共查询到20条相似文献,搜索用时 15 毫秒
1.
B Nordén 《Biophysical chemistry》1976,6(1):31-45
Linear dichroism (LD) results when pseudoisocyanine=PIC (1,1-diethy1-2,2-cyannine iodide) binds to flow-oriented DNA. LD may be used to follow the complexation both stoichmetrically and structurally, since when specified to unti complex concentration LD provides a measure of the average orientation of the absorbing transition dipole. Two different types of complexes can be distinguished: I. One strong, ionic-strength insensitive complex with monomeric PIC with an orientation indication intercalation. II. Several weaker complexes of electrostatic nature (only observed at I less than 0.2M Na Cl) among which those with dimeric (IIa) and with polymeric (IIb) PIC are concluded both from LD and from circular dichroism (CD). The dimer is probably formed by employing one intercalated PIC as a second site. The polymer complex is characterized by a very sharp absorption band at 553 nm polarized parallel to the DNA-axis (with positive LD and positive CL). The structure, a right-handed helical array of PIC molecules, is discussed in terms of exciton theory in relation to that of polymeric free PIC ("Scheibe polymer") which is also shown to interact with DNA (IIc) yielding a large aggregate which is degraded at a distinct flow force field... 相似文献
2.
DNA tris-intercalation: first acridine trimer with DNA affinity in the range of DNA regulatory proteins. Kinetic studies 总被引:4,自引:0,他引:4
A trimer made up of three acridine chromophores linked by a positively charged poly(aminoalkyl) chain was synthesized as a potential tris-intercalating agent. The length of the linking chain was selected to allow intercalation of each chromophore according to the excluded site model. 1H NMR studies have shown that, at 5 mM sodium, pH 5, the acridine trimer occurred under a folded conformation stabilized by stacking interactions between the three aromatic rings. DNA tris-intercalation of the dye at a low dye/base pair ratio was shown by measurements of both the unwinding of PM2 DNA and the lengthening of sonicated rodlike DNA. The trimer exhibits a high DNA affinity for poly[d(A-T)] (Kapp = 8 X 10(8) M-1, 1 M sodium) as shown by competition experiments with ethidium dimer. Kinetic studies of both the association with poly[d(A-T)] and the exchange between poly[d(A-T)] and sonicated calf thymus DNA have been performed as a function of the ionic strength. In 0.3 M sodium the on-rate constant (k1 = 2.6 X 10(7) M-1 s-1) is similar to that reported for other monoacridines or bis(acridines), whereas the off-rate constant is much smaller (k-1 = 1.2 X 10(-4) s-1), leading to an equilibrium binding constant as large as Kapp = 2.2 X 10(11) M-1. A plot of log (k1/k-1) as a function of log [Na+] yielded a straight line whose slope shows that 5.7 ion pairs (out of 7 potential) are formed upon the interaction with DNA. From this linear relationship a Kapp value of 10(14) M-1 in 0.1 M sodium can be estimated. Such a value reaches and even goes beyond that of some DNA regulatory proteins. This acridine trimer appears to be the first synthetic ligand with such a high DNA affinity. 相似文献
3.
The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect lo optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K1 ca 105 M?1; site density per nucleotide base n1 ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50° to the local DNA helix axis. The complex with fuchsin is weaker (104M?1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C2v symmetry) XXXre angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. Tne optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler's hydrol blue, a perpendicular orientation was observed, possibly due to intercaiation. 相似文献
4.
E Buszman T Wilczok B Witman G Siebert 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1977,358(7):819-824
Colchicine (7.5 X 10-6M or 3.3 X 10-5M) was incubated at pH 7.0, 10.0, or 12.0 with high-molecular DNA from salmon sperm (7 X 10-5M or 3.5 X 10-5M DNA-P) at 40 degrees C. Interaction was monitored by UV spectrophotometry; Amax/Amin ratios, difference and additive spectra, and the quantity of colchicine bound to DNA were presented as functions of time, ionic strength and DNA concentration. Using NMR techniques, delta deltav 1/2/deltac values, chemical shifts and half-line widths of colchicine protons were analyzed as a function of the DNA/colchicine ratio, thus proving the interaction between alkaloid and DNA. An intercalation of the tropolone moiety of colchicine between the nitrogenous bases of DNA is suggested. 相似文献
5.
Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers. 总被引:5,自引:4,他引:1
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Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered. 相似文献
6.
Bengt Norden 《Biophysical chemistry》1978,8(4):385-391
Linear dichroism (LD) gives useful information on the interaction between DNA and the directly acting carcinogen N-acetoxy-N-2-acetylaminofluorene (AAAF). In 50% methanol solvent with low ionic strength only a weak complex (van der Waals) appears. However, above 40° C strand separation takes place and a covalent aminofluorene complex forms. After renaturation a characteristic positive LD.band is observed at 306 nm. The average angular orientation of the longaxis of the fluorene moiety (47° to the local helix axis) is inconsistent with intercalation- It can be explained for instance by a free rotation around a C(DNA)-N (aminofluorene) bond or by a major groove site. The occupation density was 1–2 aminofluorene residues per 100 bases. With native DNA, AAAF slowly forms a covalent complex which has a negative LD at 307 nm. The orientation (70–90° ) is consistent with steric direction by the strand. 相似文献
7.
C Bailly N Helbecque J P Hénichart P Colson C Houssier K E Rao R G Shea J W Lown 《Journal of molecular recognition : JMR》1990,3(1):26-35
The binding to DNA of a mixed function ligand (NETGA) is described, in which a potential intercalating group, an acridine moiety, is incorporated at the carboxyl terminus of the minor groove binding oligopeptide netropsin skeleton. Scatchard analysis of absorption data provided evidence of two modes of binding to DNA with K1 = 9.1 x 10(5) M-1 at low r values (0.003-0.1), and a binding site size n = 10, indicative of binding of both moeities. At high binding ratios (greater than 0.1), K2 = 0.9 x 10(5) M-1 and n = 5 corresponding to external binding. Complementary strand MPE footprinting on a pBR322 restriction fragment showed NETGA binds to 5'-AAAT like netropsin. It causes enhanced cleavage by MPE, particularly at G-C rich sequences and remote from the preferred binding sites. Viscometry measurements provided evidence for biphasic modes of the two binding portions of NETGA. Fluorescence polarization and linear dichroism measurements were in accord with distinct modes of interaction of the acridine (intercalation) and oligopeptide (minor groove binding) portions of NETGA. LD measurements on NETGA indicate that the oligopeptide moiety (netropsin-like) has an orientation typical of minor groove binders, whereas the degree of intercalation of the acridine group is decreased by association of the oligopeptide moiety. 相似文献
8.
We have studied the structure of nuclease-solubilized chromatin from Ehrlich ascites cells by flow linear dichroism (LD) using the anisotropic absorption of the DNA bases and of two intercalated dyes, ethidium bromide and methylene blue. It is confirmed that intercalation occurs preferentially in the linker part of the chromatin fiber, at binding ratios (dye/base) below 0.020. Using this information, we determined the orientation of the linker in relation to the average DNA organization in chromatin. The LD measurements indicate that the conformation of chromatin is considerably changed in the ionic strength interval 0.1-10 mM NaCl: with increasing salt concentration, the LD of the intrinsic DNA base absorption changes signs, from negative to positive, at approximately 2.5 mM NaCl. The LD of the intercalated dyes also changes signs, however, at a somewhat higher salt concentration. The results are analyzed in terms of possible allowed combinations of tilt angles of nucleosomes and pitch or tilt angles of linker DNA sections relative to the fiber axis, at different salt concentrations in the interval 0.1-10 mM NaCl. Two models for the salt-induced structural change of chromatin are discussed. 相似文献
9.
F Watanabe 《Nucleic acids research》1986,14(8):3573-3585
The cooperative binding of histone H1 with DNA was studied using a fluorescently labelled histone H1. The titration data were analysed in terms of the large ligand model. The stoichiometric number, n = 65 +/- 10 bases/H1, was independent of NaCl concentration (0.02 - 0.35 M). The nucleation and the cooperative binding constants, K' and K, and the cooperativity parameter q were sensitive to salt concentration; K = 3.6 +/- 0.8 X 10(7) M-1 and q = 1.1 +/- 0.4 X 10(3) at 0.2 M NaCl. The dependence of K' on NaCl concentration revealed that 6 Na+ ions were released from DNA upon complex formation. An extrapolation of K' to 1M NaCl yielded a small value, K' = 5 +/- 2 M-1. Thus the binding of H1 is essentially electrostatic, being compatible with its independence of temperature. A calculation of K' based on the counterion release reproduced the salt concentration dependence of K'. Therefore, the binding of H1 is of an electrostatic territorial type. Thus, H1 may move along the DNA chain to a certain extent, when both salt concentration and the degree of saturation are sufficiently low. The condition is so restricted that the sliding would not play an important role in vivo. It was concluded from the DNA concentration independent binding isotherm that H1 can cooperatively bind onto a single DNA molecule. A simple power law dependence of the cooperativity parameter q upon NaCl concentration was found; q oc[NaCl]h with h = 0.72, though the physical basis of this dependence remains unknown. 相似文献
10.
The binding of Ru(phen)(2)dppz(2+) (dppz=dipyrido[3,2-a:2',3'-c]phenazine) to DNA was investigated at pH 7.0 and 25 degrees C using stopped-flow and spectrophotometric methods. Equilibrium measurements show that two modes of binding, whose characteristics depend on the polymer to dye ratio (C(P)/C(D)), are operative. The binding mode occurring for values of C(P)/C(D) higher than 3 exhibits positive cooperativity, which is confirmed by kinetic experiments. The reaction parameters are K=2 x 10(3)M(-1), omega=550, n=1, k(r)=(1.9+/-0.5) x 10(7)M(-1)s(-1) and k(d)=(9.5+/-2.5)x10(3)s(-1) at I=0.012 M. The results are discussed in terms of prevailing surface interaction with DNA grooves accompanied by partial intercalation of the dppz residue. The other binding mode becomes operative for C(P)/C(D)<3 and the equilibria analysis shows this is an ordinary intercalation mode (K=1.3 x 10(6) M(-1), n=1.5 at I=0.012 M and K=2 x 10(5) M(-1), n=1.2 at I=0.21 M). Similar behaviour is displayed by double-stranded poly(A). 相似文献
11.
Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have examined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5 X 10(-8) M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1 X 10(-7) M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5 X 10(-9) M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K'd1 = (0.82 +/- 0.36) X 10(-10) M and K'd2 = (3.2 +/- 0.60) X 10(-8) M. The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1 X 10(-8) M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5 X 10(-8) M T3 showed a K'd1 = (0.15 +/- 0.10) X 10(-10) M and K'd2 = (3.5 +/- 0.10) X 10(-8) M. We conclude that T3 inhibits the action of aldosterone on sodium transport at a site after receptor binding in the nucleus. 相似文献
12.
The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities 总被引:9,自引:0,他引:9
W M Mackin C K Huang E L Becker 《Journal of immunology (Baltimore, Md. : 1950)》1982,129(4):1608-1611
F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity. 相似文献
13.
N E Geacintov V Ibanez A G Gagliano S A Jacobs R G Harvey 《Journal of biomolecular structure & dynamics》1984,1(6):1473-1484
The conformation of adducts derived from the reactions and covalent binding of the (+) and (-) enantiomers of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) with double-stranded calf thymus DNA in vitro were investigated utilizing the electric linear dichroism technique. The linear dichroism and absorption spectra of the covalent DNA complexes are interpreted in terms of a superposition of two types of binding sites. One of these conformations (site I) is a complex in which the plane of the pyrene residue is close to parallel (within 30 degrees) to the planes of the DNA bases (quasi-intercalation), while the other (site II) is an external binding site; this latter type of adduct is attributed to the covalent binding of anti-BaPDE to the exocyclic amino group of deoxyguanine (N2-dG), while site I adducts are attributed to the O6-deoxyguanine and N6-deoxyadenine adducts identified in the product analysis of P. Brookes and M.R. Osborne (Carcinogenesis (1982) 3, 1223-1226). Site II adducts are dominant (approximately 90% in the covalent complexes derived from the (+) enantiomer), but account for only 50 +/- 5% of the adducts in the case of the (-)-enantiomer. The orientation of site II complexes is different by 20 +/- 10 degrees in the adducts derived from the binding of the (+) and the (-) enantiomers to DNA, the long axis of the pyrene chromophore being oriented more parallel to the axis of the DNA helix in the case of the (+) enantiomer. These findings support the proposals by Brookes and Osborne that the difference in spatial orientation of the N2-dG adducts of (-)-anti-BaPDE together with their lower abundance may account for the lower biological activity of the (-) enantiomer. The external site II adducts, rather than site I adducts, appear to be correlated with the biological activity of these compounds. 相似文献
14.
Synthesis, crystal structure and DNA-binding studies of the Ln(III) complex with 6-hydroxychromone-3-carbaldehyde benzoyl hydrazone 总被引:2,自引:0,他引:2
A novel 6-hydroxy chromone-3-carbaldehyde benzoyl hydrazone ligand (L) and its Ln(III) complexes, [Ln=La(1) and Sm(2)], have been prepared and characterized. The crystal and molecular structures of complexes 1 and 2 were determined by single-crystal X-ray diffraction. Antioxidative activity tests in vitro showed that L and its complexes have significant antioxidative activity against hydroxyl free radicals from the Fenton reaction and also oxygen free radicals, and that the effect of the La(III) complex 1 is stronger than that of mannitol and the other compounds. The compounds were tested against tumor cell lines including HL-60 and A-549. The data shows that the suppression rate of complexes 1 and 2 against the tested tumor cells are superior to the free ligand (L). The interactions of complexes 1 and 2, and L, with calf thymus DNA were investigated by UV-visible (UV-vis), fluorescence, denaturation experiments and viscosity measurements. Experimental results indicated that complexes 1 and 2, and L can bind to DNA via the intercalation mode, and that the binding affinity of complex 1 is higher than that of complex 2 and of free ligand (L). The intrinsic binding constants of complexes 1 and 2, and L were (7.62+/-0.56)x10(6), (3.70+/-0.47)x10(6) and (2.41+/-0.46)x10(6)M(-1), respectively. 相似文献
15.
Phosphorus-31 nuclear magnetic resonance of highly oriented DNA fibers. 2. Molecular motions in hydrated DNA 总被引:3,自引:0,他引:3
31P nuclear magnetic resonances (NMR) of salmon sperm DNA, poly(rA).poly(rU), and poly(rA).poly(dT) fibers were measured as a function of relative humidity. The results indicated that the spectra were strongly perturbed by the molecular motions occurring in the hydrated fibers. The humidity dependence of the spectra at a number of orientations of the fibers relative to the magnetic field was reasonably explained by taking into account at least three motional modes, namely, conformational fluctuations, restricted rotation about a tilted axis, and rotational diffusion about the helical axis. The rotational diffusion about the helical axis was found to perturb the spectral line shapes most strongly, and its constants were 1.5 X 10(4) and 5.0 X 10(4) S-1 for DNA fibers at 92% and 98% relative humidities, respectively. A DNA-RNA hybrid, poly(rA).poly(dT), has been shown to adopt different conformations on two strands at high relative humidity [Zimmerman, S. B., & Pheiffer, B. H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 78-82], which was unquestionably confirmed in the present study: that is, the 31P NMR spectra from the hydrated form of this polymer were clearly explained by assuming that one strand had an A-like conformation and the other a B-like conformation. 相似文献
16.
A method for the determination of estrogen and progesterone receptor levels in human mammary cell lines (MCF-7, Cama-1, ZR-75-1, Evsa-T and HBL-100) is described. Cells cultured as monolayers were incubated with the tritiated steroids, [3H]-17 beta-Estradiol or [3H] ORG-2058. Binding of steroids to receptors was a function of cellular uptake. Incubation periods of 50 min were sufficient to attain maximum intracellular incorporation. The binding of 17 beta-E2 and ORG-2058 to MCF-7 cells, a phenomenon which is saturable at low concentrations for the radioactive ligand, is a linear function of the number of cells assayed (Interval: 2.5 X 10(4) to 1.5 X 10(6) cells per well). Binding data and their Scatchard plot allowed for the calculation of affinity and capacity values. Thus, for ER, Kd = 2.0 +/- 0.5 X 10(-10) M and n = 3.76 +/- 0.91 Fmol/microgram DNA, and for PgR Kd = 2.0 +/- 0.2 X 10(-10) M and n = 14.02 +/- 2.30 Fmol/microgram DNA (Mean +/- SD). Binding specificity of 17 beta-Estradiol and ORG-2058 to MCF-7 cells was analysed by means of study on the inhibitory effect of increasing concentrations of unlabelled competitors: 17 beta-Estradiol, ORG-2058, Estrone, DES, R-5020, Cortisol, Androsterone and Testosterone. Only pharmacological doses of some of the mentioned molecules produce displacement of the hormonereceptor binding. This phenomenon appears to be related to the affinity of these chemical compounds for the receptor macromolecules to which estrogens and progesterone bind. 相似文献
17.
Norepinephrine-stimulated vascular prostacyclin synthesis. Receptor-dependent calcium channels control prostaglandin synthesis 总被引:1,自引:0,他引:1
D Stewart E Pountney D Fitchett 《Canadian journal of physiology and pharmacology》1984,62(11):1341-1347
Norepinephrine-stimulated prostacyclin synthesis was studied in rat aortic rings by measuring 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Norepinephrine (10(-6) M) results in a 10- to 20-fold increase in 6-keto-PGF1 alpha synthesis by rat aortic rings (54 +/- 11 to 437 +/- 260 pg X mg wet weight-1 X 20 min-1). The maximal stimulation of 6-keto-PGF1 alpha synthesis was observed with a norepinephrine concentration of 10(-5) M at a mean effective concentration (EC50) of 9.5 +/- 3.2 X 10(-7) M which is similar to the contractile response (Emax = 10(-5) M, EC50 = 6.5 +/- 1.8 X 10(-7) M). Potassium chloride (30 mM), although causing a similar maximal contractile response as 10(-6) M norepinephrine, did not increase 6-keto-PGF1 alpha synthesis. Norepinephrine-stimulated 6-keto-PGF1 alpha synthesis was dependent upon extracellular calcium. Norepinephrine stimulation in Ca2+-free medium did not lead to a significant increase in 6-keto-PGF1 alpha synthesis. However, on the introduction of Ca2+, 6-keto-PGF1 alpha synthesis was restored to its initial level. Phentolamine (10(-6) M) (an alpha-adrenergic antagonist) and trifluroperazine (2.5 X 10(-4) M) (a calmodulin inhibitor) completely inhibited norepinephrine-stimulated 6-keto-PGF1 alpha synthesis, whereas verapamil 3 X 10(-6) M (a calcium channel blocking drug) only partially inhibited synthesis (control, 74 +/- 12; norepinephrine, 437 +/- 260; norepinephrine + verapamil, 123 +/- 8 pg X mg wet weight-1 X 20 min-1).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor 总被引:8,自引:0,他引:8
We have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block alpha-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit alpha-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds alpha-BuTx with a forward rate constant of 0.98 X 10(5) M-1 s-1, d-tubocurarine (dTC) with a KD of (6.8 +/- 0.3) X 10(-8) M, and pancuronium with a KD of (1.9 +/- 1.0) X 10(-9) M. The site recognized by B mAbs binds alpha-BuTx with a forward rate constant of 9.3 X 10(5) M-1 s-1, dTC with a KD of (4.6 +/- 0.3) X 10(-6) M, and pancuronium with a KD of (9.3 +/- 0.8) X 10(-6) M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by alpha-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site. 相似文献
19.
Eric R. Taylor Kenneth J. Miller Anthony J. Bleyer 《Journal of biomolecular structure & dynamics》2013,31(4):883-904
Abstract A theoretical model is proposed for the covalent binding of (+) 7 β,8α-dihydroxy-9α, 10α- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene denoted by BPDE I(+), to N2 on guanine. The DNA must kink a minimum of 39° to allow proper hybrid configurations about the C10 and N2 atoms involved in bond formation and to allow stacking of the pyrene moiety with the non-bonded adjacent base pair. Conservative (same sugar puckers and glycosidic angles as in B-DNA) and non-conservative (alternating sugar puckers as in intercalation sites) conformations are found and they are proposed structures in pathways connecting B-DNA, an intercalation site, and a kink site in the formation of a covalently intercalative bound adduct of BPDE I(+) to N2 on guanine. Stereographic projections are presented for (3′) and (5′) binding in the DNA. Experimental data for bending of DNA by BPDE, orientation of BPDE in DNA and unwinding of superhelical DNA is explained. The structure of a covalent intercalative complex is predicted to result from the reaction. Also, an anti ? syn transition of guanine results in a structure which allows the DNA to resume its overall B-form. The only change is that guanine has been rotated by 200° about its glycosidic bond so that the BPDE I(+) is bound in the major groove. The latter step may allow the DNA to be stored with an adduct which may produce an error in the genetic code. 相似文献
20.
The dissociation constant for GDP binding to the E site of tubulin isolated by chromatography on Sepharose 6B is equal to 6.1 X 10(-8) M, as determined by the Hummel-Dryer procedure. This is smaller than any previously reported value, and the discrepancy with earlir results is analyzed.By use of a recently described column centrifugation procedure [Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899], it was established that GDP and GTP bind to the same site. GTP is bound 2.8-fold tighter than GDP, and the dissociation constant is 2.2 X 10(-8) M. A new method for the determination of dissociation constants for a protein-bound ligand, based on a quantitative analysis of the loss of ligand during exclusion chromatography, is presented. This has been used to determine that the dissociation constant for GDP bound to tubulin is equal to 5.5 X 10(-8) M, in excellent agreement with that determined independently from the Hummel-Dryer method. A previous theoretical treatment [Dixon, H. B. F. (1976) Biochem. J. 159, 161-162] of ligand loss during exclusion chromatography is discussed. 相似文献