Specific binding sites for d-α-tocopherol on human erythrocytes

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 K_a = 2.6·10⁷ M^?1), low capacity (7600 sites/cell) and the second with low affinity (K_a = 1.24·10⁶ M^?1), high capacity (150000 sites/cell). These sites are at least partly protein in nature.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

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 K_d = 10^?7 or 2 · 10^?7, respectively. The fluorescence emission (λ_max 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.     

Uptake and binding of β-ecdysone in imaginal discs of Drosophila melanogaster

The kinetics of uptake and retention of β-ecdysone by imaginal discs from late third instar larvae of Drosophila melanogaster correspond well with those of the first synthetic response of discs to hormone, an increase in RNA synthesis.Competition studies indicate the presence of two types of hormone binding sites, specific and non-specific. The specific sites are saturated at hormone concentrations which fully induce morphogenesis. Results are consistent with the hypothesis that analogs which induce morphogenesis at differing concentrations bind to the same sites. Experiments with the inhibitors N-ethylmaleimide, actinomycin d, and cycloheximide suggest that the binding sites are pre-existing in the cell and require functional sulfhydryl groups for binding.Specific binding, binding that is competed by excess unlabeled β-ecdysone, is saturable (70–80 nM). Kinetic rate constants for this specific binding were estimated to be k_a = 1.5 × 10⁵M^?1 min^?1, k_d = 3 × 10^?2 min^?1. The equilibrium dissociation constant calculated from the kinetic rate constants was K_eq = 2 × 10^?7M compared to 1.7 × 10^?7M β-ecdysone required to induce morphogenesis in vitro and 2.5 × 10^?7M determined to be the in vivo concentration at the time of induction of morphogenesis.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

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 K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, 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.     

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa₃ and to the (porphyrin) cytochrome c-cytochromeaa₃ complex.2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2 · 10⁸ M^?1 · s^?1 and about 10⁷ M^?1 · s^?1 (I = 8.8 mM, pH 7.0, 10° C), respectively.3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa₃ reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa₃. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa₃ has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa₃ has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.     

Characterization of lactoferrin binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin technique

• 1.1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line.
• 2.2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable.
• 3.3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete.
• 4.4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (K_a) of 2.36 × 10⁷ and 3.36 × 10⁶M⁻¹.
• 5.5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

     

High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

The binding of high specific activity, radioactive Concanavalin A to cultured normal human fibroblasts was investigated. We report the presence of two classes of Concanavalin A binding sites on the plasma membranes of these cells. These classes of binding sites are distinguished by their affinities for the lectin. Scatchard analysis of the binding data indicates the presence of a class of high affinity sites which are saturated at about 0.25 μg/ml of Concanavalin A. The other, lower affinity binding sites are not saturated until 50–100 μg/ml Concanavalin A levels are achieved. At 4°C the K_a for the high affinity sites varies between 1.5 – 5 × 10⁹ M^?1 depending on the method used to label the Concanavalin A. For the lower affinity sites K_a varies between 1 – 4 × 10⁶ M^?1. The average number of high affinity sites per cell is 8 × 10⁵ representing less than 1% of the total receptor sites for the lectin.     

Vicia graminea lectin binding to human M and N erythrocytes

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 $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ 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.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

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.     

The stimulation of coupling factor 1 ATPase by tentoxin

Tentoxin at 10–1000 μM causes a marked species-selective stimulation of coupling factor 1 Ca²⁺-dependent ATPase activity (K_a 6.3 · 10³ M^?1). This effect decreases the K_m for ATP to about 0.3 mM and increases V 2.75-fold. Above 1.6 μM tentoxin the rate of coupled electron transport was reduced to basal without uncoupling.     

Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.     

Follitropin receptors in rat testis characterization with enzymatically 125I-labeled human follitropin

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The ¹²⁵I-labeled human follitropin exhibited high binding activity, with specific binding of up to 17% in the presence of an excess of testis homogenate.Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (K_a) determined at 24°C were not significantly different in immature and adult rat testis, and the mean value for K_a was 3.9 · 10⁹ M^?1. At 37°C, the K_a value obtained using immature rat testis was 1.3 · 10¹⁰ M^?1. The association of ¹²⁵I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30–60% of the preformed hormone · receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnant mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Properties of human growth hormone binding sites solubilized from female rabbit kidney

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, K_d, 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.     

Hydrophobic interactions are the driving force for the binding of peptide mimotopes and <Emphasis Type="Italic">Staphylococcal </Emphasis>protein A to recombinant human IgG1

We studied the interaction of several nona-peptide mimotopes of different sequence and Staphylococcal protein A (SpA) with a recombinant human IgG1 antibody using isothermal titration calorimetry (ITC). The amino acid primary structure of the peptides was varied in order to identify the specific antibody-peptide binding sites. Additionally, the influence of temperature and salt concentration was investigated. An attempt was made to elucidate the structural changes upon complex formation using the determined thermodynamic parameters. The amino acid composition of the mimotopes determined their binding affinity. The binding constant K _a of the mimotopes was in the range 1 × 10⁴ to 1 × 10⁶ M⁻¹. The binding constant of SpA was on the average about three orders of magnitude higher than that of the peptides. The binding constant of the peptides and of SpA decreased with temperature and the binding process was connected with negative changes in enthalpy, entropy, and heat capacity. The binding of the mimotopes to the Fab part of the IgG1 antibody and binding of SpA to the Fc part of the IgG1 antibody were mainly driven by hydrophobic effects and associated with a relatively large change in water-accessible surface area. Determinants for a strong/reduced antibody-peptide binding were identified.     

Porcine IgG: structure,genetics, and evolution

Eleven genomic porcine Cγ gene sequences are described that represent six putative subclasses that appear to have originated by gene duplication and exon shuffle. The genes previously described as encoding porcine IgG1 and IgG3 were shown to be the IgG1^a and IgG1^b allelic variants of the IGHG1 gene, IgG2a and IgG2b are allelic variants of the IGHG2 gene, while “new” IgG3 is monomorphic, has an extended hinge, is structurally unique, and appears to encode the most evolutionarily conserved porcine IgG. IgG5^b differs most from its putative allele, and its C_H1 domain shares sequence homology with the C_H1 of IgG3. Four animals were identified that lacked either IgG4 or IgG6. Alternative splice variants were also recovered, some lacking the C_H1 domain and potentially encoding heavy chain only antibodies. Potentially, swine can transcribe >20 different Cγ chains. A comparison of mammalian Cγ gene sequences revealed that IgG diversified into subclasses after speciation. Thus, the effector functions for the IgG subclasses of each species should not be extrapolated from “same name subclasses” in other species. Sequence analysis identified motifs likely to interact with Fcγ receptors, FcRn, protein A, protein G, and C1q. These revealed IgG3 to be most likely to activate complement and bind FcγRs. All except IgG5^a and IgG6^a should bind to FcγRs, while all except IgG6^a and the putative IgG5 subclass proteins should bind well to porcine FcRn, protein A, and protein G. An erratum to this article can be found at     

The oxidation of ferrocytochrome c by Br−2, (SCN)−2, N3 and OH radicals studied by pulsed-electron and γ-ray radiolysis

The reactions of ferrocytochrome c with Br^?₂, (SCN)^?₂, N₃ and OH radicals were followed by measuring the change in the optical spectra of cytochrome c on γ-irradiation as well as the rate of change of absorbance upon pulse irradiation.Ferrocytochrome c is oxidized to ferricytochrome c by Br^?₂, (SCN)^?₂ or N₃ radical with an efficiency of about 100% through a second-order process in which no intermediates were observed. The rate constants in neutral solutions at I = 0.073 are 9.7 · 10⁸ M^?1 · s^?1, 7.9 · 10⁸ M^?1 · s^?1, 1.3 · 10⁹ M^?1 · s^?1 for the oxidation by Br^?₂, (SCN)^?₂ and N₃ radicals, respectively. The rate constants do not vary appreciably in alkaline solutions (pH 8.9). The ionic strength dependence was observed for the rate constants of the oxidation by Br^?₂ and (SCN)^?₂. Those rate constants estimated on the assumption that the radicals react only with the amino acid residues with the characteristic steric correction factors were less than one-tenth of the observed ones. These results suggest that the partially exposed region of the heme is the probable site of electron transfer from ferrocytochrome c to the radical.Hydroxyl radicals also oxidize ferrocytochrome c with a high rate constant (k > 1 · 10¹⁰ M^?1 · s^?1), but with a very small efficiency (5%).     

Synthesis,characterization, DNA interaction,and cytotoxicity of novel Pd(II) and Pt(II) complexes

Four complexes [Pd(L)(bipy)Cl]·4H₂O (1), [Pd(L)(phen)Cl]·4H₂O (2), [Pt(L)(bipy)Cl]·4H₂O (3), and [Pt(L)(phen)Cl]·4H₂O (4), where L = quinolinic acid, bipy = 2,2’-bipyridyl, and phen = 1,10-phenanthroline, have been synthesized and characterized using IR, ¹H NMR, elemental analysis, and single-crystal X-ray diffractometry. The binding of the complexes to FS-DNA was investigated by electronic absorption titration and fluorescence spectroscopy. The results indicate that the complexes bind to FS-DNA in an intercalative mode and the intrinsic binding constants K of the title complexes with FS-DNA are about 3.5?×?10⁴ M^?1, 3.9?×?10⁴ M^?1, 6.1?×?10⁴ M^?1, and 1.4?×?10⁵ M^?1, respectively. Also, the four complexes bind to DNA with different binding affinities, in descending order: complex 4, complex 3, complex 2, complex 1. Gel electrophoresis assay demonstrated the ability of the Pt(II) complexes to cleave pBR322 plasmid DNA.     

Low- and high-affinity concanavalin A binding to thymocyte plasma membrane vesicles

Binding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (～6 × 10⁶/cell equivalent) low-affinity sites (K_a = 1.3 × 10⁵ M^?1) and (b) fewer (～0.4 × 10⁶/cell equipment) specific receptors (K_a = 6.8 × 10⁶ M^?1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively-cooperative, with a Hill coefficient of～1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively-cooperative sites by high-affinity sites. It is proposed that concanavalin A-binding induces multimerization of the 55,000 D glycoprotein.     

Binding of meso-tetra(4-sulfonatophenyl)porphine to haemopexin and albumin studied by spectroscopy methods

• 1.1. The interaction of haemopexin and albumin with TPPS₄ was studied by measuring the absorption and fluorescence spectra. Haemopexin was found to have one strong TPPS₄ binding center (K_a = 3 × 10⁷M⁻¹).
• 2.2. Haem-haemopexin complex appears to have no specific binding site for TPPS₄. Occupation of the specific binding center of haemopexin molecule by a haem abolishes TPPS₄ binding.
• 3.3. Albumin was found to possess one strong TPPS₄ binding center (K_a = 3 × 10⁶M⁻¹) besides two or three weak binding sites (K_a = 2 × 10⁵M⁻¹).
• 4.4. Haern-albumin complex possesses only one weak TPPS₄ binding site (K_a = 7 × lO⁵M⁻¹). These observations suggest identity of primary binding sites of TPPS₄ and haem on albumin molecule.

     

α-Bungarotoxin binding properties of a central nervous system nicotinic acetylcholine receptor

High-affinity, specific binding of radiolabeled α-bungarotoxin to particulate fractions derived from rat brain shows saturability (B_max ≈ 37fmol/mg, K_D^app = 1.7 nM) and insensitivity to ionic strength, and is essentially irreversible (K_on = 5 · 10⁶ min^?1 · mol^?1; K_displacement = 1.9 · 10^?4 min^?1, τ_1/2 = 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.