Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes

Summary The specific binding of [³H]cortisol to plasma membranes purified from mouse liver, studied by the ultrafiltration method, shows the existence of specific binding sites for cortisol. The kinetic parameters of this binding areK _D=4.4nm andB _max=685 fmol/mg protein in presence of 1 m of corticosterone. With respect to the binding of 4nm [³H]cortisol to the membrane, the affinities of the steroids decreased in the following order: deoxycorticosterone>corticosterone>progesterone>cortisol >prednisolone>testosterone>20-hydroxyprogesterone >cortisone. Estradiol, dexamethasone, ouabain and triamcinolone acetonide do not have affinity for this binding site. Neither Ca²⁺ nor Mg²⁺ affected the binding of [³H]cortisol to the plasma membranes. Likewise, the presence of agonists and antagonists of alpha and beta-adrenergic receptors did not modify the binding of [³H]cortisol. The results suggest that the plasma membrane binding site characterized is more specific for corticoids and is different from nuclear glucocorticoid and progesterone receptors.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

Steroid hormone specifically binds to rat kidney plasma membrane

A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using anin vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 µM corticosterone. Under standardized conditions of pH 7.4 at 2°C and 30 min incubation time, the binding was saturable and showedK _d=13±3 nM andB _max=616±34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane.     

G-proteins of rat liver membranes. Subcellular compartmentation and disposition in the plasma membrane

Summary The distribution of the alpha- and beta-subunits of G-proteins and their disposition in rat liver plasma and intracellular membranes was investigated. Western blotting, using antibodies that recognised the alpha-subunit of the inhibitory and the beta-subunits of most G-proteins, identified 41 and 36 kDa polypeptides respectively in all plasma membrane functional domains, in endosomes as well as in Golgi membranes. Lysosomes were devoid of these subunits. The highest levels of G-protein subunits were found in bile canalicular plasma membranes prepared by density gradient centrifugation followed by free-flow electrophoresis. Separation of membrane proteins into extrinsic and intrinsic components was carried out by extraction of the membranes at pH 11.0 and by partitioning the membranes in Triton X-114/aqueous phases. The results demonstrated that the alpha- and beta-subunits were tightly associated with the hepatic membranes but they could be solubilised by extraction with detergent, e.g. SDS. Prolonged incubation in the presence of GTP analogues also released up to approximately 50% of the alpha-subunit of inhibitory G-proteins from membranes. The beta-subunit was still associated with membranes after alkaline extraction. The results emphasise the strong association of G-protein subunits with liver membranes, and show that these proteins are distributed widely in the plasma membrane and along the endocytic pathways of hepatocytes.     

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.     

The fluidity of plasma membranes from ethanol-treated rat liver

Summary Male Wistar rats were maintained for 35–40 days on a liquid diet containing 36% of calories as ethanol. Ethanol was replaced by carbohydrates in the isocaloric diet fed to control animals. The effect of ethanol consumption has been studied on the fluorescence polarization of rat liver plasma membranes and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization in both membranes and vesicles was determined using DPH and TMA-DPH as fluorescence markers; from these data, the polarization term (r_o/r–1)^–1 and flow activation energy (E) were calculated. The ethanol consumption induces a more fluid environment within the membrane core of liver plasma membranes; the ethanol-fed rat membranes are more resistant to the in vitro effect of ethanol disordering the membrane structure. Vesicles obtained with lipids from either control membranes or ethanol-fed rat membranes were treated with ethanol and the changes in polarization paralleled to those exhibited by the membranes. The absence of phase transitions and of E changes was also shown in temperature-dependence studies. The lower cholesterol content found in ethanol-fed rat plasma membranes might be responsible for observed variations in the microviscosity.Abbreviation OG octyl -D-glucopyranoside     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Acyl-CoA: 1-acyl-glycero-3-phosphoethanolamine O-acyltransferase and liver plasma membrane fluidity

Investigations have been carried out on the influence of membrane lipid composition and physical state on acyl-CoA: 1-acyl-glycerol-3-phosphoethanolamine O-acyltransferase activity in rat liver plasma membranes. The lipid composition of the membranes was modified either by way of lipid transfer proteins or by partial delipidation with exogenous phospholipases and subsequent enrichment of the membranes with different phospholipids. The results indicated that membrane rigidification by enrichment of the membranes with DPPC or SM reduced the transfer of oleic and palmitic acid to lysophosphatidylethanolamine, whereas all phospholipids inducing membrane fluidization lead to acyltransferase activation. The eventual role of membrane fluidity in the deacylation-reacylation cycle is discussed.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

High-affinity folate binding in human liver membranes

High-affinity binding of³H-folate in Triton X-100 solubilized membranes of human liver displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. Ultrogel^® AcA 44 chromatography of solubilized membranes saturated with³H-folate revealed a major peak of 100 kDa and a minor peak of 25 kDa. The 100 kDa peak could represent a hydrophobic membrane associated molecular form of the protein. This notion was supported by the fact that the two peaks had identical molecular weights as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with immunoblotting.     

Properties of Na+, K+-ATPase of liver plasma membranes in the hamster

This study was designed to establish the properties of liver plasma membranes (LPM) Na+,K+-ATPase in the hamster and to determine whether a similar assay may be used to measure enzyme activity in the hamster and in the rat. Maximal Na+,K+-ATPase activity was obtained when the assay medium contained 5 mM Mg APT2- with or without 1 mM free Mg2+, 120 mM Na+, 12,5 mM K+. The incubation must be performed at 37 degrees C, pH 7.4. In the absence of free Mg2+, the saturation curve with respect to the substrate Mg ATP2- resulted in biphasic complex kinetics with a maximal activity at a substrate concentration of 5 mM. In the presence of 1 mM free Mg2+ activation of Na+,K+-ATPase and modification of the kinetics were observed: the biphasic curve tended to disappear and to become of the Michaelis-Menten type. The apparent Km for Mg APT2- was 0.36 mM and the Vmax 34.5 mumol.h-1.mg protein-1. In the presence of 10 mM free Mg2+ a decrease in the Vmax was observed without any effect on the apparent Km for Mg APT2-. It is concluded that the same incubation medium may be used to assay LPM N+,K+-ATPase from hamster and rat and that the addition of 1 mM free Mg2+ to the incubation medium is recommended to obtain Michaelis-Menten kinetics in order to eliminate complex kinetics due to the absence of free Mg2+.     

Suppression of inositol phosphate release by cardiac myocytes isolated from fish oil-fed pigs

Apo C-III plays an important role in the metabolism of plasma triglyceride, which can delay the catabolism of triglyceride-rich lipoproteins by interfering with apo E-mediated receptor clearance of remnant particles from plasma. The mechanism of the interference has not yet been defined. To further explore the role of apo C-III, we first injected mice with ¹²⁵I-apo C-III. The measurement of radioactivity showed that liver took up 3.3-10 fold as much radioactivity as other organs such as heart, spleen, lung, kidney, stomach, large intestine, small intestine, and muscle. This was confirmed by incubating the tissue homogenates of the organs with ¹²⁵I-apo C-III that the radiolabeled apo C-III specifically bound to only hepatic homogenate. To investigate which subcellular part or parts of hepatic cells play the role of binding to apo C-III, hepatic cell components of nucleus, mitochondria, microsomes and plasma membranes were then incubated with ¹²⁵I-apo C-III. The radiolabeled apo C-III could specifically bind to only hepatic plasma membranes. Finally hepatic plasma membranes were purified to study the characteristics of the specific binding with apo C-III. Addition of increasing concentration of ¹²⁵I-apo C-III to human hepatic plasma membranes revealed saturable binding to membranes with a Kd of 0.31±0.07 mol/l. The maximum specific binding capacity was 1.74±0.45 apo C-III/mg membrane protein. In competition studies using unlabeled apo C-III and isolated lipoproteins HDL, LDL and VLDL, only apo C-III and VLDL effectively competed with ¹²⁵I-apo C-III for membrane binding. The binding of 125I-apo C-III to human liver plasma membranes was Ca²⁺-independent, and was abolished when plasma membranes were treated with trypsin. The characteristics of ¹²⁵I-apo C-III binding to mouse liver plasma membranes were similar to those of human liver plasma membranes with the exception of a binding maximum of 1.52±0.39 apo C-III/mg membrane protein. We conclude that apo C-III exhibits high-affinity binding to hepatic plasma membranes, which is saturable, reverse and specific.     

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--I. Binding and transport.

To gain insight into the mechanisms governing cellular uptake of glucocorticoids, we studied the binding and membrane transport of corticosterone (B) on a highly purified plasma membrane fraction from rat liver that was homogenized using a gentle, isotonic procedure. The fraction was mostly in the form of right-side out and osmotically active vesicles that were free of intracellular glucocorticoid receptors (GCR), transcortin (CBG) and ATP. Our uptake and binding studies carried out at 22 degrees C with [3H]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [3H]B for uptake by the membrane vesicles; half-maximal competition of specific uptake was achieved with a 10- to 11-fold molar excess of unlabeled B. (2) [3H]B uptake was a saturable process of unusual kinetics (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent Kd-values of 1.3, 4.7 and 17.3 nM, corresponding to free B in the blood of non-stressed rats (4-16 nM). (3) Osmotic shrinkage of the vesicles led to a linear decrease in specific uptake, while non-specific uptake was independent of vesicle volume. Passive diffusion of [3H]B took place in leaky, but not in intact, vesicles. Reversible binding to, and mediated transport through, the membrane were interdependent parts of a strongly linked process. B was accumulated inside the vesicle up a concentration gradient by an active transport that followed first-order kinetics (Kt:3.9 nM); for its statistically reliable mathematical formulation and kinetic analysis, a replot was developed that revealed that relative accumulation increased with decreasing external hormone concentration. (4) Comparative binding studies disclosed that the apparent Kd-values (86.5 +/- 7.3 and 77.0 +/- 14.3 nM, respectively) of the [3H]B interactions with CBG and GCR did not differ (P greater than 0.3). These findings permit the conclusion that a plasma membrane-inserted carrier for B, effectively operating at physiological concentrations in the blood, is involved in a functional and regulatory manner in the biological action of glucocorticoids.     

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--II. Comparative influx and efflux.

To elucidate the initial step in the interaction between glucocorticoids (GC) and the hepatocyte, we examined at 22 degrees C further kinetic properties of active corticosterone (B) transport mediated by a putative, plasma membrane-inserted carrier for GC (GCC) as previously reported [Alléra and Wildt, J. Steroid Biochem. Molec. Biol. 42 (1992) 737-756]. We used a purified, well-characterized, osmotically active vesicle fraction of plasma membrane (PM), free of ATP, isolated from rat liver and a method developed by us to describe transport processes mathematically: (1) uptake (U) of 7 nM B into the vesicles (influx, I) occurred very rapidly whereby T1/2 = 8.3 s, the time (S) required for half maximum transport; the influx velocity (dU/dS = V) decreased degressively with time following second-order kinetics characterized by an initial transport V (VT0) of 177.7 fmol/mg membrane protein/s. (2) VToI of B-influx rose with temperature biphasically (P less than 0.025): activation energy above and below 15 degrees C (at PM phase transition) amounted to 9.5 and 26.5 kJ/mol. Neither at 45 nor at 60 degrees C did transport take place, revealing the high thermolability of GCC. (3) Efflux (E) of 6.5 nM B, i.e. transport out of the vesicles preincubated with the steroid, showed that influx had resulted in a 19.6-fold intravesicular hormone accumulation, indicating active ("uphill") transport. (4) The efflux velocity (dE/dS = V) exhibited almost the same kinetic quality as that of influx: it decreased following mainly second-order kinetics whereby T1/2 = 8.0 s. However, its whole time-course was much slower and the VT0 of efflux (VToE) was 6.3 lower than VToI. (5) Using physics and thermodynamics, we deduced that the affinity (AF) between B and GCC is proportional to the square of VT0. (6) Thus, because AF approximately (1/6.3)2, AF of the B-GCC interaction after completion of influx was calculated to be 40 times lower (Kd = 708 nM; delta G degrees = -34.9 kJ/mol) than at outset of influx, whereby delta G degrees = -44.0 kJ/mol. Concluding from these and previous findings, we present a new hypothesis on B transport into the hepatocyte: There is no difference (P greater than 0.3) in free enthalpy between transcortin (CBG) and the intracellular GC receptor interacting with B (delta G degrees = -40.1 and -40.4 kJ/mol). The GCC, however, is characterized by its ability to switch from a high- to lower-affinity when interacting with B (and vice versa due to metabolic energy input).(ABSTRACT TRUNCATED AT 400 WORDS)     

Amino acid transport in plasma membrane vesicles from isolated rat liver parenchymal cells

Plasma membrane vesicles were prepared from isolated rat liver parenchymal cells. The transport of several amino acids was studied and found to be identical to that in membrane vesicles from whole liver tissue.     

Ionic requirements for taurocholate transport in rat liver plasma membrane vesicles

As part of the enterohepatic circulation, taurocholate is taken up by hepatocytes by a Na⁺-gradient-dependent, carrier-mediated process. The dependence of taurocholate uptake on the presence of a Na⁺ gradient, outside greater than inside, has been studied in isolated rat liver plasma membranes. The uptake is specific for sodium, and a cotransport stoichiometry of 2 Na⁺ per taurocholate taken up was found. The presence of K⁺ ions inside the vesicles was also found to be essential for maximum Na⁺-stimulated uptake of taurocholate, although a K⁺ gradient is not required. Mg²⁺ was almost as effective as K⁺ in this regard. The symport of Na⁺ and taurocholate during uptake was shown to be electrogenic, so that K⁺ may act as an exchange counterion preventing the accumulation of positive charge within the vesicles.Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.