顺铂与小鼠艾氏腹水肝癌细胞膜的相互作用

本文研究了顺铂对小鼠艾氏腹水肝癌细胞膜蛋白内源性荧光的淬灭作用和测定了其在膜上的结合量。结果表明顺铂能与癌细胞膜结合。按存在两类结合部位,得到表观结合常数和结合部位数为: K_1=1.35×10~5L/mol n_1=6.80×10~(-4)mol/g(protein) K_2=2.50×10~3L/mol n_2=1.92×10~(-3)mol/g(protein)     

Phospholipid binding of synthetic talin peptides provides evidence for an intrinsic membrane anchor of talin

Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21-39 (S19), 287-304 (H18), and 385-406 (H17) have been proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/m). S19 partitions into charged monolayers/bilayers with a penetration area A(p) = 140 +/- 30 A(2) and a free energy of binding of DeltaG(0) = -5.7 kcal/mol, thereby forming a partially alpha-helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with A(p) = 148 +/- 23 A(2) and A(p) = 160 +/- 15 A(2), respectively, forming an alpha-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is DeltaG(0) = -9. 4 kcal/mol with a hydrophobic contribution of DeltaG(h) = -7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of DeltaG(h) = -1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor.     

Quantification and characterization of P-glycoprotein-substrate interactions

It is generally accepted that P-glycoprotein binds its substrates in the lipid phase of the membrane. Quantification and characterization of the lipid-transporter binding step are, however, still a matter of debate. We therefore selected 15 structurally diverse drugs and measured the binding constants from water to the activating (inhibitory) binding region of P-glycoprotein, K(tw(1)) (K(tw(2))), as well as the lipid-water partition coefficients, K(lw). The former were obtained by measuring the concentrations of half-maximum activation (inhibition), K(1) (K(2)), in living NIH-MDR-G185 mouse embryo fibroblasts using a Cytosensor microphysiometer, and the latter were derived from surface activity measurements. This allowed determination of the membrane concentration of drugs at half-maximum P-glycoprotein activation (C(b(1)) = (0.02 to 67) mmol/L lipid), which is much higher than the corresponding aqueous concentration (K(1) = (0.02 to 376) microM). Moreover we determined the free energy of drug binding from water to the activating binding region of the transporter (DeltaG degrees (tw(1)) = (-30 to -54) kJ/mol), the free energy of drug partitioning into the lipid membrane (DeltaG degrees (lw) = (-23 to -34) kJ/mol), and, as the difference of the two, the free energy of drug binding from the lipid membrane to the activating binding region of the transporter (DeltaG degrees (tl(1)) = (-7 to -27) kJ/mol). For the compounds tested DeltaG degrees (tl(1)) was less negative than DeltaG degrees (lw) but varied more strongly. The free energies of substrate binding to the transporter within the lipid phase, DeltaG degrees (tl(1)), are consistent with a modular binding concept, where the energetically most efficient binding module comprises two hydrogen bond acceptor groups.     

Interaction of a mitochondrial presequence with lipid membranes: role of helix formation for membrane binding and perturbation

The binding of a peptide to a biological membrane is often accompanied by a transition from a random coil structure to an amphipathic alpha-helix. Recently, we have presented a new approach which allows the determination of the thermodynamic parameters of membrane-induced helix formation [Wieprecht et al. (1999) J. Mol Biol. 294, 785]. It involves a systematic variation of the helix content of a given peptide by double D-substitution and a correlation of the binding parameters with the helicity. Here we have used this method to study membrane-induced helix formation for the presequence of rat mitochondrial rhodanese (RHD). The thermodynamic parameters of binding of the peptide RHD and of four of its double D-isomers were determined for 30 nm (SUVs) and 100 nm (LUVs) unilamellar vesicles composed of phosphatidylcholine/phosphatidylglycerol (3:1) using circular dichroism spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry. The incremental changes of the thermodynamic parameters of helix formation were found to be very similar for SUVs and LUVs. Membrane-induced helix formation of RHD entailed a negative enthalpy of Delta H(helix) = -0.5 to -0.6 kcal/mol/residue and was opposed by an entropy of about Delta S(helix) = -1 to -1.4 cal/mol K/residue. The free energy of helix formation, Delta G(helix), was about -0.2 kcal/mol, and helix formation accounted for 50-60% of the total free energy of membrane binding. Dye-release experiments were used to assess the role of helix formation for the membrane perturbation potential of the peptides. While helix formation plays a major role for membrane binding, it appears to have little importance for inducing membrane leakiness.     

The receptor binding of 3H-corticosterone by rat hepatocytes]

The binding of 3H-corticosterone was studied on rat hepatocytes both in presence of unlabeled corticosterone, obsidan and their absence at 0 degrees-4 degrees C. The analysis of binding by the method of Scatchard showed that there are two types of specific binding sites for 3H-corticosterone. Possible existence of proper glucocorticoid receptors (Ka = 4 x 10(9)M-1, n = 0.52 x 10(-14) mol/mg prot.) has been shown, as well as possibility of 3H-corticosterone interaction with beta-adrenoreceptors (Ka = 1.2 x 10(9)M-1, n = 0.9 x 10(-14) mol/mg prot.) have been demonstrated on hepatocytes.     

Determination of the activation volume of PLCbeta by Gbeta gamma-subunits through the use of high hydrostatic pressure

Activation of phospholipase Cbeta (PLCbeta) by G-proteins results in increased intracellular Ca(2+) and activation of protein kinase C. We have previously found that activated PLCbeta-Gbetagamma complex can be rapidly deactivated by Galpha(GDP) subunits without dissociation, which led to the suggestion that Galpha(GDP) binds to PLCbeta-Gbeta gamma and perturbs the activating interaction without significantly affecting the PLCbeta-Gbeta gamma binding energy. Here, we have used high pressure fluorescence spectroscopy to determine the volume change associated with this interaction. Since PLCbeta and G-protein subunits associate on membrane surfaces, we worked under conditions where the membrane surface properties are not expected to change. We also determined the pressure range in which the proteins remain membrane bound: PLCbeta binding was stable throughout the 1-2000 bars range, Gbeta gamma binding was stable only at high membrane concentrations, whereas Galpha(s)(GDP) dissociated from membranes above 1 kbar. High pressure dissociated PLCbeta-Gbeta gamma with a DeltaV = 34 +/- 5 ml/mol. This same volume change is obtained for a peptide derived from Gbeta which also activates PLCbeta. In the presence of Galpha(s)(GDP), the volume change associated with PLCbeta-Gbeta gamma interaction is reduced to 25 +/- 1 ml/mol. These results suggest that activation of PLCbeta by Gbeta gamma is conferred by a small (i.e., 3-15 ml/mol) volume element.     

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.     

An ESR study of the effect of an electrostatic field on binding of divalent cations to the surface of serum low-density lipoproteins

The ESR technique has been used to study binding of Mn(II) ions to low-density lipoprotein (LDL) in solutions of various electrolyte ionic strengths. A model of the binding has been proposed which describes all the observations in electrolytes of ten different concentrations in terms of two types of binding sites and two corresponding sets of intrinsic binding parameters (n1 = 8, Kd1 = 1.31 X 10(-3) mol X l-1 and n2 = 170, Kd2 = 5.71 X 10(-2) mol X l-1). These parameters, together with the values of the potential (phi 0) responsible for binding of the ions to specific charged sites on the surface, reproduce the observed binding curves well in all the systems studied. The phi 0 values are obtained as an appropriate solution of the Poisson-Boltzmann equation.     

Calcium translocation and storage of isolated intact cattle rod outer segments in darkness.

Bovine rod outer segments (rods), isolated with an intact plasma membrane and a stable calcium exchange and storage capacity, contain 2-3 mol endogenous calcium/mol rhodopsin. By means of 45Ca accumulation experiments and concomitant 40Ca analysis, the calcium metabolism of these organelles has been studied with the following results: 1. The majority of endogenous calcium is localized within disks. 2. In the presence of the ionophore A23187 the intradiskal binding sites can be titrated with external calcium. 3. The Scatchard plot of calcium binding of rods indicates the presence of a single set of intradiskal binding sites with a maximal capacity of 8-9 mol calcium/mol rhodopsin and an affinity constant of 55 microM to calcium. 4. Without A23187 more than 99% of the rod calcium appears in a bound state in equilibrium with a free calcium concentration of 15-25 microM. 5. External calcium exchanges with endogenous calcium in a fast (t 1/2 = 12 s) process with a uniform rate constant, whereas net calcium transport is very slow (t 1/2 greater than 2 h). 6. Intact rods contain a calcium translocation system, presumably located in the plasma membrane, which performs Ca-Ca exchange with a high unidirectional flux of 2 . 10(6) calcium ions/rod per s. 7. This translocation system can be saturated by external calcium (Km = 0.5 -1 microM) and has a low Q10 (1.08). Both the calcium translocation system and the calcium binding system appear to depend on the structural integrity of the stacked disks and are very sensitive to the experimental conditions. The relevance of these findings is discussed in relation to the proposed role of calcium ions as the intracellular transmitter in vertebrate rod photoreceptor cells.     

Properties of red cell membrane proteins: mechanism of spectrin and band 4.1 interaction

Interactions between human red cell's band 4.1 and spectrin were studied by fluorescence resonance energy transfer and batch microcalorimetry techniques. The association constant (Ka = 8.6 X 10(7) M-1), the stoichiometry (one molecule of band 4.1 to one molecule of spectrin), the reversibility, and the enthalpy (delta H = -6 kcal/mol) were determined. A proton uptake was observed to take place as a result of the spectrin-band 4.1 complex formation. In addition to the protonation of the reaction products, the entropic contribution (-T delta S) has been observed to be responsible for approximately 50% of the binding free energy. We concluded that the environment plays a significant role in the stabilization of the complex. Since band 4.1 has been required for the maintenance of the cytoskeletal stability, small alterations of the binding energies or the degree of interaction could have a pronounced effect on the structure of the erythrocyte membrane.     

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues.     

Temperature- and concentration-dependence of urea permeability of human erythrocytes determined by NMR

The temperature- and concentration-dependence of [13C]urea self-exchange across the human red cell membrane has been determined by NMR measurements of T1 (spin-lattice) relaxation times. T1 for intracellular label is 17 s, which is much longer than the urea exchange time across the cell membrane (about 0.5 s). T1 for urea in extracellular solution is quenched with 17 mM of impermeable Mn2+ in less than 2 ms. Hence the observed T1 (corrected for intracellular decay) is a measure of urea exchange across the cell membrane. The method is tested by showing both PCMBS and increasing concentrations of urea lengthen T1. Urea exchange permeability, defined as Purea = flux/conc, can be described by Purea = Vmax/(K1/2 + conc). Studies of temperature-dependence showed that activation energies were strongly dependent on both temperature and concentration. However, this apparently anomalous behavior was resolved into two well-behaved functions, K1/2 and Vmax, with linear Arrhenius plots and apparent 'activation energies' of 15.5 and 12.4 kcal/mol, respectively. These were used to construct an equation for calculating Purea at any concentration and temperature. Assuming a simple channel model with single binding, K1/2 becomes the dissociation equilibrium constant for the site with delta H degree = 15.5 kcal/mol and delta S degree = 51.8 cal/(mol.deg); dissociation is entropically driven.     

Binding of nucleotides to purified coupling factor-latent ATPase from Mycobacterium phlei.

Binding studies of various nucleotides to the purified coupling factor-latent ATPase from Mycobacterium phlei have been carried out using gel filtration, equilibrium dialysis, and ultrafiltration methods. The purified latent ATPase binds 3 mol of ADP per mol of the enzyme with an apparent dissociation constant of 68 muM. Binding of nucleotides occurred in the decreasing order: ADP, epsilon-ATP, epsilon-ADP, UDP, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), IDP, and adenosine 5'-(alpha,beta-methylene)diphosphate (AdoP(CH2)P). AMP-P(NH)P inhibits both soluble (Ki = 77 muM) and membrane-bound latent ATPase activity. However, AMP-P(NH)P does not affect oxidative phosphorylation in membrane vesicles of M. phlei. AMP-P(NH)P exhibits one binding site per molecule of the enzyme with a dissociation constant of 71 muM. After trypsin treatment of the enzyme, the binding of ADP decreases 35%, while AMP-P(NH)P binding remains unchanged. Moreover, AMP-P(NH)P binding was not displaced by ADP. Studies with sulfhydryl agents showed that, in contrast to AMP-P(NH)P, binding of at least 1 mol of ADP requires the participation of sulfhydryl groups. The results indicate that AMP-P(NH)P and ADP do not share a common binding site and that the latent ATPase enzyme has separate sites for ATP hydrolysis and ATP synthesis.     

Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

Fucoidan-dependent conformational changes in annexin II tetramer

Fucoidan, a sulfated fucopolysaccharide, mimics the fucosylated glycans of glycoproteins and has therefore been used as a probe for investigating the role of membrane polysaccharides in cell-cell adhesion. In the present report we have characterized the interaction of fucoidan with the Ca(2+)- and phospholipid-binding protein annexin II tetramer (AIIt). AIIt bound to fucoidan with an apparent K(d) of 1.24 +/- 0.69 nM (mean +/- SD, n = 3) with a stoichiometry of 0.010 +/- 0.001 mol of fucoidan/mol of AIIt (mean +/- SD, n = 3). The binding of fucoidan to AIIt was Ca(2+)-independent. Furthermore, in the presence but not the absence of Ca(2+), the binding of fucoidan to AIIt caused a decrease in the alpha-helical content from 32% to 7%. A peptide corresponding to a region of the p36 subunit of AIIt, F(306)-S(313), which contains a Cardin-Weintraub consensus sequence for heparin binding, was shown to undergo a conformational change upon fucoidan binding. This suggests that heparin and fucoidan bound to this region of AIIt. The binding of fucoidan but not heparin by AIIt also inhibited the ability of AIIt to bind to and aggregate phospholipid liposomes. These results suggest that the binding of AIIt to the carbohydrate conjugates of certain membrane glycoproteins may have profound effects on the structure and biological activity of AIIt.     

Isolation and characterization of a folate receptor from human placenta

While folate binding proteins have been described in serum and a variety of tissues, the function of these proteins is unknown. A particulate folate binding protein from human placenta has been isolated and characterized following solubilization with Triton X-100. The protein was purified 61,000-fold using affinity chromatography on pteroylglutamic acid-Sepharose as the major purification step. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein gave a single band with a Mr = 38,500. Stoichiometry of binding indicated that 1 mol of folate was bound per mol of protein. The protein was a glycoprotein that contained 12% carbohydrate. Antiserum was raised in a rabbit, and on immunodiffusion, gave a single precipitin line with the purified placental folate binding protein. Immunoprecipitation studies using this antiserum indicated that the purified placental folate binding protein shared antigenic determinants with both the large Mr and small Mr folate binding proteins from human milk. Immunofluorescent studies with this antiserum and human erythrocytes revealed the presence of an immunologically similar protein on the plasma membrane of these cells suggesting that this protein may function as a folate receptor.     

Glucose transporter oligomeric structure determines transporter function. Reversible redox-dependent interconversions of tetrameric and dimeric GLUT1.

This study investigates the relationship between human erythrocyte glucose transport protein (GLUT1) oligomeric structure and glucose transporter function. Oligomeric structure was analyzed by hydrodynamic studies of cholate-solubilized GLUT1, by chemical cross-linking studies of membrane-resident GLUT1 and by using conformation-specific antibodies. Transporter function (substrate binding) was analyzed by equilibrium cytochalasin B and D-glucose binding measurements. Erythrocyte-resident glucose transporter is a GLUT1 homotetramer, binds 1 mol of cytochalasin B/2 mol of GLUT1, and presents at least two binding sites to D-glucose. Native structure and function appear to be stabilized by intramolecular disulfide bonds and are preserved during GLUT1 purification by the omission of reductant. Native structure is independent of in vitro and in vivo membrane GLUT1 density but is transformed to dimeric GLUT1 by alkaline reduction. Dimeric GLUT1 binds 1 mol of cytochalasin B/mol of GLUT1, presents a single population of binding sites to D-glucose, and is obtained upon GLUT1 purification in the presence of reductant. Native structure and function are restored by treatment of dimeric GLUT1 with glutathione-disulfide (K0.5 glutathione disulfide = 29 microM). We propose that native structure is established prior to transporter translocation to the plasma membrane and that intrasubunit disulfide bonds promote cooperative subunit interactions that stabilize transporter structure and function.     

p-(Chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes

The binding of [203Hg]-p-(chloromercuri)benzenesulfonate to the membrane proteins of human erythrocytes and erythrocyte ghosts was examined under conditions where binding to the bulk of membrane sulfhydryl groups was blocked by N-ethylmaleimide. Binding was essentially complete within 90 min when approximately 40 nmol was bound per milligram of membrane protein. This binding was correlated with the inhibition of water transport measured by an NMR technique. Maximal inhibition was observed with the binding of approximately 10 nmol of p-(chloromercuri)benzenesulfonate/mg of membrane protein. Under these conditions, both band 3 and band 4.5 bound 1 mol of inhibitor/mol of protein. In contrast to previous experiments, these results indicate that band 4.5 proteins as well as band 3 have to be considered as playing a role in water transport.     

Thermodynamics of fusion peptide-membrane interactions

The fusion peptides of viral membrane fusion proteins play a key role in the mechanism of viral spike glycoprotein mediated membrane fusion. These peptides insert into the lipid bilayers of cellular target membranes where they adopt mostly helical secondary structures. To better understand how membranes may be converted to high-energy intermediates during fusion, it is of interest to know how much energy, enthalpy and entropy, is provided by the insertion of fusion peptides into lipid bilayers. Here, we describe a detailed thermodynamic analysis of the binding of analogues of the influenza hemagglutinin fusion peptide of different lengths and amino acid compositions. In small unilamellar vesicles, the interaction of these peptides with lipid bilayers is driven by enthalpy (-16.5 kcal/mol) and opposed by entropy (-30 cal mol(-1) K(-1)). Most of the driving force (deltaG = -7.6 kcal/mol) comes from the enthalpy of peptide insertion deep into the lipid bilayer. Enthalpic gains and entropic losses of peptide folding in the lipid bilayer cancel to a large extent and account for only about 40% of the total binding free energy. The major folding event occurs in the N-terminal segment of the fusion peptide. The C-terminal segment mainly serves to drive the N-terminus deep into the membrane. The fusion-defective mutations G1S, which causes hemifusion, and particularly G1V, which blocks fusion, have major structural and thermodynamic consequences on the insertion of fusion peptides into lipid bilayers. The magnitudes of the enthalpies and entropies of binding of these mutant peptides are reduced, their helix contents are reduced, but their energies of self-association at the membrane surface are increased compared to the wild-type fusion peptide.     

A recognition system for sex-hormone-binding protein-estradiol complex in human decidual endometrium plasma membranes

We have studied the specific binding of human serum sex-hormone-binding protein and its complexes with estradiol and testosterone to an enriched membrane fraction isolated from human decidual endometrium. The specific binding of sex-hormone-binding protein to these membranes has been found to be dependent on it being bound with estradiol. When estradiol concentration in the medium is high enough to provide for the saturation of sex-hormone-binding protein with the steroid, the protein exhibits specific binding to the membranes with an apparent equilibrium constant, Kd = (3.5 +/- 2.0) X 10(-12) M. In the absence of estradiol, the binding protein devoid of steroid or complexed with testosterone does not interact with the membranes. At low concentrations of sex-hormone-binding protein and estradiol, when their complex is almost completely dissociated in solution, it can be reconstituted on the membranes with the optimum estradiol to binding protein ratio being 1:1 (mol/mol). It is proposed that, in plasma membranes of the estradiol target cells, there is a recognition system for the sex-hormone-binding protein-estradiol complex which may allow these cells to take up from blood not only free estradiol, but also estradiol complexed with the binding protein.