共查询到20条相似文献,搜索用时 15 毫秒
1.
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
2.
Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a Km and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (Ki) of approx. 5·10?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (Kd) of approx. 1·10-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·106 molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a Kd of 4–6·10?7 M, represents approx. 60% of the total saturable binding (14·106 molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a Kd of 2–6 · 10?8 M, represents less than 10% of the total sites (2 · 106 molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved. 相似文献
3.
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
4.
Leonor P. Roguin Juan S. Bonifacino Alejandro C. Paladini 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,715(2):222-229
Human growth hormone binding sites from female rabbit kidney microsomes were solubilized by treatment with the nonionic detergent Triton X-100. The binding of 125I-labelled human growth hormone to the solubilized sites retains many of the properties observed in the particulate fraction, such as saturability, reversibility, high affinity and structural specificity. The association and the dissociation process are time- and temperature-dependent. The association rate constant, k1, is 1.6·107 mol?1·l·min?1 at 25°C, and the dissociation rate constant, k?1, is 2.8·10?4 min?1 at 25°C. Solubilization causes an increase in affinity as well as in binding capacity. Scatchard plots from saturation curves suggest the presence of a single class of binding site with a dissociation equilibrium constant, Kd, of 1.3·10?11 M and a binding capacity of 133 fmol/mg of protein. Similar results were obtained from competition experiments. Specificity studies revealed the lactogenic characteristics of the solubilized sites. The Stokes radii of the free binding sites and of the 125I-labelled human growth hormone-binding site complex, determined on a Sepharose CL-6B column, are 57 and 53 Å, respectively. 相似文献
5.
Ishayahu Friedman Herzl Schwalb Haifa Hallaq Arie Pinson Michael Heller 《生物化学与生物物理学报:生物膜》1980,598(2):272-284
Electron microscopic and biochemical studies revealed a salient difference in the response to toxic doses of ouabain by cultured cardiac muscle and non-muscle cells from neonatal rats. Progressive cellular injury in myocytes incubated with 1 · 10?4–1 · 10?3 M ouabain ultimately leads to swelling and necrosis. The morphological damage in myocytes was accompanied by a drastic decrease in 14CO2 formation from 14C-labeled stearate or acetate but not glucose. Neither morphological nor biochemical impairments were observed in non-muscle cells. The interaction between ouabain and the cultured cells, using therapeutic doses of ouabain (i.e., <1 · 10?7 M), was characterized. Two binding sites were described in both classes of cells, one site is a saturable K+-sensitive site whereas the other is non-saturable and K+-insensitive. The complexes formed between the sarcolemma receptor(s) and ouabain, at low concentrations of the drug (e.g., 7.52 · 10?9 M), had Kd values of 8.9 · 10?8 and 2.3 · 10?8 M for muscle and non-muscle cells, respectively. The formation and dissociation of the complexes were affected by temperature and potassium ions. 相似文献
6.
The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 104 to 12.8 · 104 M?. s?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant Kap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression Kap = KaK(Ka + [H+])?1. The values of Ka, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10?4 M and 1 · 10?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C. 相似文献
7.
Since vitamin E deficiency is associated with increased susceptibility of erythrocytes to hemolysis, we investigated the presence of tocopherol binding sites in human red blood cells. Erythrocytes were found to have specific binding sites for d-α-[3H]tocopherol with properties of receptors. Kinetic studies of binding demonstrated two binding sites: one with high affinity (equilibrium association constant Ka = 2.6·107 M?1), low capacity (7600 sites/cell) and the second with low affinity (Ka = 1.24·106 M?1), high capacity (150000 sites/cell). These sites are at least partly protein in nature. 相似文献
8.
Ronald J. Lukasiewicz Edward L. Bennett 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,544(2):294-308
High-affinity, specific binding of radiolabeled α-bungarotoxin to particulate fractions derived from rat brain shows saturability (Bmax ≈ 37fmol/mg, KDapp = 1.7 nM) and insensitivity to ionic strength, and is essentially irreversible (Kon = 5 · 106 min?1 · mol?1; Kdisplacement = 1.9 · 10?4 min?1, τ1/2 = 62 h). Subcellular distribution of specific sites is consistent with their location on synaptic junctional complex and post-synaptic membranes. These membrane-bound binding sites exhibit unique sensitivity to cholinergic ligands; pretreatment of membranes with cholinergic agonists (but not antagonists) induces transformation of α-bungarotoxin binding sites to a high affinity form toward agonist. The effect is most marked for the natural agonist, acetylcholine. These results strongly support the notion that the entity under study is an authentic nicotinic acetylcholine receptor. 相似文献
9.
David C. Pang 《生物化学与生物物理学报:生物膜》1980,598(3):528-542
Ca2+ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10?5 and 1.2 · 10?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca2+ binding were studied. All cations tested inhibited Ca2+ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na+ > K+ > Li+ ? Cs+ and for the divalent and trivalent ions: La3+ ? Mn2+ > Sr2+ ? Ba2+ > Mg2+. 1 · 10?3 M caffeine and 1 · 10?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10?7 M verapamil, acidosis (pH 6.4), 1?10?5 M Mn2+ and 1 · 10?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents. 相似文献
10.
Marie-José Prigent Patrice Boquet 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,717(3):445-452
The mode of binding of Vicia graminea125I-labelled lectin to human M and N erythrocytes at 4°C has been investigated. The labelled lectin retained the full activity of native lectin. Lectin association at 4°C was characterized by a of 3 to 5 min, reaching steady-state within 15 min. Incubation of cells for 15 min at 4°C with increasing concentrations of Vicia graminea125I-labelled lectin showed that saturation binding occurred. Scatchard analysis of equilibrium data determined over a wide range of lectin concentrations yielded a curvilinear plot with an upward concave slope; this representation indicated that there was not a single homogeneous class of noninteracting binding sites. This result could indicate two or more independent classes of binding sites or one class of interacting sites exhibiting negative cooperativity. Since unlabelled lectin, which at the concentration used, rapidly binds to available receptors, did not affect the dissociation rate of the labelled lectin and since identical Scatchard plots were found using native and formaldehyde-fixed erythrocytes we conclude that there are two classes of independent Vicia graminea binding sites on human erythrocytes. Computer analysis of the Scatchard plots gave high- and low-affinity constant (7.07±1.1) · 107 M?1 and (0.2±0.01) · 107 M?1, respectively, for N erythrocytes and (1.13±0.18) · 107 M?1 and (0.24±0.01) · 107 M?1, respectively for the M cells. N erythrocytes were estimated to have 0.085 · 105 high-affinity and 2.1 · 105 low-affinity sites and M erythrocytes, 0.011 · 105 high affinity and 0.13 · 105 low-affinity sites. N cells therefore have 10-times as many sites as M cells. Studies of the dissociation of 125I-labelled lectin from N and M cells in the presence of unlabelled lectin gave dissociation rate constants of 51 · 10?4 s?1 and 1.97 · 10?4 s?1 for the high- and low-affinity sites of N cells and 13 · 10?4 s?1 and 1.6 · 10?4 s?1 for the high- and low-affinitym sites of M cells, indicating that the binding of Vicia graminea lectin to human erythrocytes is reversible. 相似文献
11.
The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-3H]cytochalasin B and transport of into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a KD of 2.86 · 10?7 M, while the low-affinity site had a KD of 1.13 · 10?5M. Sugar transport was monitored by uptake and it was found that cytochalasin B (10?5M) drastically inhibited transport. However, D-glucose (10?5M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier. 相似文献
12.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1982,710(3):324-331
Mn(II) ions were used to study ion-binding properties of human low density lipoproteins (LDL). From the intensity of the EPR lines corresponding to the unbound Mn(II) ions the percentage of the ions bound to LDL is determined. By the titration of LDL with Mn(II) the binding parameters, dissociation constant, Kd, and the number of binding sites, n, could be derived. It has been found that there are at least two types of binding site on the LDL surface: ‘strong’ sites characterized by n = 6, Kd = 1.5 · 10−5M · l−1, and ‘weak’ sites characterized by n = 145 and Kd = 6.6 · 10−3M · l−1 for the sample in 0.01 M Tris-HCI buffer at 10°C. At very low Mn(II) concentrations binding to the ‘strong’ sites exhibits a cooperative behaviour. In the 0.1 M buffer the ‘strong’ sites are almost completely occupied or blocked by the monovalent buffer cations. The number of the ‘weak’ sites remains unaltered and Kd is decreased slightly (Kd = 4.9 · 10−3M · l−1). The location, chemical nature and the structural and functional relevance of the binding sites are discussed. 相似文献
13.
[3H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent Ka of 1.9 · 106 and 1.2 · 105 LM?. 1.4 · 10?4 M Norepinephrine covalently-linked to agarose beads reduced [3H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [3H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10?7-2 · 10?6 M. Dopamine and dopa were potent inhibitors of [3H]norepinephrine binding at 8.5 · 10?7 M and 2.0 · 10?6 M respectively, but did not stimulate lipolysis even at 10?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [3H]norepinephrine binding at 10?4 M but completely inhibited catecholamine-stimulated lipolysis at 10?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [3H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect. 相似文献
14.
The binding of 45Ca2+ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg2+ (no ATP) binding was half-maximal at 1.4 · 10?6M Ca2+ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 106 M?1) and the Hill coefficient was 1.0. In the absence of Mg2+, binding was half-maximal at 3 · 10?7 M Ca2+ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 107 and 2 · 106 M?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg2+ binding to about half of the total Ca2+ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites. 相似文献
15.
William McChesney Moore Robert N. Brady 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,444(1):252-260
Specific binding of 125I-labeled α-bungarotoxin to a 34 800 × g pellet of a whole rat brain homogenate has been obtained at levels 2 pmol toxin per g of whole brain with a Kd of 8·10?9 M. Binding is reduced 90% by 10?5 M (+)- tubocurarine chloride and 10?4 M nicotine, whereas concentrations of 10?4 M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with 125I-labeled α-bungarotoxin and soluble acetylcholine receptor protein preparations form Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character.Extraction of the 34 800 × g pellet with 1% Emulphogene yields a soluble fraction with specifically binds 125I-labeled α-bungarotoxin with a Kd of 5·10?9 M. Nicotine and α-bungarotoxin at concentrations of 10?5 M abolish toxin- receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35–40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10?5 M. 相似文献
16.
Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF1 were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000.In the presence of 1.25 mM MgCl2, 1 mol of ATP or FTP is bound to the latent enzyme, with Kd = 10?7 or 2 · 10?7, respectively. The fluorescence emission (λmax 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with K1 = 2.4 · 104 M?1 · s?1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with k?1 = 3 · 10?3 s?1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed.The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another.The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 106 M?1 · s?1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation. 相似文献
17.
《BBA》1986,849(1):121-130
The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF1 and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (KD(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The KD(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The KD(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF1 binds 2 mol 1-naphthoyl-ATP per mol enzyme with KD = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 104 M−1s−1. (6) The rate constant for 1-naphthoyl-ATP binding to TF1 is 6.6 · 103 M−1 · s−1 (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (kA−1 = 3 · 10−3s−1) and a slower second phase (kA−2 < 0.2 · 10−3s−1). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF1 are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex. 相似文献
18.
Ouabain binding was studied in isolated adult dog heart myocytes. The binding was correlated with the inhibition of K+-activated para-nitrophenylphosphatase (K+-PNPPase) activity and the beating response. It was shown that: (i) the specific binding was dependent upon Mg2+ and was inhibited by K+; (ii) the maximal binding capacity (Bmax) was 7.4 × 105 ouabain molecules per cell, or 410 pmol ouabain/K+-PNPPase unit (μmol/min); (iii) in the presence of Mg2+ (5 mm), there were two components in the Scatchard plot, i.e., a high-affinity component with a Kd value of 5.6 × 10?8m and a low-affinity component with a Kd value of 6.7 × 10?7m; (iv) the Hill coefficient (n′) for ouabain binding was 0.72 with a S0.5 value of 7.1 × 10?7m; these values were compatible with the values obtained from studies of K+-PNPPase inhibition by ouabain (n′ = 0.55, S0.5 = 3.6 × 10?7 m) and remained unchanged in the presence of physiological concentrations of Na+ plus K+; (v) in the presence of Mg2+ and K+, the high-affinity component tended to conform to the low-affinity component with an apparent decrease in Bmax; (vi) in the presence of Mg2+ and para-nitrophenylphosphate, the low-affinity component was changed to the high-affinity component with no change in Bmax; (vii) the dissociation rate of the labeled ouabain in the highly dilute medium was not altered in the presence of excess amounts of unlabeled ligand; this eliminated the possibility that the apparent negative cooperativity was due to a site-to-site interaction between receptors; (viii) ouabain increased the number of beating cells and the frequency of beating. Based on these findings, it is concluded that: (i) isolated myocytes possess functional receptors for ouabain; (ii) the binding of ouabain is associated with its inhibition of K+-PNPPase activity; (iii) ouabain receptors in isolated myocytes are of one class with at least two interconvertible conformational states. 相似文献
19.
Birendra Singh Anna M. Blom Can Unal Bo Nilson Matthias Mörgelin Kristian Riesbeck 《Molecular microbiology》2010,75(6):1426-1444
The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (KD) for vitronectin binding to UspA2 was 2.3 × 10?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a KD of 7.9 × 10?8 M. Electron microscopy verified that the active binding domain (UspA230–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA230–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance. 相似文献
20.
Masao Sasaki B.L. Larson D.R. Nelson 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,497(1):160-170
Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using 125I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (Ka) of about 5 · 108?10 · 108 M?1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding of IgG1 (Ka about 45 · 108 M?1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7:1 ratio of these immunoglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions. 相似文献