High affinity thyroxine binding to purified rat liver plasma membranes.

Two sets of high-affinity thyroxine binding sites (K_D 0.39 ± 0.06 nM and 23 ± 5 nM) were detected on purified rat liver plasma membranes. Thyroxine is bound with high stereospecificity regarding iodine substituents and alanine side chain modifications of the molecule. Thyroxine binding is inhibited by -SH blocking agents and proteases. The highest affinity thyroxine binding site is also affected by phospholipase A and is distinct from triiodothyronine binding sites present in the membrane preparations; arguments are given for its plasmalemma origin.     

High affinity thyroid hormone binding sites on purified rat liver plasma membranes.

Two orders of saturable binding sites for L-T₃ were detected on purified rat liver plasma membranes--a high affinity, low capacity binding site with a Kd of 3.2 ± 0.5 nM, and a lower affinity, higher capacity site with a Kd of 220 ± 50 nM. Competition-inhibition studies revealed that both D-T₃ and L-T₄ (two compounds with lower biological potencies than L-T₃) were also less potent than L-T₃ in competing for these binding sites. The present studies demonstrate, therefore, the presence of specific thyroid hormone binding sites on rat liver plasma membranes. In addition, they suggest that these sites may have a role both in mediating the known effects of thyroid hormones on membrane functions, and in regulating the entry of thyroid hormones into target cells.     

High affinity L-triiodothyronine binding sites on washed rat erythrocyte membranes

Two orders of saturable binding sites for L-triiodothyronine were found on washed rat erythrocyte membranes. The high affinity, low capacity site showed values of Kd 0.19 X 10(-10) M. This value was in the range of concentration of free L-triiodothyronine found in the plasma and was several orders of magnitude higher than the Kd values previously reported for other L-triiodothyronine membrane-binding systems. The binding site also showed a high stereospecificity for L-triiodothyronine. D-3,5,3'-triiodothyronine and L-thyroxine were less potent (about 1000-fold) than L-triiodothyronine in competing for these sites. L-3,3,5'-triiodothyronine and triiodothyroacetic acid were inactive. The physiological relevance of this site is considered.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes

The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.     

High affinity ryanodine binding sites in rat liver endoplasmic reticulum

The binding of [3H]ryanodine to liver microsomal subfractions was investigated. The smooth microsomal membranes were enriched with ryanodine binding sites and also with a polypeptide of 360 kDa. Caffeine completely inhibited [3H]ryanodine binding. Ryanodine also affected the membrane Ca2+ permeability. At low concentrations (less than 10 microM) ryanodine stimulated Ca2+ efflux and at higher concentrations (greater than 50 microM) it blocked Ca2+ efflux. These results suggest that hepatic microsomes contain ryanodine binding sites which can modify the membrane permeability for Ca2+.     

Partial purification of rat liver glucocorticoid binding proteins by affinity chromatography

Dexamethasone (9-fluoro-16α-methyl-116,17,21-trihydroxy-1,4-pregnadiene-3, 20-dione) binding proteins from rat liver cytosol were purified approximately 6470 fold by the use of an affinity column in which deoxycorticosterone was linked to CH-Sepharose 4B through a disulfide linkage. The receptor proteins were eluted from the column by washing with β-mercaptoethanol. A preliminary Sephadex G-200 filtration step of the cytosol was necessary in order to separate the dexamethasone binding proteins from other glucocorticoid receptors.     

High affinity binding sites for basic fibroblast growth factor in rat hepatic plasma membranes

The binding of [125I]-recombinant basic FGF (rec bFGF) to rat hepatic plasma membranes was investigated. [125I] rec bFGF bound to an apparent single class of high affinity binding sites (KD = 69 pM; Bmax = 9.61 fmoles/mg proteins). The absence of low affinity sites was confirmed by the inability of sulphated polysaccharides and heparinase to interfere with FGF binding. A good correlation existed between the ability of bovine pituitary-derived bFGF, rec bFGF and bovine brain-derived aFGF to displace [125I]rec bFGF from these binding sites and their in vitro potency on bovine aortic endothelial cell proliferation.     

Small GTP-binding proteins on rat liver lysosomal membranes.

GTP-binding proteins have been identified on the membranes of highly purified dextran-filled lysosomes (dextranosomes) and Triton-filled lysosomes (tritosomes) obtained from rat liver. Autoradiography of blots of lysosomal membrane proteins incubated with [alpha-32P]GTP revealed the presence of several specific GTP-binding proteins with a relative molecular mass (M(r)) predominantly in the range of 26-30 kDa. These GTP-binding proteins migrated slower in polyacrylamide gels than purified c-Ha-ras protein expressed in E. coli, whose apparent M(r) was 23 kDa in the same blot. The relative contents of GTP-binding proteins in lysosomal membranes were comparable or greater than that of plasma membranes and of microsomes. Chemical extraction showed that lysosomal GTP-binding proteins were more tightly associated with the membranes than with microsomal GTP-binding proteins. The possible involvement of lysosomal GTP-binding proteins in cellular functions including vacuolar (lysosomal) acidification and organellar dynamics are discussed.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [³H]estradiol-17β are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [³H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4–5 S and the other had a sedimentation coefficient of 8–9 S. The two components differed from each other regarding steroid specicity and various physiocochemical parameters. [³H]-estradiol binding to the 4–5 S component was not inhibited by estrogens, 5α-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appera to be saturable and lavel was rapidly stripped from it by cahrcoal. Estradiol bindng to the 8–9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4–5 S moiety. The specific binding protein has a K_d of 3.05 · 10⁻¹⁰ M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incbuation of [³H]estradiol with mature male liver cytosol at 0–5°C polar metabolites of estradiol are produced.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Specific adenosine binding proteins from rat liver.

Specific adenosine-binding proteins from homogenates of rat liver have been fractionated on a DEAE-cellulose column. Three major peaks have been identified with respect to histone phosphokinase and cAMP and adenosine-binding activities. Peak I contains only histone phosphokinase activity not stimulated by cAMP. Peak II contains histone phosphokinase slightly stimulated by cAMP. Both cAMP- and adenosine-binding activities are found in this fraction. The major adenosine-binding protein is associated with Peak III. Histone phosphokinase in Peak III which also binds cAMP is stimulated 2-fold by 2.5 muM cAMP whereas adenosine at 2.5 X 10(-4)M inhibits these enzymes equally well in each of three peaks. The specificity of adenosine binding is discussed.     

High affinity binding of [3H] cocaine to rat liver microsomes

[3H]Cocaine bound reversibly, with high affinity (KD 2.3 +/- 1.1 nM) and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow (T1/2 for association, 6 min and for dissociation 17 min), and the kinetically calculated KD was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in [3H]cocaine binding. On the other hand, chronic administration of cocaine reduced [3H]cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of [3H]cocaine to rat liver microsomes was insensitive to monovalent cations and greater than 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced [3H]cocaine binding to liver with a different rank order of potency than their displacement of [3H]cocaine binding to striatum. This high affinity [3H]cocaine binding protein in liver is not likely to be a monooxygenase, but may have a role in cocaine-induced hepatotoxicity.     

Binding characteristics of 4-S proteins from rat and mouse liver. High affinity of ellipticines

The binding characteristics of 4-S components (carcinogen-binding protein) from livers of Sprague-Dawley rats, C57BL/6 and DBA/2 mice have been examined before and after separation from other binding components presents in the cytosol. Competitive potency of 3-methylcholanthrene, benzo[alpha]pyrene, beta-naphthoflavone and 20 ellipticines, a series of compounds differently substituted on the dimethyl-pyrido-carbazole nucleus and deprived of carcinogenic activity, has been determined with [3H]3-methylcholanthrene and/or [3H]benzo[a]pyrene as radioligands. The inducing ability of the same compounds for aryl hydrocarbon hydroxylase and for ethoxyresorufin-O-deethylase has been compared to their affinity for the 4-S protein and the Ah receptor respectively. The main results of this study are as follows. 1. The intrinsic binding characteristics of 4-S proteins were dependent on both the nature of the radioligand used and the presence or absence of other cytosolic binding components. 2. The heterocyclic ellipticines were revealed as strong ligands for the carcinogen-binding protein (stronger than benzo[alpha]pyrene for five derivatives substituted in the A ring of ellipticine), with IC50 values ranging from 0.047 microM (8-hydroxyellipticine) to 5.8 microM (N2-ethyl-9-hydroxyellipticinium). 3. When the affinity of ellipticines was plotted versus their inducing ability of aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase, it appears that a good correlation exists for the Ah receptor but not for the 4-S protein. It is concluded that these data, as well as the lack of enzymatic induction after benzo[alpha]pyrene treatment of DBA/2 mice, which display a high level of 4-S protein, do not support the implication of this binding component in the positive control of cytochrome P-450 induction.     

Receptor binding of pancreatic spasmolytic polypeptide in intestinal mucosal cells and membranes

Rat intestinal mucosal cells contain receptors for pancreatic spasmolytic polypeptide (PSP). The binding of 125I-PSP was rapid, saturable, reversible and specific. PSP competed with 125I-PSP for binding to the receptors and 10(-7) M of PSP half-maximally inhibited 125I-PSP binding. The normalized PSP dose-response graphs in intact cells and crude membranes were superimposable. Scatchard plots of PSP binding to membranes were curvilinear, indicating multiple classes of binding sites, negative cooperative interaction between sites or a combination of both. PSP increased the rate of dissociation of the 125I-PSP-receptor complex compared to the rate observed by dilution only, thus giving evidence that negative cooperative interaction may occur between PSP binding sites. The half-life of the fast dissociating complex was about 1.5 min and that of the slow dissociating complex 38 min. These values were independent of the receptor occupancy. The increased rate of dissociation at high receptor occupancy stemmed from a shift in the ratio of the pool sizes of fast and slow dissociating receptor complexes.