共查询到20条相似文献,搜索用时 31 毫秒
1.
Synthetic myosin filaments of rabbit were prepared. Electron microscopy showed that the number-average length (Ln = 470 nm) and sharpness in length distribution (Lw/Ln = 1.036) were independent of ionic strengths of 134, 74, and 44 mM, whereas the number ratio of M-filaments (about 15 nm in diameter at the bare zone) to m-filaments (about 10 nm) strongly depended on ionic strength (IS); the major filaments were M-filaments at IS = 134 mM, m-filaments at IS = 74 mM, and almost exclusively m-filaments at IS = 44 mM. Dynamic light scattering showed that the change in diameter with the change in ionic strength by 2-h dialysis was reversible. Combination of dynamic light scattering and sedimentation studies suggested a dynamic equilibrium between M- and m-filaments. Dynamic light-scattering spectra at IS = 134 and 74 mM could be analyzed by a theory for rigid rods, whereas those at IS = 44 mM only by introducing semiflexibility of filaments; m-filaments are more flexible at IS = 44 than at 74 mM. 相似文献
2.
Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite Km of 170 μM at very high ionic strength indicates a ratio of of about 5000. It is proposed that anions either modify the E10 of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form. 相似文献
3.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1988,952(1):122-130
Two methods for extracting calelectrin, a Ca2+-regulated membrane-binding protein from the electric organ ofTorpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7–8 mg · kg−1 wet weight of tissue, that is 4–5-times greater than the original method. The calelectrin so obtained could be resolved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% β-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32 000) than the H-form (34 000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of Mr 34 000 and 32 000. The addition of 2 mM Ca2+ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3–5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3 nm, corresponding to an apparent Mr of 44 000. It was unaffected by changes in ionic strength, pH or Ca2+ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent Mr of 36 000. Measurements of circular dichroism indicated that 78% of the molecule was in the α-helix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation. 相似文献
4.
5.
Direct measurement of the melting temperature of supported DNA by electrochemical method 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Meunier-Prest R Raveau S Finot E Legay G Cherkaoui-Malki M Latruffe N 《Nucleic acids research》2003,31(23):e150
The development of biosensors based on DNA hybridization requires a more precise knowledge of the thermodynamics of the hybridization at a solid interface. In particular, the selectivity of hybridization can be affected by a lot of parameters such as the single-strand (ss)DNA density, the pH, the ionic strength or the temperature. The melting temperature, Tm, is in part a function of the ionic strength and of the temperature and therefore provides a useful variable in the control of the selectivity and sensitivity of a DNA chip. The electrochemical technique has been used to determine the Tm values when the probe is tethered by a DNA self-assembled monolayer (SAM). We have built a special thin layer cell, which allows the recording of the cyclic voltammogram under controlled temperature conditions. Tm has been determined by recording the thermogram (current versus temperature) of a redox indicator on a double-stranded hybrid (dsDNA) modified electrode and comparison with the corresponding ssDNA response. Tm of supported DNA varies linearly with the ionic strength. The stability of the SAMs has been considered and comparison between Tm in solution and on a solid support has been discussed. 相似文献
6.
Per Foss 《Carbohydrate polymers》1987,7(6):421-433
The aim of the present study was to determine the influence of the ionic strength on the thermal stability of xanthan, i.e. xanthan resistance to chain breaking at high temperatures. Xanthan solutions of various ionic strengths were kept at 80, 90 and 95°C for periods up to 95 h. The thermal stability was determined by measuring the intrinsic viscosity after the heating periods. The experiments showed a critical ionic strength for the thermal stability of xanthan between 10 and 100 mm NaCl or KCl in this temperature range. Below the critical ionic strength the intrinsic viscosity was rapidly reduced, whereas above the critical ionic strength the intrinsic viscosity was virtually unaffected by heating.We then looked for a possible correlation between thermal stability and secondary structure of xanthan. The transition ionic strength (Im) of xanthan solutions, i.e. where xanthan is midway between an ordered and a disordered structure, was determined by NMR at constant temperatures. Im was found to be in the range of 24 mm at 80°C to 60 mm NaCl at 95°C, thus lying in the range of the critical ionic strength of the thermal stability. This suggests a close relationship between thermal stability and secondary structure of xanthan, indicated by the enhanced thermal stability in the ordered state. We believe this enhanced thermostability arises from a double-stranded conformation in the ordered state, as in DNA. The presence of double-stranded xanthan is also indicated by electron micrographs taken at both high and low ionic strengths.The transition temperature (Tm) of xanthan was determined by NMR and optical rotation measurements. At the ionic strength of 7·5 mm the two methods resulted in Tm values of 67 and 52°C respectively. This difference in Tm can possibly be due to the fact that the observed NMR and optical rotation (OR) effects are caused by different molecular phenomena. 相似文献
7.
Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside 总被引:5,自引:1,他引:4
The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)o depolarized the cell. The potential is related to the logarithm of (K)o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)o. Diminutions of (Na)o and (Cl)o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10-3 and 10-5
M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10-3
M K-strophanthoside was not observed when (K)o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)o of 30 mM, 10-2
M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process. 相似文献
8.
Keiko Yokoyama Brian S. Leigh Katsumi Niki Hiroyuki Ohno Harry B. Gray John H. Richards 《Inorganica chimica acta》2008,361(4):1095-1099
Robust voltammetric responses were obtained for wild-type and Y72F/H83Q/Q107H/Y108F azurins adsorbed on CH3(CH2)nSH:HO(CH2)mSH (n = m = 4, 6, 8, 11; n = 13, 15 m = 11) self-assembled-monolayer (SAM) gold electrodes in acidic solution (pH 4.6) at high ionic strengths. Electron-transfer (ET) rates do not vary substantially with ionic strength, suggesting that the SAM methyl headgroup binds to azurin by hydrophobic interactions. The voltammetric responses for both proteins at higher pH values (>4.6-11) also were strong. A binding model in which the SAM hydroxyl headgroup interacts with the Asn47 carboxamide accounts for the relatively strong coupling to the copper center that can be inferred from the ET rates. Of particular interest is the finding that rate constants for electron tunneling through n = 8, 13 SAMs are higher at pH 11 than those at pH 4.6, possibly owing to enhanced coupling of the SAM to Asn47 caused by deprotonation of nearby surface residues. 相似文献
9.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,721(1):127-134
A micellar electrokinetic capillary chromatography (MECC) method was developed for the separation of the 3-O-glucuronides of entacapone and its (Z)-isomer, the two main urinary metabolites of entacapone in humans. Entacapone is a novel, potent inhibitor of catechol-O-methyltransferase (COMT) intended for use as an adjunct in the treatment of Parkinson’s disease. Urine samples spiked with synthetic 3-O-glucuronides were used to study the effects of running buffer pH, composition and applied voltage on separation of the closely migrating glucuronides. The 3-O-glucuronide of nitecapone, was used as internal standard. The greatest improvement in separation was achieved by increasing the running buffer ionic concentration. Changes in pH had little effect on the separation, whereas increase in sodium dodecyl sulfate (SDS) concentration slightly improved resolution. Baseline separation and good selectivity relative to urine components were achieved by using a phosphate (25 mM)–borate (50 mM)–SDS (20 mM) running buffer, pH 7.0, in a 75 μm×60/67 cm fused-silica capillary at 15 kV and a 335 nm cut-off filter in the UV detector. The limits of detection (LOD) at a signal-to-noise ratio of 3 were about 0.25 μg/ml (5.2·10 −7M) (injection 0.5 p.s.i./8 s). The linear detection range was 2–100 μg/ml (r2>0.999). Good repeatability of injection and relative migration times were obtained. 相似文献
10.
Annalisa La Gatta 《Analytical biochemistry》2010,404(1):21-1246
Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC-TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights (Mw and Mn, respectively), polydispersity index (Mw/Mn), hydrodynamic radius (Rh), and intrinsic viscosity ([η]). The Mark-Houwink-Sakurada (MHS) curves (log [η] versus log Mw) were then derived directly. Fragments covering a whole range of Mw (10-900 kDa) and size (Rh = 4-81 nm) and presenting a rather narrow distribution of molar masses (Mw/Mn = 1.6-1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the Mw. HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC-TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions. 相似文献
11.
Dan Yu Nan Dai Eric J. Wolf Ivan R. Corrêa Jr. Jujun Zhou Tao Wu Robert M. Blumenthal Xing Zhang Xiaodong Cheng 《The Journal of biological chemistry》2022,298(4)
The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2′O-methyladenosine (Am), generating N6, 2′O-dimethyladenosine (m6Am) when Am is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and Am), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2′O-methylated Am over unmodified A nucleosides is due mainly to increased binding affinity for Am. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5′-Am oligonucleotide with approximately the same affinity as that of a cap analog (KM = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5′-Am with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs. 相似文献
12.
Henning Juhl 《Molecular and cellular biochemistry》1979,26(1):19-27
Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had Mr 250 000, 7.2S and the peak A enzyme Mr 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl2. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K
m
app
4.2 m for muscle glycogen synthease I and K
m
app
45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca2+, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP
adenosine cyclic 3:5-monophosphate
- DTT
dithiothreitol
- EGTA
ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid
- PMSF
phenylmethylsulfonylfluoride
- PKI
protein kinase inhibitor
- RI
ratio of independence for glycogen synthase
- SDS
sodium dodecyl sulphate 相似文献
13.
Ultrathin (black) lipid membranes were made from sheep red cell lipids dissolved in n-decane. The presence of aliphatic alcohols in the aqueous solutions bathing these membranes produced reversible changes in the ionic permeability, but not the osomotic permeability. Heptanol (8 mM), for example, caused the membrane resistance (Rm) to decrease from >108 to about 105 ohm-cm2 and caused a marked increase in the permeability to cations, especially potassium. In terms of ionic transference numbers, deduced from measurements of the membrane potential at zero current, T
cat/T
Cl increased from about 6 to 21 and T
K/T
Na increased from about 3 to 21. The addition of long-chain (C8ndash;C10) alcohols to the lipid solutions from which membranes were made produced similar effects on the ionic permeability. A plot of log Rm vs. log alcohol concentration was linear over the range of maximum change in Rm, and the slope was -3 to -5 for C2 through C7 alcohols, suggesting that a complex of several alcohol molecules is responsible for the increase in ionic permeability. Membrane permselectivity changed from cationic to anionic when thorium or ferric iron (10-4
M) was present in the aqueous phase or when a secondary amine (Amberlite LA-2) was added to the lipid solutions from which membranes were made. When membranes containing the secondary amine were exposed to heptanol, Rm became very low (103–104 ohm-cm2) and the membranes became perfectly anion-selective, developing chloride diffusion potentials up to 150 mv. 相似文献
14.
Two isoenzymes of lactate dehydrogenase have been purified from Homarus americanus: One is found predominantly in the tail muscles; the other, in the walking leg muscles. This is the first demonstration of multiple forms of l-specific lactate dehydrogenase in an invertebrate organism. These proteins contain four essential sulfhydryl groups titratable by p-hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). The molecular weights of these isoenzymes are dependent upon ionic strength. The native tetramer (Mr 145,000) exists in low ionic strength solutions; the active dimer (Mr 75,000), in high ionic strength solutions; this is the only example of lactate dehydrogenase disaggregation without concomitant loss in enzymatic activity. Microcomplement fixation studies suggest that there may be less than 4% difference in the primary structures of these two proteins. 相似文献
15.
Kenneth S. Schmitz Nambi Parthasarathy Jacqueline C. Kent Jennifer Gauntt 《Biopolymers》1982,21(7):1365-1382
Quasielastic light scattering and electrophoretic light scattering experiments were performed on chicken erythrocyte polynucleosome solutions at various temperatures and ionic strengths. The apparent diffusion coefficient, Dapp, was found to depend on the scattering vector K. In general, Dapp can be described as a damped oscillatory function of K in the ionic strength range of 10 to 60 mM and over the temperature range of 10 to 40°C. Electrophoretic light scattering studies on total digest chromatin samples indicate the apparent charge on the polynucleosomes increases as the ionic strength is lowered from 10 to 1 mM. These data are interpreted in terms of fluctuations in the surface charge distribution of the polyion and subsequent inducement of an asymmetric distribution of small ions about the polyion. These fluctuation components lead to the formation of “clusters” of polyions. 相似文献
16.
In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (Mr 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs−1, with a Km value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs−1 and 0.0766 µMs−1, with a Km value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII kcat/Km ratios were 0.12 µM−1s−1and 0.13 µM−1s−1 and 0.216 µM−1s−1 and 0.094 µM−1s−1 for for cis- and trans-dienelactone, respectively. The kcat/Km ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though Km value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms. 相似文献
17.
18.
Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises Tm, the melting temperature of free base pairs. The linear dependence of Tm on log Na+ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na+ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs Tm′ remains constant at various ionic strengths. On the other hand, the Tm′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of Tm′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na+ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers Tm′. This explanation is supported by circular dichroism (CD) results. 相似文献
19.
《Experimental mycology》1989,13(4):380-391
The thermophilic fungusPenicillium duponti has an active sulfate permease system derepressible by growth in cysteic acid or by sulfur starvation. The system is sensitive to ionic strength and is optimal at 45°C, pH 6.5. It is saturable with an apparent Km for sulfate of 55 μM. It is activated strongly by divalent cations and inhibited by inhibitors of mitochondrial ATP production and the group VI oxyanions. 相似文献
20.
The linear dichroism (LD) has been measured for DNA molecules 239–164,000 base pairs long oriented in shear flow over a large range of velocity gradients (30–3,000 s ?1) and ionic strengths (2–250 mM). At very low gradients, the degree of DNA orientation increases quadratically with the applied shear as predicted by the Zimm theory [J. Zimm, (1956) Chemical Physics, Vol. 24, p. 269]. At higher gradients, the orientation of fragments ≥ 7 kilobase pairs (kbp) increases linearly with increasing shear, whereas the orientation of fragments ≥ 15 kbp shows a more complicated dependence. In general, the orientation decreases with increasing ionic strength throughout the studied ionic strength interval, owing to a decrease in the persistence length of the DNA. The effect is most dramatic at ionic strengths below 10 mM, and is more pronounced for longer DNA fragments. For fragments ≥ 15 kbp and velocity gradients ≥ 100 s?1, the orientation can be adequately described by the empirical relation: LDr= –(k1-G)/(k2 + G), where k1is a linear function of the square root of the ionic strength and k2 depends on the DNA contour length. Since the DNA persistence length can be represented as a linear function of the reciprocal square root of the ionic strength [D. Porschke, (1991) Biophysical Chemistry, Vol. 40, p. 169], extrapolation of the empirical relation provides information about the stiffness of the DNA fibers. © 1993 John Wiley & Sons, Inc. 相似文献