Nuclear reverse T3 binding sites: an artefact of isolation?

1985 ◽  
Vol 108 (4) ◽  
pp. 525-531 ◽  
Author(s):  
R. Hartong ◽  
W. M. Wiersinga
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Reverse T3 ◽  
Nuclear Extracts ◽  
Ping Pong ◽  
Ionic Detergent ◽  
Enzymatic Nature ◽  
Triton X ◽  
Sulfhydryl Compounds

Abstract. Binding of [125I]rT3 to rat liver nuclear extracts prepared in 0.25 m sucrose could be abolished by a prior wash of the nuclei with 2.4 m sucrose. Analysis of mixtures containing [125I]rT3 and nuclear extracts (0.25 m sucrose) showed that after incubation for 2 h at 22°C, degradation of rT3 into 3,3'-T2 and I- had taken place. It appears that the presence of 125I- in these mixtures can account for the previously observed 'binding' of [125I]rT3 to these nuclear extracts. Further characterization of the deiodination process in nuclear extracts showed: 1) inactivation by heating, 2) production of equimolar amounts of I- and 3,3'-T2, 3) stimulation by sulfhydryl compounds and inhibition by propylthiouracil in a fashion similar to the microsomal iodothyronine 5'-deiodinase (ping-pong mechanism). We conclude that the observed deiodination of rT3 in hepatic nuclear extracts is of enzymatic nature, due to contamination of the nuclear preparation by microsomal iodothyronine 5'-deiodinase. However, since the nuclei are prepared in the presence of the non-ionic detergent Triton X-100, a nucleus associated deiodinase activity cannot be totally excluded.

Characterization of thyroid hormone receptors in human IM-9 lymphocytes

1988 ◽  
Vol 117 (3) ◽  
pp. 327-332 ◽  
Author(s):  
John W. Barlow ◽  
Philippe De Nayer
Keyword(s):  
Thyroid Hormone ◽  
Binding Sites ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptors ◽  
Finite Capacity ◽  
Thyroid Hormone Action ◽  
Nuclear Extracts ◽  
Triton X ◽  
Scatchard Plots

Abstract. Although putatively identified more than 10 years ago, thyroid hormone receptors in human tissues remain poorly characterized. As a first step towards understanding the mechanism of thyroid hormone action in man we have characterized T3 binding sites in nuclei of the human lymphoblastoid line, IM-9 cells. In whole cell experiments at 37°C, nuclear binding of [125I]T3 was saturable (Kd 34 ± 6 pmol/l) and of finite capacity (≈ 350 sites/cell). The binding sites were extracted from a nuclear pellet by treatment with 0.4 mol/l KCl and sonication. Separation of bound from free [125I]T3 in the extracts was achieved using the calcium phosphate matrix, hydroxyapatite at a concentration of 0.3 ml of a 150 g/l slurry. Rectilinear Scatchard plots were obtained only when the hydroxyapatite was washed with a buffer containing 0.5% Triton X-100. Under these conditions T3 binding sites in the nuclear extracts were present at a concentration of 22.4 ± 8.6 fmol/mg protein and showed an affinity of (Kd, room temperature) 140 ± 10 pmol/l. The same assay system was used to determine the hierarchy of affinities for a range of natural and synthetic analogues. Calling T3 100, the order of potencies observed was: Triac, 500; 3,5-diiodo-3′-isopropylthyronine, 89; T4, 32; 35-dimethyl-3′-isopropylthyronine, 2; 3,2-T2, 0.7, rT3, 0.4; 3′5′-T2, < 0.01. These results suggest that the T3 binding sites present in human IM-9 lymphocyte nuclei and extracts thereof are thyroid hormone receptors. These cells may be a useful tool to increase our understanding of human T3 receptors. The use of hydroxyapatite to separate bound from free hormone may be adapted for use with extracts of other tissues to characterize these receptors further.

The red cell band 3 protein: its role in anion transport

1982 ◽  
Vol 299 (1097) ◽  
pp. 497-507 ◽  
Keyword(s):  
Binding Sites ◽  
Band 3 ◽  
Anion Transport ◽  
Anion Binding ◽  
Hydrophobic Residues ◽  
Ping Pong ◽  
Hydrophilic Residues ◽  
Aqueous Core ◽  
Positively Charged

Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60 % of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

scholarly journals Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes

1991 ◽  
Vol 280 (1) ◽  
pp. 171-178 ◽  
Author(s):  
L M Traub ◽  
E Shai ◽  
R Sagi-Eisenberg
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Trypsin Treatment ◽  
Putative Receptor ◽  
Microsomal Membranes ◽  
Direct Binding ◽  
Triton X ◽  
Binding Sequence

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.

scholarly journals Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in β-linkage to mannose: role in assembly of sialyl-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides

1996 ◽  
Vol 319 (1) ◽  
pp. 209-216 ◽  
Author(s):  
Robert G SPIRO ◽  
Yuichiro YASUMOTO ◽  
Vishnu BHOYROO
Keyword(s):  
Liver Enzyme ◽  
Rat Liver ◽  
Nitrous Acid ◽  
Bovine Milk ◽  
Ph Optimum ◽  
Golgi Membranes ◽  
Triton X

Rat liver Golgi membranes were found to contain an enzyme that can transfer sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of the terminal GlcNAc in β-linkage to mannose and has properties indicating that it is involved in the synthesis of the NeuAcα2-3(6)Galβ1-4GlcNAc(6-SO4) sequences observed in the N-linked carbohydrate units of various glycoproteins. Assays performed with [35S]PAPS (Km 0.67 µM) and GlcNAcβ1-6Manα1-O-Me (GnMaMe) acceptor (Km 0.71 mM) indicated that the sulphotransferase had a pH optimum of approx. 7.0 and is markedly stimulated by Mn2+ ions (maximum approx. 15 mM) and Triton X-100 (0.05-0.1%). Hydrazine/nitrous acid/NaBH4 treatment of the 35S-labelled product yielded radiolabelled 2,5-anhydromannitol(6-SO4). The sulphated GnMaMe product of the GlcNAc-6-O-sulphotransferase could be galactosylated by a rat liver Golgi enzyme that was shown to have the same properties as the UDP-Gal:GlcNAc β-1,4-galactosyltransferase from bovine milk. Competition studies performed with GlcNAc and GlcNAc-6-SO4 furthermore indicated that the same liver enzyme acted on both acceptors to produce Galβ1-4GlcNAc and Galβ1-4GlcNAc(6-SO4) with Km values of 1.04 and 1.68 mM respectively. Because the sulphated N-acetyl-lactosamine could in turn serve as an acceptor for rat liver sialyltransferase, it seems that this enzyme, together with the Golgi galactosyltransferase and the GlcNAc-6-O-sulphotransferase, could act in concert in assembling the NeuAcα2-3(6)Galβ1-4GlcNAc(6-SO4) branches of complex N-linked oligosaccharides.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

1979 ◽  
Vol 92 (3) ◽  
pp. 512-521 ◽  
Author(s):  
B. Czarnocka ◽  
J. Nauman ◽  
G. Adler ◽  
W. Kiełczyński
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Soluble Proteins ◽  
The Other ◽  
High Affinity ◽  
Triton X ◽  
Maximal Capacity

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

Characterization of the binding sites for [3H]glibenclamide in rat liver membranes

1995 ◽  
Vol 284 (1-2) ◽  
pp. 77-82 ◽  
Author(s):  
Yasushi Inoue ◽  
Masahiro Emoto ◽  
Hiroshi Inoue ◽  
Kohei Kaku ◽  
Toshio Kaneko
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Liver Membranes
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