Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Affinity Labels”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,567 records · Page 87Linked to original sources
PubMedDate not supplied

A site-specific bifunctional protein labeling system for affinity and fluorescent analysis.

Most covalent protein labeling schemes require a choice between visual and affinity properties, requiring the use of multiple fusion systems where both attributes are needed. While not disruptive at the single experiment level, this detail becomes critical when addressing high-throughput experimentation. Here we develop a uniform site-specific protein tag for use in both fluorescent and affinity screening. Covalent protein tagging with a stilbene reporter via promiscuous phosphopantetheinyltransferase (PPTase) modification enables a switchable, antibody-elicited fluorescent response in solution or on affinity resin. For demonstration purposes, VibB, a natural fusion protein harboring a carrier protein domain, was labeled with a stilbene tag through PPTase modification with a stilbene-labeled coenzyme A analogue. Analysis of the resulting stilbene-tagged VibB was accomplished by fluorescent and Western blot analysis with anti-stilbene monoclonal antibody EP2-19G2. The illustration of this method for general application to fusion protein analysis offers a dual role in assisting both solution-based fluorescent analysis and surface-based affinity detection and purification.

Affinity Labels↗
PubMed1986

Molecular characteristics of receptors for atrial natriuretic factor.

Specific, high-affinity receptors for atrial natriuretic factor (ANF) have been identified on membranes from a variety of tissues and cultured cells. By affinity labeling procedures, radioactivity from 125I-labeled ANF was specifically incorporated into three different polypeptides of ca. 120,000, 70,000, and 60,000 daltons, which may represent the binding subunits of ANF receptors. These polypeptides were present in varying amounts in different target tissues. In rat adrenal membranes, the 120,000- and 70,000-dalton peptides were specifically labeled whereas in A10 rat smooth muscle cells, only the 60,000-dalton peptide was labeled. Membranes from rat kidney and rabbit aorta contain all three peptides. Gel filtration chromatography of solubilized receptors suggested that intact ANF receptors are large molecular complexes with apparent molecular masses in the range of 250,000-350,000 daltons. The differential labeling pattern observed with the various tissues suggested that there might be at least two different receptors composed of unique ANF-binding polypeptides.

Adrenal Glands↗
PubMed1993

Site-directed cross-linking studies on the E. coli tRNA-ribosome complex: determination of sites labelled with an aromatic azide attached to the variable loop or aminoacyl group of tRNA.

tRNA(Phe) from E. coli, modified with the photoreactive label N-(p-azidobenzoyl)-glycine (ABG) either at the naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine (acp3U47) or the alpha-amino group of Phe-tRNA(Phe), was bound nonenzymatically to 70S ribosomes in the presence of poly (U) or short synthetic mRNA molecules prepared by T7 transcription. The noncovalent complexes were subjected to a mild ultraviolet irradiation treatment and the sites of photo-incorporation were analysed. When the photo-affinity label was attached to the aminoacyl group cross-linking was observed from both A- and P-site bound tRNA and involved exclusively the 50S subunit. In both cases the major target of cross-linking was a single site in 23S RNA, localized to position A-2439. A lower yield of cross-linking to L27 from both P- and A-sites was also observed. In contrast, cross-linking from the acp3U47 derivative was specific for P-site bound tRNA and involved mainly (but not exclusively) the 50S subunit. In this case rRNA and ribosomal protein were labelled in approximately equal yields, the sites of cross-linking involving A-2309 in 23S RNA and L33. These results are discussed in the light of our present knowledge concerning the structural arrangement of the tRNA-ribosome complex.

Affinity Labels↗
PubMed1989

[Localization of a lysine residue near the site of initiating substrate binding of T7 bacteriophage RNA polymerase].

A highly selective affinity label was introduced into the T7 phage RNA polymerase by means of GMP ortho-formylphenyl ester and [alpha-32P]UTP nearby the enzyme's active site, which was located using limited cleavage technique. Hydroxylamine, bromine, N-chlorosuccinimide, and cyanogen bromide were employed as the reagents. Analysis of gel-electrophoretic patterns of the cleavage products led to a conclusion that Lys631 is the target of labelling. The region nearby this residue has a high degree of sequence homology with regions of RNA polymerases from T3 and SP6 phages and yeast mitochondria.

Affinity Labels↗
PubMed1987

205Tl+ as a spectroscopic probe of the monovalent cation binding sites of bovine plasma activated protein C and des-1-41-light-chain-activated protein C.

The kinetic properties of the stimulation by Tl+ of the amidase activity of bovine plasma activated protein C (APC) and a limited-proteolytic derivative of this enzyme, des-1-41-light chain APC (GDAPC), which has no remaining gamma-carboxyglutamic acid residues, have been compared, along with a 205Tl+ NMR analysis of the interaction of this cation with these enzymes, at 6 degrees C. In contrast to other monovalent cations, the productive kinetic complex of Tl+ and APC involves only a single Tl+ site, or class of sites, and is similar to GDAPC in this regard. In the case of each enzyme, the kinetic mechanism that best describes the participation of Tl+ is a rapid equilibrium type with random addition of the cation and substrate to the enzyme. The dissociation constants of the Tl+ X APC and Tl+ X GDAPC complexes have been determined by NMR analysis and have been found to be very similar to the same constants as calculated by kinetic means. These cation sites are also present intact on each zymogen, demonstrating that they are not generated as a result of activation. Our results also show that the Ca2+ binding sites of these proteins are exclusive of the T1+ site and that some interference with Tl+ binding is exercised by an active site-directed affinity label. We conclude that Tl+ can be effectively employed as a spectroscopic probe of the monovalent cation sites that serve an extensive stimulatory role in the amidolytic and esterolytic activities of APC.

Affinity Labels↗
PubMed1992

Not all insulin-like growth factor-binding proteins (IGFBPs) are detectable by western ligand blotting: case studies of PC12 pheochromocytoma and rat anterior pituitary IGFBPs and proteolyzed IGFBP-3.

We studied the limitations of the Western ligand blot (WLB) for detecting insulin-like growth factor-binding proteins (IGFBPs). PC12 rat pheochromocytoma cells and rat anterior pituitary cells (AP) secrete IGFBPs that cannot be detected by WLB. We used affinity labeling, WLB, dot blotting, competitive binding, ion exchange chromatography, and deglycosylation to characterize these IGFBPs. These IGFBPs were compared with pregnancy protease-derived IGFBP-3 fragments that also bind insulin-like growth factors (IGFs), but are not detectable by WLB. We showed that PC12 IGFBP is cationic, not glycosylated, with 25,500 mol wt reduced (18,500 unreduced), with high affinity for IGF-II and low affinity for IGF-I. It cannot be detected by WLB and is not a proteolytic derivative of other IGFBPs or IGF-II receptors. Its binding activity is not destroyed by sodium dodecyl sulfate (SDS) and heating. It binds to nitrocellulose and IGF-II after dot blotting, but not to IGF-II during WLB. AP also secrete an IGFBP(s) that was not detectable by WLB. AP IGFBPs, unlike those of PC12, have a higher mol wt, and at least one component is glycosylated. The failure of WLB to detect these proteins remains unexplained. Pregnancy protease-derived IGFBP-3 fragments also bind IGFs and are not detectable by WLB. However, they do electrotransfer to nitrocellulose. The failure of WLB to detect these fragments is probably due to proteolysis rendering the binding site susceptible to irreversible denaturation (under conditions of WLB) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that WLB, while valuable, may have significant limitations in specific cases. Other techniques must complement WLB for detection of IGFBPs in conditioned media and other biological specimens.

Affinity Labels↗
PubMed2000

Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease.

The presence, microenvironment, and proximity of an essential Trp with the essential His and Cys residues in the active site of an alkaline protease have been demonstrated for the first time using chemical modification, chemo-affinity labeling, and fluorescence spectroscopy. Kinetic analysis of the N-bromosuccinimide- (NBS) or p-hydroxymercuribenzoate- (PHMB) modified enzyme from Conidiobolus sp. revealed that a single Trp and Cys are essential for activity in addition to the Asp, His, and Ser residues of the catalytic triad. Full protection by casein against inactivation of the enzyme by NBS and quenching of Trp fluorescence upon binding of the enzyme with NBS, substrate (sAAPF-pNA), or inhibitor (SSI) confirmed participation of the Trp residue at the substrate/inhibitor binding site of the alkaline protease. Comparison of the K(sv) values for the charged quenchers CsCI (1.66) and KI (7.0) suggested that the overall Trp microenvironment in the protease is electropositive. The proximity of Trp with His was demonstrated by the sigmoidal shape of the pH-dependent fluorometric titration curve with a pK(F) of 6.1. The vicinity of Trp with Cys was indicated by resonance energy transfer between the intrinsic fluorophore (Trp) and 5-iodoacetamide-fluorescein labeled Cys (extrinsic fluorophore). Our results on the proximity of Trp with essential His and Cys thus confirm the presence of Trp in the active site of the alkaline protease.

Affinity Labels↗
PubMed1985

Insulin-like growth factor II binding to cultured human chondrosarcoma cells.

Cultured cells originally derived from a human chondrosarcoma (A1684) were used to investigate somatomedin binding in terms of kinetics and specificity. In this study, the rat somatomedin, multiplication-stimulation activity (MSA) was utilized. While the human chondrosarcoma cells did not exhibit a mitogenic response to MSA, the rate of transport of glucose and amino acids was significantly increased. In competitive binding experiments a specific insulin-insensitive MSA receptor was identified which showed half maximal displacement of tracer at a concentration of 250 ng/ml of MSA using whole cells. This receptor had an affinity constant of 4.8 X 10(7) M-1. Kinetic analysis of MSA binding to membrane preparations and to Triton X-100 solubilized membranes revealed an increase in the binding affinity to 1.28 X 10(8) M-1 and 2.8 X 10(8) M-1, respectively. Of particular significance is the observation that these cells have especially high levels of MSA receptors. Determination of binding capacity revealed that these cells contain approximately 1.9 X 10(6) MSA receptors per cell and therefore are an excellent model system for the characterization and purification of somatomedin receptors. Affinity labeling of the MSA receptor using the chemical crosslinking reagent, disuccinimidyl suberate, confirmed that this receptor was of the type II class of somatomedin receptors and exhibited a molecular weight of 218,000 under nonreducing conditions.

Affinity Labels↗
PubMed1985

Structural similarities between the plasma membrane binding sites for L-thyroxine and 3,3',5-triiodo-L-thyronine in cultured cells.

Using 125I-labeled L-thyroxine (T4), the binding of [125I]T4 to GH3 rat pituitary tumor cells was studied. At 15 degrees C, the binding of [125I]T4 to cells is saturable and specific. Least squares analysis of binding data showed two classes of binding sites with apparent dissociation constants of 4.3 +/- 0.3 nM and 350 +/- 30nM and binding capacities of (3.8 +/- 0.5) X 10(4) and (9.1 +/- 0.35) X 10(6) sites/cell, respectively. Affinity labeling of cells or purified plasma membranes with N-bromoacetyl-[125I]T4 (BrAc[125I]T4) showed a major specifically labeled protein band with an apparent molecular mass of 55 kilodaltons (kDal). Digestion of the 55-kDal protein from cells and plasma membrane by Staphylococcus aureus V8 protease or elastase gave similar peptide fragments. Thus, the 55-kDal protein labeled from intact cells is the same protein as that from purified plasma membranes. Peptide mapping was further used to compare the 55-kDal protein specifically labeled by either N-bromoacetyl-3,[125I]3',5-triiodo-L-thyronine (BrAc[125I]T3) or BrAc[125I]T4 in intact cells and highly purified plasma membranes. Very similar patterns were obtained. These results indicate that plasma membrane T3 and T4 binding sites have similar hormone binding domains. In addition the plasma membrane T3 and T4 binding sites of Swiss 3T3-4 mouse fibroblasts and A431 human epithelioid carcinoma cells are structurally similar to the T3 and T4 binding sites of GH3 cells.

Affinity Labels↗
PubMed1984

The effect of aminoacyl- or peptidyl-tRNA at the A-site on the arrangement of deacylated tRNA at the ribosomal P-site.

Photoreactive derivatives of E. coli tRNAPhe bearing arylazido groups on guanine residues (azido-tRNA) were used for affinity labelling of E. coli ribosomes in the region of the P-site when the A-site was either free or occupied by aminoacyl- or peptidyl-tRNA. Corresponding complexes of azido-tRNA with ribosomes and poly(U) were obtained both nonenzymatically and with the use of elongation factors. UV-irradiation of the complexes resulted in labelling of ribosomal proteins (preferentially of 30 S subunit). Proteins S9 and S21 were labelled only when the A-site was free; S14 - only when it was occupied; S11, S13, S19 - in both cases; S5, S7, S12, S20 - in some states.

Affinity Labels↗
PubMed1984

GTP interacts with the gamma-subunit of eukaryotic initiation factor eIF-2.

Eukaryotic initiation factor eIF-2 is an oligomeric protein consisting of three different subunits. During initiation of protein synthesis eIF-2 interacts with GTP, Met-tRNAf and 40 S ribosomal subunit. By affinity labeling with a photo-reactive GTP analogue it was shown that in the binary complex [eIF-2 X GTP] GTP is in contact with the gamma-subunit of eIF-2.

Affinity Labels↗
PubMed2000

Dual contacts between peptide agonist ligands and the secretin receptor directly established by photoaffinity labeling.

Structural analysis of secretin in solution has demonstrated extended helical domains within both amino- and carboxyl-terminal halves, with a possible turn in between. However, the conformation of this peptide as it resides in its binding site within the receptor has not been established. In the work reported here, we performed affinity labeling of the secretin receptor with radioiodinated secretin analogues having photolabile benzoyl-phenylalanine residues positioned in each half of the peptide. The probes had sites of covalent attachment in positions 6 and 22, and have been recently synthesized and characterized to represent high affinity agonist ligands. Both covalently labeled the secretin receptor in a saturable, specific, and efficient manner. After purification of the labeled receptor, we used a series of chemical and enzymatic cleavage techniques to define the domain of labeling. We complemented this by receptor mutagenesis, followed by additional cleavage and Edman degradation sequencing to refine our insights into the labeled residues. This has allowed us to demonstrate that sites of attachment were both within the extracellular aminoterminal domain of the receptor. Of particular interest, both probes labeled residues within the amino-terminal thirty residues at the distal end of the receptor. It will be particularly interesting to use these molecular approximations to model the binding domain of this important receptor.

Affinity Labels↗
PubMed1993

Identification of an essential glutamate residue in the active site of endoglucanase III from Trichoderma reesei.

n-Propyl, n-butyl and n-pentyl beta-cellobiosides with a reactive omega-epoxide in their aglycon completely and irreversibly inactivate endoglucanase III from Trichoderma reesei. The pentyl derivative was found to be most effective. From these affinity labeling experiments evidence was found for the implication of Glu329 in the reaction mechanism. This is discussed in relation to other structural/functional data known for endoglucanase III and several other family A glycanases.

Affinity Labels↗
PubMed1986

Differential human interferon alpha receptor expression on proliferating and non-proliferating cells.

The expression of interferon-alpha (IFN-alpha) receptors was studied on a variety of human cells, using monoiodinated IFN-alpha 2 probes. Steady-state binding at 4 degrees C revealed a single class of non-interacting IFN receptor on peripheral blood lymphocytes, and tonsillar B lymphocytes, which are both known to be G0/G1 resting cell populations. The binding affinity of this class of receptor was found to be on the order of 5 X 10(-10) M, expressed as an apparent dissociation constant (Kd). However, cells proliferating either in culture or in vivo were found to express a heterogeneity in IFN-alpha 2 binding. Such binding could be objectively resolved (by a version of the LIGAND program of P. Munson) into a two-site receptor model. Hill plots of binding to proliferating cells indicated a negative cooperativity in the interaction of IFN and receptor. The high-affinity component, expressed on proliferating cells, typically exhibits a Kd of (1-10) X 10(-11) M, while the lower-affinity component indicates a Kd of (1-10) X 10(-9) M. Furthermore, the low-affinity component is apparently expressed on the order of 10-200 times the copy number, per cell, of the high-affinity site. Affinity-labeling experiments revealed that, in addition to the 140-160-kDa IFN-binding complex reported by others, both the proliferating and non-proliferating cell populations possess a novel IFN-binding component of 60 kDa.

Affinity Labels↗
PubMed1990

Mapping of the region of the tick-borne encephalitis virus replicase adjacent to initiating substrate binding center.

Affinity labelling with aldehyde-containing analogs of initiation substrates of nuclear fraction of tick-borne encephalitis virus (TBEV) infected cells results in a labelling of a single polypeptide with a molecular mass of 68 kDa which was immunologically identified as TBEV NS3 protein. A single-hit hydroxylamine hydrolysis, using limited and long-term CNBr cleavages allowed one to identify Lys1800 and/or Lys1803 as the label attachment sites. These amino acid residues are situated in the proximity of the 'B'-site of NTP-binding motif of viral RNA replicase.

Adenosine Triphosphate↗
PubMed1991

Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger.

The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.

Affinity Labels↗
PubMed1990

Biologically active aromatic retinoids bearing azido photoaffinity-labeling groups and their binding to cellular retinoic acid-binding protein.

Retinoids bearing azido photoaffinity-labeling groups (azidoretinoids) have potential as probes for investigating the molecular mechanisms of action of all-trans-retinoic acid (RA) as mediated by its cellular retinoic acid-binding protein (CRABP) and nuclear receptor proteins. Two new azidoretinoids, 3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1E- propen-1-yl]-benzonic acid and 4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)be nzoic acid were synthesized, and evaluated for their in vitro biological potency, and binding affinity for CRABP. Like RA, these aromatic azides had significant activity in modulating cell differentiation in retinoid-deficient hamster tracheal organ culture (ED500.02 nM and 0.03 nM, respectively) and in the inhibition of the induction of ornithine decarboxylase in mouse epidermis (ED50 7.0 nmol and 0.5 nmol, respectively). They also possessed high binding affinity for CRABP (ID50 0.9 microM and 0.85 microM, respectively). The tritiated aromatic azides were further evaluated for their ability to bind covalently to CRABP after photolysis. On photolysis at -78 degrees C, the two radiolabeled azidoretinoids formed stable adducts with CRABP. Treatment of the adducts with either RA or p-chloromercuriphenylsulfonic acid (CMPS) and subsequent dialysis did not cause any dissociation, indicating the formation of a covalent bond. In contrast, treatment of the unirradiated complexes with RA or CMPS led to dissociation of the complex. Synthesis of affinity labels and characterization of CRABP-retinoid complexes should provide useful information on the ligand-binding regions and insights into the mechanism of action of RA.

Affinity Labels↗
PubMed1997

Neurotoxic phospholipases A2 ammodytoxin and crotoxin bind to distinct high-affinity protein acceptors in Torpedo marmorata electric organ.

We studied the binding of radioiodinated ammodytoxin C, a monomeric phospholipase A2 neurotoxin from Vipera ammodytes, and of radioiodinated crotoxin, a dimeric phospholipase A2 neurotoxin from Crotalus durissus terrificus, to presynaptic membranes from the electric organ of Torpedo marmorata. In both cases, two different families of specific binding sites were identified and characterized. The high-affinity binding sites for both toxins have been shown to be proteins. The low-affinity binding sites were not affected by proteinases or heat, suggesting the involvement of certain lipid structures in this type of binding. By affinity-labeling, [125I]ammodytoxin C was shown to be associated predominantly with membrane proteins of apparent molecular masses of 70,000 and 20,000 Da and to a lesser extent with several proteins of apparent molecular masses ranging between 39,000 and 57,000 Da. [125I]crotoxin, on the other hand bound primarily to a 48,000 Da membrane protein. All phospholipases A2 tested, except beta-bungarotoxin, inhibited the low-affinity specific binding of ammodytoxin C, whereas only neurotoxic phospholipases A2 prevented the high-affinity binding and the cross-linking of ammodytoxin C and crotoxin. The inhibition profiles of high-affinity binding for [125I]crotoxin and for [125I]ammodytoxin C were quite different. Ammodytoxin C and crotoxin did not inhibit each other on their respective high-affinity binding sites. These observations indicate that at least high-affinity binding sites of these two toxins are different. In contrast with crotoxin, the isolated basic subunit CB of crotoxin was able to completely inhibit the high-affinity binding of [125I]ammodytoxin C. Therefore, the acidic subunit CA of crotoxin does not simply act as a chaperone for CB subunit, but it also confers a distinct binding specificity to the crotoxin.

Affinity Labels↗