A wide range of concentrated random coil polysaccharide solutions have been assessed for textural attributes by a trained sensory panel. The only textural terms invoked to describe these model systems were ‘thickness’ and ‘stickiness’, which were shown to be highly correlated, and essentially identical numerically, using a ratio scaling technique. Viscosity (η) measurements over a wide range of shear rates (γ) for all these samples gave flow curves (log η versus log γ) of the same form. Differences in flow behaviour between samples could then be characterised completely by two parameters, the maximum viscosity at low shear rates (η0), and the shear rate () at which . A simple linear relationship was demonstrated between these two parameters and perceived thickness (T) or stickiness (S), irrespective of polysaccharide type. For Newtonian liquids, log T (or log S) varied linearly with log η. Hence the effective ‘in-mouth’ thickness of random coil polysaccharide solutions, in normal viscosity units, may be predicted directly from η0 and by the simple relationship: where ηN is the viscosity of a Newtonian solution which would be perceived as identical in thickness (and stickiness) to the polysaccharide solution. 相似文献
A thermodynamic characterization of the Na+-H+ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na+-H+ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, , was in the range of 850–1150 nmol H+·(mg protein)?1·h?1·(pH unit)?1 for four different preparations; for the total Na+ permeability, , it was 1620–2500 nmol Na+·(mg protein)?1·h?1·(pNa unit)?1; and for the proton ‘cross-permeability’, , it was 220–580 nmol H+·(mg protein)?1·h?1·(pNa unit)?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na+-H+ exchange (ηmax), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; ηmax ? 5%; 0.36 ? r ? 2; ?JNa+ ? 1.3 · 105μmol · (RT unit)?1 at JNa = 1 μmolNa+ · (mgprotein)?1 · h?1; and ?ΔpNa ? 5 · 104 ΔpNa · (mg protein) · h · (RT unit)?1 at ΔpNa = 1 unit, for different preparations. 相似文献
Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pKa's are ΔH0 = 8.4 ± 0.3 kcal mol?1, for 6-Cy and ΔH0 = 10.0 ± 0.1 kcal mol?1, for 7-Cy. Intrinsic 13C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D2O (). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 20-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding. 相似文献
Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston3 at 20 °C in the absence of phosphate revealed a high affinity (P50 = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Böhr effect (). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P50 = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Böhr effect (). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′4, of ~ 6 × 106m?1s?1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ~ 0·5 × 106m?1s?1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k4~ 11 s?1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α2βAβCranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell. 相似文献
Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]0 ? [S]0 and [S]0 ? [E]0 conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant (. The limiting values of Ks′ (dissociation constant of E · S), K2 (acylation rate) and k3 (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10?5 m, 360 ± 15 s?1 and 29.3 ± 0.8 s?1, respectively. Likewise small values were observed for Ki of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and Km of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k2 value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters. 相似文献
The transport of sucrose by selected mutant and wild-type cells of Streptococcus mutans was studied using washed cocci harvested at appropriate phases of growth, incubated in the presence of fluoride and appropriately labelled substrates. The rapid sucrose uptake observed cannot be ascribed to possible extracellular formation of hexoses from sucrose and their subsequent transport, formation of intracellular glycogen-like polysaccharide, or binding of sucrose or extracellular glucans to the cocci. Rather, there are at least three discrete transport systems for sucrose, two of which are phosphotransferases with relatively low apparent values and the other a non-phosphotransferase (non-PTS) third transport system (termed TTS) with a relatively high apparent . For strain 6715-13 mutant 33, the values are 6.25·10?5 M, 2.4·10?4 M, and 3.0·10?3 M, respectively; for strain NCTC-10449, the values are 7.1·10?5 M, 2.5·10?4 M and 3.3·10?3 M, respectively. The two lower systems could not be demonstrated in mid-log phase glucose-adapted cocci, a condition known to repress sucrose-specific phosphotransferase activity, but under these conditions the highest system persists. Also, a mutant devoid of sucrose-specific phosphotransferase activity fails to evidence the two high affinity (low apparent ) systems, but still has the lowest affinity (highest ) system. There was essentially no uptake at 4°C indicating these processes are energy dependent. The third transport system, whose nature is unknown, appears to function under conditions of sucrose abundance and rapid growth which are known to repress sucrose-specific phosphotransferase activity in S. mutans. These multiple transport systems seem well-adapted to S. mutans which is faced with fluctuating supplies of sucrose in its natural habitat on the surfaces of teeth. 相似文献
The interaction of |CnH2n+1N+(CH3)3| · I? (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by 31P-NMR spectroscopy. It is shown that the effective anisotropy of 31P chemical shift (?Δσeff) of the lamellar phospholipid liquid-crystalline phase Lα increases with increasing concentration and alkyl chain length of the drug. Addition of or |C9H19N+(CH3)3|·I? to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n 12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells. 相似文献
Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A0 and At are activities at time zero at t, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg2+ (5.0 mm) to a solution of this protein. Addition of Ba2+ or Mn2+ brings about partial recovery. Other metal ions are not effective. 相似文献
A theoretical model which can account for both the dynamic and steady responses is proposed based on the occupation theory. The reaction scheme used is; Here, S and A are stimulus chemicals and receptor sites unbound, respectively. The binding of S to A leads to an active complex (SA)active, which is successively transformed into an inactive complex (SA)active. The response is assumed to be proportional to number of (SA)active. When a stimulating solution is applied instantaneously at t = 0, the solution to the set of differential equations based on the above scheme is obtained as follows; where p and C stand for the fraction of (SA)active to the total number of receptor sites and stimulus concentration, respectively, and αi, and ωi (i = 1, 2) are numerical parameters depending on the rate constants and on C. The steady response is expressed as the third term in the above equation, which indicates that the response accords with the Beidler taste equation. Mathematical analysis of the above scheme shows that the dynamic response appears when k1C > k?2, and the calculated results for the dynamic response agree approximately with the Hill equation. The Hill coefficient lays within 1·00 and 0·79 and reaches unity with increasing , which implies that the dynamic response under this condition satisfies the Beidler taste equation. For the case of gradual application of stimuli, i.e. the experimental condition, the time course of p is simulated with use of an analogue computer rather than with a numerical solution to the above equation. The results indicate that the dynamic response diminishes with decreasing the application speed of stimulus solution. The present theory accounts consistently for various experimental data observed in the chemoreceptor systems. 相似文献
The observed equilibrium constants (Kobs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg2+]. Using Σ and square brackets to indicate total concentrations . The value of Kobs has been found to be relatively sensitive to pH. At 38 °C, K+] = 0.2 m and free [Mg2+] = 0; Kobs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔGobs0 = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H2O] = 1). The effect of the free [Mg2+] on Kobs was relatively slight; at pH 7.0 ([K+] = 0.2 m) Kobs = 52.0 m at free [Mg2+] = 10?3, m and 47.8 m at free [Mg2+] = 10?2, m. Kobs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔGobs0 = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H2O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 104, m [ΔGobs0 = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. . Values of Kobs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg2+], being calculated to be 668 [ΔGobs0 = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg2+] = 0; 111 [ΔGobs0 = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg2+] = 10?3, m; and 9.1 [ΔGobs0 = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg2+] = 10?2, m). Kobs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of Kobs are 4000 [ΔGobs0 = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg2+] = 0; and 97.4 [ΔGobs0 = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg2+] = 10?3, m. Combining Kobs for the l-phosphoserine phosphatase reaction with Kobs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of Kobs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. The value of Kobs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K+ as the monovalent cation) is 1.34 × 10?2, m at free [Mg2+] = 0 and 1.27 × 10?2, m at free [Mg2+] = 10?3, m. 相似文献
1. In mitochondrial particles antimycin binds to two separate specific sites with dissociation constants and , respectively.2. The concentrations of the two antimycin binding sites are about equal. The absolute concentration for each binding site is about 100 – 150 pmol per mg of mitochondrial protein.3. Antimycin bound to the stronger site mainly inhibits NADH- and succinate oxidase. Binding of antimycin to the weaker binding site inhibits the electron flux to exogenously added cytochrome c after blocking cytochrome oxidase by KCN.4. Under certain conditions cytochrome b and c1 are dispensible components for antimycin-sensitive electron transport.5. A model of the respiratory chain in yeast is proposed which accounts for the results reported here and previously. (Lang, B., Burger, G. and Bandlow, W. (1974) Biochim. Biophys. Acta 368, 71–85). 相似文献
The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of 125I-labeled toxin B-IV by native toxin B-IV have shown specific binding of 125I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [3H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association () and dissociation () shows a nearly identical of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding. 相似文献
(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots ( and ). It is not necessary to postulate a third reaction of . (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only a single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed. 相似文献
Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (BzGlyNHCH2CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters and their pH dependencies are , decreasing with either acid or base (PKE1 = 5.64, pKE2 = 9.55), and kcat = 1.02 × 102 min?1, decreasing with acid (pKES = 6.61). The thiopeptide is less efficiently cleaved by native (zinc) carboxypeptidase A. This cadmium-sulfur synergism supports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio) carbonyl. 相似文献
Sunflower pectin has been fractionated on Sepharose 2B/Sepharose 4B. Molecular weights were measured within the eluate by light scattering and intrinsic viscosities to establish a universal calibration line, e.g. a plot of the logarithm of the product of the weight-average molecular weight and the intrinsic viscosity against the elution volume. It was found that the universal calibration line of pectin differs, if only modestly, from those of dextran and dextran sulphate. Intrinsic viscosities and molecular weights do not correlate in the region of high molecular weights concerning about 15% of the sample. In most instances with molecular weights below 100 000 a Mark-Houwink relation of is valid. 相似文献
Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (ka) for complex formation (ka = 54,000 s?1m? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k?a = 6.2 s?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio (0.9 × 104m?1) was in reasonable agreement with value of 1.1 ± 0.1 × 104m?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln() and ln() vs were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins. 相似文献
Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10?8 M and was half-maximal at . The increase at 1·10?5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10?9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10?5 M dopamine was . Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The value for high affinity binding sites was and for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was noradrenaline, 2·10/t-7 M epinine, fluphenazine, haloperidol, cis-flupenthixol, trans-flupenthixol, apomorphine, sulpiride, naloxone and isoproterenol. 相似文献
Isometric force levels, ranging between 0 and 100% of maximal force P0 at 2 to 3 °C, were elicited in frog sartorius muscle by means of rapidly cooling a Ringer solution containing 1·25 to 2·0 mm-caffeine. Equatorial X-ray diffraction patterns were obtained in the resting state and during contraction. The ratio of the intensities increased with force almost linearly, with a slight upward curvature. The individual intensities for the contracting state were normalized relative to both the intensity of the undiffracted beam and the intensity of each reflection in the resting state. These normalized intensities were found to vary in a reciprocal way: I10 decreased while I11 increased throughout the range of forces studied.The gradual change in with force level indicates that this ratio is a sensitive measure of the number of cross-bridges in the isometric state. A two-state model in which myosin projections are either in a resting or attached state and in which force is proportional to the fraction of projections in the attached state was applied to the experimental data of the individual reflections. I10 deviates from this model in a way that suggests that formation of the first few cross-bridges may decrease the regularity of the remaining unattached myosin projections. 相似文献