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1.
Jan Holm Steen Ingemann Hansen Jørgen Lyngbye 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,629(3):539-545
Binding of [3H]folate to proteins in serum of pregnant women was studied in equilibrium dialysis experiments (pH 7.4, 37°C). A Scatchard analysis revealed the presence of high-affinity (Kass = 1010M?1, N = 0.4 nM folate) and low-affinity sites. The high-affinity folate binding protein (Mr ≈ 30 000–35 000) appeared in front effluent after application of serum to a DEAE-Sepharose CL-6B column equilibrated with 0.05 M imidazole buffer (pH 6.3)/ 30 mM NaCl. Low-affinity binding protein eluted from the column after a rise in NaCl concentration to 1 M was mainly similar to albumin. A minor part was, however, associated with a large molecular size (Mr > 200 000) protein, probably α2-macroglobulin.High-affinity binding which displayed positive cooperatively was saturated at folate concentrations above 10?10 M. Folate dissociation was a complex process consisting of an initial rapid phase (terminated within 48 h) followed by a slow release. At pH 3.5 dissociation became rapid and complete. Purified methotrexate had no effect on high-affinity binding, whereas N10-methylfolate (an impurity in the methotrexate preparation) acted as a potent inhibitor. Low-affinity binding was proportional to the folate concentration within the range 10?10–10?7 M. Dissociation of folate was rapid. 相似文献
2.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,886(1):76-87
The presence of three distinct populations of folate binding sites on the surface of Dictyostelium discoideum cells has been reported recently. The A sites (consisting of two subtypes) recognize folic acid, 2-deaminofolic acid and methotrexate with equal affinity and appear to be linked to cAMP synthesis. B and C sites (the latter is composed of two interconvertable subtypes; rapidly equilibrating, CF, and more slowly equilibrating, CS) preferentially bind N10-methylfolic acid and folic acid and may both be involved in the chemotactic response (De Wit, R.J.W. and Van Haastert, P.J.M. (1985) Biochim. Biophys. Acta 814, 199–213; De Wit, R.J.W., Bulgakov, R., Pinas, J.E. and Konijn, T.M. (1985) Biochim. Biophys. Acta 814, 214–226). The present study gives two lines of evidence that the B and C sites are interconvertable. (i) Occupancy of the B-sites by N10-methylfolic acid proceeds at a rate identical to that of the association of this ligand to CS at all concentrations tested. This suggests that association to CS and formation of occupied B sites share a common pathway. (ii) After preincubation with ligand, removal of free ligand results in the reappearance of unoccupied CF sites with kinetics identical to the disappearance of occupied B sites. Furthermore, the existence of a third type of C site, which is formed out of CS and may be converted to B, is proposed. This is based on competition studies using folate analogs with a different affinity for CS and B. 相似文献
3.
Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei 总被引:2,自引:2,他引:0
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Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (Kd) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. Kd measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca2+, Mg2+, and ethylenediammonium2+ ions). Monovalent cations (e.g., Na+, K+, and Tris+) were also effective, but only at concentrations in the millimolar range. The Kd for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl2. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism. 相似文献
4.
R A Greiner D Jacobs-Krahnen R Mutzel D Malchow B Wurster 《The Journal of biological chemistry》1992,267(8):5096-5101
A 138-kDa glycoprotein comprising folate deaminase activity was purified to apparent homogeneity from membranes of Dictyostelium discoideum. Deaminase activity could be effectively inhibited by p-chloromercuriphenylsulfonate. This treatment protected folate from deamination and thus allowed investigation of folate binding to deaminase fractions. Two types of folate binding sites, differing in affinity and specificity, were detected on the folate deaminase glycoprotein. One type displays high affinity and binds folate stronger than N10-methylfolate. This binding site appears to be identical with the catalytic site of folate deaminase. The other type of binding site shows lower affinity but prefers N10-methylfolate relative to folate. A similar preference for N10-methylfolate was observed in chemotaxis tests pointing to the possibility that the second type of binding site is involved in chemotactic perception of folate compounds. Folate perception and deamination could thus be performed by activities residing on the same polypeptide. 相似文献
5.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,886(1):88-95
The ligand binding properties of folate chemotactic receptors on isolated membranes of Dictyostelium discoideum were analyzed. Three out of the four receptor states (BF, BS and BSS) were detected, showing rate constants and Kd values similar to those obtained for intact cells. Guanine nucleotides changed the proportioning of the receptor states as well as the rates of several conversions. (i) The transformation of BF into BS was inhibited by GDP but not by guanylyl imidodiphosphate (GuaPP[NH]P) or GTP. (ii) The number of BS sites was lowered by GTP and GuaPP[NH]P. (iii) The binding to BSS was lowered by GTP and GDP, but increased by GuaPP[NH]P. (iv) The rate of disappearance of BSS was increased by GTP, but not by GuaPP[NH]P. Effects of guanine nucleotides were not observed after treatment of the membrane preparations with 15 mg/ml bovine serum albumin. This treatment caused the detection of a binding type different from the types described previously. The affinity of this binding site was extremely high (Kd ≤ 0.2 nM for N10-methylfolic acid), while the dissociation was relatively slow (k−1 ≤ 3·10−4 s−1). It is proposed that bovine serum albumin uncouples the folate receptor from a guanine nucleotide regulatory (G) protein in an irreversible manner. A model is presented in which the four receptor states correspond to distinct interactions with a G protein and GDP or GTP. 相似文献
6.
Zvetanka D. Zhivkova Veska N. Russeva 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,714(2):1173
A chiral stationary phase based on immobilized human serum albumin (HSA) was used to study the stereoselective binding of ketoprofen enantiomers by means of high-performance liquid affinity chromatography. The technique of zonal elution was applied together with a novel mathematical approach describing attachment to more than one type of binding site. Phenylbutazon (PBZ) and diazepam (DAZ) were used as markers for the major believed binding regions on HSA. Both R- and S-ketoprofen (KTR and KTS) display high affinity to the primary PBZ- and DAZ-binding sites and low-affinity to the secondary DAZ sites. The binding to high-affinity regions is accepted to be a stepwise process initiated by the binding to the primary DAZ sites and followed by the attachment to the primary PBZ sites. The chiral recognition is attributed to the high-affinity PBZ-binding sites and to the low-affinity DAZ-binding sites. 相似文献
7.
Characterization of Multiple [3 H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue 总被引:2,自引:0,他引:2
Abstract: High-affinity [3H]5-hydroxytryptamine ([3H]5-HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [3H]5-HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [3H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high-affinity binding appears to be to multiple sites, since displacement studies using 2 nM [3H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (nH): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [3H]5-HT, since unlabeled 5-HT did not discriminate between them. Thus, the high-affinity [3H]5-HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5-HTIA, 5-HTIB). In addition to the multiple high-affinity spinal cord binding sites, a low-affinity [3H]5-HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [3H]5-HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high-and low-affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [3H]5-HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [3H]5-HT in the spinal cord is associated with the 5-HT uptake carrier, since fluoxetine, an inhibitor of 5-HT uptake, does not alter binding characteristics. In addition, a 5-HT binding site analogous to the site designated 5-HT, was not apparent in the spinal cord. Ketanse-rin and cyproheptadine, drugs that are highly selective for 5-HT, sites, did not displace [3H]5-HT from spinal tissue, and [3H]spiperone, a radioligand that binds with high affinity to 5-HT2 sites, did not exhibit saturable binding in the tissue. Thus, the 5-HT2 binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord. 相似文献
8.
E. K. Nénonéné F. Radja M. Carli L. Grondin T. A. Reader 《Journal of neurochemistry》1994,62(5):1822-1834
Abstract: The selective serotonin (5-HT) agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5-HT1A receptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [3H]8- OH-DPAT (free ligand from 200 pM to 160 nM) revealed high-affinity 5-HT1A receptors (KH= 0.7–0.8 nM) and lowaffinity (KL= 22–36 nM) binding sites. The kinetics of [3H]8-OH-DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high- and low-affinity binding sites. When the high-affinity sites were labeled with a 1 nM concentration of [3H]8- OH-DPAT, the competition curves of agonist and antagonist drugs were best fit to a two-site model, indicating the presence of two different 5-HT1A binding sites or, alternatively, two affinity states, tentatively designated as 5-HT1AHIGH and 5-HT1ALOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5-HT1A sites are modulated by 5′-guanylylimidodiphosphate, inhibition experiments with 5-HT were performed in the presence or in the absence of 100 μM 5′-guanylylimidodiphosphate. The binding of 1 nM [3H]8-OH-DPAT to the 5-HT1AHIGH site was dramatically (80%) reduced by 5′-guanylylimidodiphosphate; in contrast, the low-affinity site, or 5-HT1ALOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high-affinity 5-HT1AHIGH site corresponds to the classic 5-HT1A receptor, whereas the novel 5-HT1ALOW binding site, labeled by 1 nM [3H]8-OH-DPAT and having a micromolar affinity for 5-HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5-HT1A sites and the 5-HT innervation, rats were treated with p-chlorophenylalanine or with the neurotoxin p-chloroamphetamine. The inhibition of 5-HT synthesis by p-chlorophenylalanine did not alter either of the two 5-HT1A sites, but deafferentation by p-chloroamphetamine caused a loss of the low-affinity [3H]8-OH- DPAT binding sites, indicating-that these novel binding sites may be located presynaptically on 5-HT fibers and/or nerve terminals. 相似文献
9.
Inhibition of HCO(3) Binding to Photosystem II by Atrazine at a Low-Affinity Herbicide Binding Site 总被引:1,自引:1,他引:0
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In maize chloroplasts, the ratio of HCO3− (anion) binding sites to high-affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO3− to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO3− and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO3− binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO3− with a dissociation constant near 80 micromolar. The location of the HCO3− binding site, and the low-affinity atrazine binding site, is not known. 相似文献
10.
Gary B. Henderson Barbara Grzelakowska-Sztabert Edward M. Zevely F.M. Huennekens 《Archives of biochemistry and biophysics》1980,202(1):144-149
A binding component with a high affinity for 5-methyltetrahydrofolate (KD = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 104 sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the KD values for 5-methyltetrahydrofolate and methotrexate binding and the Kt values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate. 相似文献
11.
Anna Faragó Péter Hasznos Ferenc Antoni Tibor Romhányi 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,538(3):493-504
Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site.The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP.Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [3H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (Kd about 44 · 10?8 M belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (Kd 2–5 · 10?6 M) was demonstrated by the inhibitory effect of 10?5 M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site. 相似文献
12.
At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco (Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known.Abbreviations BA
N
6-benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- IAA
indole-3-acetic acid
- Kd
equilibrium dissociation constant
- Rt
total concentration of binding sites
In partial fulfillment of the requirements for the Ph.D. degree in the Department of Botany and Plant Pathology, Michigan State University 相似文献
13.
Keita Kamikubo Shigeki Sakata Shigenori Nakamura Takashi Komaki Kiyoshi Miura 《Journal of Protein Chemistry》1990,9(4):461-465
125I-thyroxine (125I-T4) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied.125I-T4 binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19±0.53×10–6 M and 2.69±0.78×10–5 M, respectively. The number of binding sites of the high and the low affinity sites were 1.28±0.46 mol/mol and 23.5±9.7 mol/mol of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of125I-T4 to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-1-naphthalene sulfonic acid (ANS) inhibited the high affinity T4 binding via reduction of the binding capacity. Unlabeled T4 showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity T4 binding to HSA-Sepharose. 相似文献
14.
The sensitivity of acetylcholine receptor to eleven cholinergic drugs, phospholipase A, heat and pH provided evidence that the so-called high-affinity binding (Kd for acetylcholine 11 nm in 1% Triton) and low-affinity binding (Kd 562 nm) were related to two distinct binding sites. The low-affinity binding site was less sensitive to heat and several of the cholinergic drugs, but was a little more sensitive to bungarotoxin than the high-affinity site. Zinc (0.4 mm) and EDTA (10 mm) abolished acetylcholine binding to both sites; the EDTA inhibition was time-dependent. 相似文献
15.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1983,732(3):607-619
(1) The Mg2+-induced low-affinity nucleotide binding by (Na+ + K+)-ATPase has been further investigated. Both heat treatment (50–65°C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the Kd value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na+ + K+)-ATPase activity. (2) Thermodynamic parameters (ΔH° and ΔS°) for the apparent affinity in the ATPase reaction (Km ATP) and for the true affinity in the binding of AdoPP[NH]P (Kd and Ki) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the Km value, which thus is smaller than the Kd value. (3) Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the Kd value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the Kd value. (4) The nucleotide specificity of low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P > ATP > ADP > AMP. (5) The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the Kd value is not increased more than by ouabain alone. (6) It is inferred that the low-affinity site is Iocated on the enzyme, more specifically its α-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed. 相似文献
16.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,930(1):87-96
Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including β-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in α1-adrenergic receptors induced by agonists. α1-receptors were studied on DDT1 MF-2 smooth muscle cells (DDT1-MF-2 cells) by specific [3H]prazosin binding. In competition binding on membranes and on intact cells at 4°C or at 37°C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37°C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [3H]prazosin binding inhibited by 20 μM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [3H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4°C, but only 30% at 37°C. On DDT1-MF-2 cells preequilibrated with [3H]prazosin at 4°C, and then shifted to 37°C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4°C it is the native form of α1-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37°C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 μM norepinephrine. The nature of this low-affinity form was further investigated. On DDT1-MF-2 cells preincubated with the agonist and then extensively washed at 4°C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [3H]prazosin at 4°C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4°C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37°C may be due to the agonist-induced sequestration of α1-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands. 相似文献
17.
Gary B. Henderson Judy M. Tsuji Harish P. Kumar 《The Journal of membrane biology》1988,101(1):247-258
Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K
D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K
D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K
i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K
i=45nm) and methotrexate (K
i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells. 相似文献
18.
Naftalin RJ 《Biophysical journal》2008,95(9):4300-4314
Alternating access transporters with high-affinity externally facing sites and low-affinity internal sites relate substrate transit directly to the unliganded asymmetric “carrier” (Ci) distribution. When both bathing solutions contain equimolar concentrations of ligand, zero net flow of the substrate-carrier complex requires a higher proportion of unliganded low-affinity inside sites () and slower unliganded “free” carrier transit from inside to outside than in the reverse direction. However, asymmetric rates of unliganded carrier movement, kij, imply that an energy source, ΔGcarrier = RT ln (koi/kio) = RT ln (Cin/Cout) = RT ln (), where R is the universal gas constant (8.314 Joules/M/K°), and T is the temperature, assumed here to be 300 K°, sustains the asymmetry. Without this invalid assumption, the constraints of carrier path cyclicity, combined with asymmetric ligand affinities and equimolarity at equilibrium, are irreconcilable, and any passive asymmetric uniporter or cotransporter model system, e.g., Na-glucose cotransporters, espousing this fundamental error is untenable. With glucose transport via GLUT1, the higher maximal rate and Km of net ligand exit compared to net ligand entry is only properly simulated if ligand transit occurs by serial dissociation-association reactions between external high-affinity and internal low-affinity immobile sites. Faster intersite transit rates occur from lower-affinity sites than from higher-affinity sites and require no other energy source to maintain equilibrium. Similar constraints must apply to cotransport. 相似文献
19.
Abstract The pharmacological and biochemical characteristics of [3H]desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one [3H]desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The [3H]desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 μM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of ~1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromo-benzylamine (DSP4). In contrast, when 10 μM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with KD values of 0.5 and >100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (Bmax) for binding defined by 0.1 μM nisoxetine varied between brain regions, with very low density in the striatum (Bmax not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of [3H]noradrenaline uptake but not with 5-[3H]hydroxytryptamine uptake. The low-affinity sites did not correlate with the regional distribution of [3H]noradrenaline uptake. Drug inhibition studies showed that noradrenaline inhibits the binding defined by 0.1 μM nisoxetine in a competitive manner. Together, these findings suggest that only a small fraction of the [3H]desipramine binding can be regarded as “specific” binding, and this binding fraction may represent the substrate recognition site for noradrenaline uptake. Assuming that one molecule of desipramine binds to each carrier molecule, the turnover number for the noradrenaline carrier was calculated to be 20/min, i.e., the duration of one transport cycle was 3 s. 相似文献
20.
Bradford O. Fanger Dace Viceps-Madore John A. Cidlowski 《Archives of biochemistry and biophysics》1984,235(1):141-149
It is reported that receptors for epidermal growth factor (EGF) in HeLa S3 cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors (), which contains approximately 6 × 104 binding sites per cell, and a second, high-affinity class of receptors () containing approximately 5 × 103 binding sites per cell. Exposure of HeLa S3 cells to 10?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10?11 to 10?6m. EGF binding is half-maximal near 2–4 × 10?9m, a concentration equal to the Kd of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S3 cells are exposed to 10?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc. 相似文献