudin, the N alpha-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin [GDFEEIPEEY(O35SO3)L (DNFB-[35S]Hir54-64)]. DNFB-[35S]Hir54-64 was reacted at a 10-fold molar excess with human alpha-thrombin in phosphate-buffered saline at pH 7.4 and 23 degrees C for 18 h. Autoradiographs of the product in reducing SDS-polyacrylamide gels revealed a single 35S-labeled band of Mr approximately 32,500. The labeled product was coincident with a band on Coomassie Blue stained gels migrating slightly above an unlabeled thrombin band at Mr approximately 31,000. Incorporation of the 35S affinity reagent peptide was found markedly reduced when reaction with thrombin was performed in the presence of 5- and 20-fold molar excesses of unlabeled hirudin peptide, showing that a specific site was involved in complex formation. The human alpha-thrombin-DNFB-Hir54-64 complex was reduced, S-carboxymethylated, and treated with pepsin. Peptic fragments were separated by reverse-phase HPLC revealing two major peaks containing absorbance at 310 nm. Automated Edman degradation of the peptide fragments allowed identification of Lys-149 of human thrombin as the major site of DNFB-Hir54-64 derivatization. These data suggest that the anionic C-terminal tail of hirudin interacts with an anion-binding exosite in human thrombin removed 18-20 A from the catalytic apparatus.

Affinity Labels↗
PubMed1980

Immunoelectron microscopic localization of the site of photo-induced affinity labeling of the small ribosomal subunit with puromycin.

Ribosomes from Escherichia coli strain TPR201 (which lack N6,N6-dimethyladenosine) have been photoaffinity labeled with [3H]puromycin in the presence of chloramphenicol. Puromycin-modified 30S ribosomal subunits appear to be identical to untreated subunits in electron micrographs and are efficiently precipitated by antibodies to the puromycin analog N6,N6-dimethyladenosine. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of 30S subunits which appear to be crosslinked by a single IgG molecule. A predominant site of puromycin photoaffinity labeling has been identified from the apparent point of contact of antibody and ribosomal subunit. The puromycin site is localized to the small upper portion of the particle on the side opposite to the subunit platform. This location is close to that reported for ribosomal protein S14, the major puromycin-labeled protein in the small ribosomal subunit.

Affinity Labels↗