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At least 523 records · Page 29Linked to original sources
PubMed1982

Affinity labeling of a transforming growth factor receptor that does not interact with epidermal growth factor.

Membrane components that interact with epidermal growth factor (EGF) and transforming growth factors (TGFs) have been identified by covalent crosslinking to their respective 125I-labeled ligands. Under appropriate conditions, disuccinimidyl suberate or hydroxysuccinimidyl p-azidobenzoate cross-link receptor-bound 125I-labeled EGF to a 140- to 170-kilodalton (kDal) receptor species in membranes from both A431 human carcinoma cells and normal rat kidney cells. 125I-Labeled sarcoma growth factor (SGF), a TGF from virally transformed mouse 3T3 cells, also can be affinity-crosslinked to the 140- to 170-kDal EGF receptor species in membranes from A431 and rat kidney cells. The labeling of this receptor is inhibited when either excess unlabeled EGF or SGF is present during incubation of membranes with either 125I-labeled EGF or 125I-labeled SGF. In contrast, a second receptor species of 60 kDal is affinity-labeled with 125I-labeled SGF but not with 125I-labeled EGF in membranes from both A431 and rat kidney cells. SGF and a TGF from virally transformed rat embryo cells inhibit the labeling of the 60-kDal species when present in excess during incubation of membranes with 125I-labeled SGF, whereas EGF is completely ineffective in inhibiting the labeling of this receptor. The data suggest that a specific 60-kDal receptor that displays high affinity for TGFs but not for EGF may mediate induction of the transformed phenotype. In addition, SGF and other TGFs interact with the 140- to 170-kDal EGF receptor that appears to mediate normal cell growth effects.

Affinity Labels↗
PubMed1985

Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling.

The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with [125I]ovine PRL [( 125I]oPRL). PRL receptors were solubilized from mammary microsomes with 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with 125I using the chloramine-T method. Covalent labeling of PRL receptors with [125I]oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of [125I]oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five [125I]oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit. From these observations, we conclude that this predominant 32,000 mol wt component is a major binding subunit of the PRL receptor molecule and does not aggregate with itself or with other subunits through S-S linkages.

Affinity Labels↗
PubMed2004

Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue.

25-hydroxyvitamin D(3)- or 1alpha,25-dihydroxyvitamin D(3)-24R-hydroxylase (cytochromeP450C24 or CYP24) has a dual role of removing 25-OH-D(3) from circulation and excess 1,25(OH)(2)D(3) from kidney. As a result, CYP24 is an important multifunctional regulatory enzyme that maintains essential tissue-levels of Vitamin D hormone. As a part of our continuing interest in structure-function studies characterizing various binding proteins in the Vitamin D endocrine system, we targeted recombinant rat CYP24 with a radiolabeled 25-OH-D(3) affinity analog, and showed that the 25-OH-D(3)-binding site was specifically labeled by this analog. An affinity labeled sample of CYP24 was subjected to MS/MS analysis, which identified Ser57 as the only amino acid residue in the entire length of the protein that was covalently modified by this analog. Site-directed mutagenesis was conducted to validate the role of Ser57 towards substrate-binding. S57A mutant displayed significantly lower binding capacity for 25-OH-D(3) and 1,25(OH)(2)D(3). On the other hand, S57D mutant strongly enhanced binding for the substrates and conversion of 1,25(OH)(2)D(3) to calcitroic acid. The affinity probe was anchored via the 3-hydroxyl group of 25-OH-D(3). Therefore, these results suggested that the 3-hydroxyl group (of 25-OH-D(3) and 1,25(OH)(2)D(3)) in the S57D mutant could be stabilized by hydrogen bonding or a salt bridge leading to enhanced substrate affinity and metabolism.

Affinity Labels↗
PubMed1997

Affinity labelling of frog brain opioid receptors by dynorphin(1-10) chloromethyl ketone.

It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphin(1-10)-Gly11 chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the kappa-opioid binding sites labelled by [3H]ethylketocyclazocine (Ki is approximately equal to 200 nM), whereas its binding to the 1 ([3H]dihydromorphine) and to the delta sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 microM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The kappa-preferring binding properties of the compound suggest that it could serve as an affinity label for the kappa-type of opioid receptors.

Affinity Labels↗
PubMed1984

Characterization, solubilization, affinity labeling and purification of the cardiac Na+ channel using Tityus toxin gamma.

Saturable, high-affinity binding of iodinated toxin gamma from Tityus serrulatus scorpion venom (TiTx gamma) to Na+ channel receptor was identified in sarcolemma membrane of chick heart. A binding capacity of 450-600 fmol/mg of protein was found similar to that of tetrodotoxin-binding component. The enrichment of these membrane-bound toxin binding sites follows that of other sarcolemma markers. Kinetic data and displacement of 125I-TiTx gamma from its binding sites by unlabeled TiTx gamma gave an equilibrium dissociation constant (Kd) of 1-3 pM. The gating component and the selectivity filter of the voltage-sensitive Na+ channel, identified as binding sites of TiTx gamma and of tetrodotoxin respectively, have been efficiently solubilized with Nonidet P-40. Purification was achieved by ion-exchange chromatography on DEAE-Sephadex A-25, affinity chromatography on wheat-germ-agglutinin-Sepharose and sucrose density gradient centrifugation. An enrichment of 1400-fold from the original detergent extract was measured for both toxin binding sites (1120-1230 pmol/mg of protein). Sodium dodecyl sulfate gel electrophoresis reveals a single large polypeptide component of Mr230000-270000. The purified material exhibits an apparent sedimentation coefficient of 8.8S. Covalent cross-linking of 125I-TiTx gamma to its membrane-embedded cardiac receptor shows that the cross-linked material, solubilized and purified by the same procedure comprises a single polypeptide chain of the same Mr of 230000-270000. Furthermore, as seen for Electrophorus electricus electroplax and rat brain, the tetrodotoxin-binding component and the TiTx gamma-binding component are carried by the same polypeptide chain. The functional Na+ channel might be an oligomer of this subunit of Mr23000-270000.

Affinity Labels↗