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1.
The fluorescence recovery kinetics of succinyl-fluorescein Concanavalin A (S-F-ConA) in glycerol-physiological saline solutions of high viscosity and when bound to the surface of mouse fibroblasts were measured following brief photobleaching using a laser excited fluorescence microscope. In the high viscosity solutions, the recovery kinetics, interpreted on the basis of a simple diffusion model, yielded a diffusion coefficient in close agreement with the values predicted by the Stokes-Einstein equation. Recovery kinetics for S-F-ConA bound to the surface of mouse 3T3 and SV3T3 cells cultured in vitro yielded diffusion coefficients in the range of 5-10-10(-11) cm2/s, values considerably lower than those reported previously for membrane proteins. These measurements indicated that a considerable fraction of the S-F-ConA molecules bound to the cell surface are immobilized. These results are discussed in relation to current concepts of lateral motion of protein components within natural membranes. 相似文献
2.
The intrinsic viscosities, weight-average molecular weights (M?w), and radii of gyration [()≈] of Streptococcus salivarius levan in various solvents were respectively obtained from viscosity and light-scattering measurements. The data showed that the levan in water is not aggregated by hydrogen bonds, and that the values of both the refractive index and () are lower in water than in aqueous solutions of urea. Urea may break intramolecular hydrogen-bonds, e.g., between branches, allowing the molecule to expand. 相似文献
3.
The lateral diffusion coefficients () of the molecular fluorescence probe 3,3′-dioctadecylindocarbocyanine iodide (DII) in the membrane of discoid erythrocyte ghosts has been measured with the photobleaching technique between 7°C and 40°C. A fluorescence microscope which allows bleaching experiments within small local fields (approx. 1 μm2) at high magnification (X1600) has been used for these measurements. The diffusion coefficient increases from to from 7 to 40°C. An increase in membrane fluidity between 12°C and 17°C indicates a conformational change of the lipid bilayer moiety in this temperature region. The diffusion coefficient measured in the regions between the spicules of echinocytes is appreciably smaller than in the untransformed discoid ghosts. In the myelin tubes originating from cells, the lateral diffusion is somewhat larger (about a factor of 2) than in the non-transformed ghosts. With the fluorescence probe technique the rate of growth of myelin tubes of 0.3 μm diameter has been estimated. 相似文献
4.
P T Riquelme R W Fox R G Kemp 《Biochemical and biophysical research communications》1978,81(3):864-870
Phosphofructokinase isolated from mouse skeletal muscle 18 hours after intraperitoneal injection of [ contained 0.12 to 0.15 moles of covalently bound phosphate per protomer on the basis of the specific activity of radiolabel in the γ-position of ATP. Under identical conditions, muscle pyruvate kinase and aldolase had no covalently bound phosphate. 相似文献
5.
Kinetic parameters of human carbonic anhydrase B as determined from NMR linewidths of 13 C in CO 2 and HCO 3 - 总被引:1,自引:0,他引:1
The linewidths of the 13C NMR signals of CO2 and , in equilibrium aqueous solutions containing small amounts of carbonic anhydrase, are determined mainly by the rate of enzyme-induced interconversion of CO2 and . We have measured these linewidths in unbuffered solutions of human carbonic anhydrase B for several values of [CO2], at 25°C as a function of pH. From a least-squares analysis of the data, using the equations relating the linewidths to the enzyme kinetics, we have obtained values for the kinetic (Michaelis-Menten) parameters that characterize this interconversion. These preliminary results are in approximate agreement with published values for highly buffered solutions. Additionally, the results confirm that the product of the hydration reaction, and the substrate for the dehydration, is the neutral molecule H2CO3. 相似文献
6.
The rotational correlation time of melittin, obtained from the nanosecond anisotropy of the emission from its single tryptophan residue, has been found to increase considerably in phosphate solution relative to that in aqueous solution, consistent with protein aggregation. The steady-state fluorescence spectra as well as the absorption spectra in phosphate solution exhibit a very good degree of similarity with those of the protein bound to egg phosphatidylcholine (PC) and distearoylphosphatidylcholine (DSPC) bilayer liposomes. The value of the second-order rate constant for dynamic quenching, , by acrylamide in 0.5 M phosphate solution is comparable to those for the protein-phospholipids complexes (1·109 and 0.7·109 M?1·s?1 for egg PC and DSPC, respectively). Similarities are also found in the nanosecond properties. There is a much stronger and quite similar dependence of the fluorescence spectra on time in the nanosecond range and of the fluorescence decay times on the emission wavelength in both cases as compared to the case in aqueous solution. These observations support the notion that melittin binds to the phospholipids in an aggregated form. The results suggest that the reduction in the values of bound melittin relative to that in aqueous solution and the blue shift of the fluorescence spectrum (from 352 to 337 nm) are brought about by shielding of the tryptophan residue from the solvent through a combination of protein aggregation and enhancement of its α-helical content (suggested by published CD data). The magnitude of the values for bound melittin, however, is still relatively high implying the occurrence of rather frequent encounters between the tryptophan residue and the hydrophilic acrylamide molecules. Thus, the residue is found not to penetrate deep into the phospholipid bilayer. 相似文献
7.
Frederick H. Silver David E. Birk 《International journal of biological macromolecules》1984,6(3):125-132
Physical properties of pepsin-solubilized types I, II, III and V collagen have been measured in acid solution at 10°C. Our results indicate that types I, II and III collagen molecules undergo a monomer-aggregate equilibrium in solution whereas type V molecules appear to attract each other but do not undergo a similar monomer-aggregate equilibrium. Interstitial collagen monomers (I, II and III) have molecular weights between , translational diffusion coefficients between and particle scattering factors at an angle of 175.5° and wavelength of 633 nm between 0.430 and 0.460. Type V collagen molecules after pepsin digestion were found to have a higher molecular weight (), similar translational diffusion coefficient () and similar particle scattering factor at 175.5° (0.440) to the interstitial collagens. Theoretical bead models are discussed and suggest that changes in the translational diffusion coefficient were less sensitive to bending motions than were changes in the particle scattering factor at 175.5°C. Bend angles of 50° were shown to increase the particle scattering factor by 5% whereas a bend angle of greater than 125° was required to increase the translational diffusion coefficient by 5%. Models developed from idealized shapes seen by electron microscopy of rotary shadowed collagen molecules agreed best with experimental laser light scattering measurements when the bend angles were less than 90°. 相似文献
8.
Alexander P.R. Theuvenet Willem M.H. Van De Wijngaard Josephus W. Van De Rijke George W.F.H. Borst-Pauwels 《生物化学与生物物理学报:生物膜》1984,775(2):161-168
The applicability of 9-aminoacridine as a probe of the surface potential of yeast cells is examined. Yeast cells are found to quench the fluorescence of the dye and it is shown that this quenching is caused by a decrease in the dye concentration in the bulk aqueous phase. Consistent with predictions of the Gouy-Chapman theory the dye is displaced from the surface of the yeast cells by addition of salts, the effectiveness of the salts being related to the valency of the cation: . It is shown that 9-aminoacridine is predominantly bound by the plasma membrane of the cells. Only a minor part of the binding occurs in the cell wall, in line with the finding that enzymic removal does not significantly affect the binding of the dye to the cells. A single relationship for the distribution ratio of the dye between cells and medium with the ζ potential of the cells is found, irrespective of the way the ζ potential is changed, either by varying the pH or the Ca2+ concentration. It is argued that the electrostatic potentials probed by the dye are much higher than the corresponding ζ potentials and are of the same order of magnitude of the presumed discrete charge potentials experienced by cation transporters in the plasma membrane. It is concluded that 9-aminoacridine may be applied as a convenient and almost quantitative probe of the surface potential that effects the kinetics of ion uptake by the yeast cells. 相似文献
9.
This paper describes an investigation, using quasi-elastic light scattering, of the diffusion of polystyrene spheres through solutions of dextran. The diffusion coefficient, D, of the spheres is shown to vary inversely with the volume fraction, φ, of dextran according to . Changes in the molecular weight of dextran are shown to reflect changes in the macromolecular shape parameter, ν, and the interaction parameter, κ. This result differs from previous studies which suggested an dependence and no molecular weight dependence [8]. 相似文献
10.
A comparison between inosine- and guanosine-containing anticodons in ribosome-free codon-anticodon binding 总被引:1,自引:0,他引:1
Binding of the ribooligomers, AUC, AUU, AUA, AUCA, AUUA, and AUAA to the isoleucine-accepting tRNAs, tRNA (anticodon, -IAU-) and tRNA (anticodon, -GAU-) was measured by equilibrium dialysis. With the aid of Scatchard plots, AUCA was shown to bind to one site per tRNA molecule, presumably the anticodon. AUA and AUAA did not measurably attach to either anticodon in the ribosome-free system. All other oligomers were bound to tRNA about 5 to 13 times stronger than to tRNA. 相似文献
11.
P. Månsson Ö. Jacobsson K.A. Granath 《International journal of biological macromolecules》1985,7(1):30-32
The molecular weight of Na- and K-hyaluronate has been determined by low angle laser light scattering (LALLS) technique. Two preparations of hyaluronate from rooster comb () were investigated. The LALLS was carried out both in a static mode and on the effluent from a column filled with porous gel. In contrast to Sheehan et al.1, no significant difference was found in the molecular weight of viscosity of Na- and K-hyaluronate in 2.0 M salt solutions 相似文献
12.
The kinetics of isotopic Na+ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal 22Na+ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na+ flow. The electrical current and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at . Subsequent elimination of active Na+ transport mucosal amiloride permitted calculation of the rates of active Na+ transport and active and passive influx and and . The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence . was abolished at . As Δψ approached , active efflux became demonstrable. At exceeded , so that was negative. Experimental values of agreed well with theoretical values predicted by a thermodynamic formulation: . The dependence of on Δψ is curvilinear. 相似文献
13.
14.
The activity coefficient of sodium ions in the presence of acidic polysaccharides (alginates, pectins and κ-carrageenan) has been measured potentiometrically. The activity determined in limit-dilute solutions normally exceeded the values calculated from the Manning theory. The difference (ΔγNa+) was regarded as a measure of the chain flexibility. The change in ΔγNa+ when a polysaccharide is transferred from water to 8 m urea solution gave information about the contribution of hydrogen bonds to the stabilisation of the polysaccharide conformation in aqueous solutions. The function n is independent of polymer concentration at high concentration. where γNa+exp is the experimentally determined value of the activity coefficient of the counterions and γNa+NaCl is the activity coefficient of sodium ions in a NaCl solution of the same equivalent concentration as the polymer solution.This suggests that the polysaccharide solution has a microheterogeneous structure. The results of the viscosity measurements suggest that there is agreement between equilibrium thermodynamic and rheological evaluation criteria for the structure of polymer solutions. 相似文献
15.
A method for calculating the rate constant () for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: and , respectively.The proposed method has also been used for the estimation of the value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of has been found to be .The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively. 相似文献
16.
The effects of lead upon collagen synthesis and proline hydroxylation were examined in the Swiss mouse 3T6 fibroblast. The results indicate that lead reduces proline hydroxylation in stationary phase cultures of 3T6 cells, resulting in increased cellular retention of unhydroxylated procollagen. Inhibition of proline hydroxylation by lead was prevented by increasing the extracellular molar ratio. Interference by lead in the hydroxylation of proline in logarithmic phase cultures of 3T6 cells resulted in increases in the 0.5 n HClO4 soluble/insoluble hydroxyproline ratio. This was attributed to an increase in the rate of breakdown of lead-induced unhydroxylated procollagen. Kinetic analysis of the lead-iron interaction with proline hydroxylase suggests that the mechanism is competitive. 相似文献
17.
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
18.
E. Tipping B.P. Moore Carol A. Jones G.M. Cohen B. Ketterer J.W. Bridges 《Chemico-biological interactions》1980,32(3):291-304
The non-covalent interactions of benzo[a]pyrene (BP) and several of its hydroxylated metabolites with ligandin, aminoazodye-binding protein A (Z-protein, fatty acid binding protein) and lecithin bilayers have been studied by equilibrium dialysis, an adsorption technique and fluorescence spectroscopy. Binding affinities expressed as (where = moles of BP or BP metabolite bound per mole of protein or lipid and c = unbound concentration), were measured at concentrations sufficiently low that there was no self-association of the unbound compounds as judged by their fluorescence characteristics. 3-Hydroxybenzo[a]pyrene (BP-3-phenol), 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (BP-4,5-dihydrodiol) and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-dihydrodiol) bind more strongly () to all three binders than does BP itself (). 9,10-Dihydro-9,10-dihydroxybenzo[a]pyrene (BP-9,10-dihydrodiol) binds to ligandin with an affinity similar to those of the other BP metabolites studied here, but binds much less strongly to both protein A and lecithin ( and 3 · 104 l · mol?1, respectively). The low affinity of BP-9,10-dihydrodiol for lecithin would account for earlier findings that on incubation of BP with isolated rat hepatocytes, this metabolite egressed from the cells to the extracellular medium much more readily than either BP-4,5-dihydrodiol or BP-7,8-dihydrodiol.Calculations based on these results suggest that within hepatocytes BP and its metabolites, including BP-9,10-dihydrodiol, will be found almost exclusively associated (>98%) with lipid membranes. 相似文献
19.
T R Green R E Schaefer M T Makler 《Biochemical and biophysical research communications》1980,94(4):1213-1220
In many instances the effect of superoxide () trapping agents in suppressing the net rate of O2 consumption of activated PMN's is not in accordance with theoretical expectations. We offer here an alternate explanation to those previously presented by Segal and Meshulam (FEBS Letters 100, 27–32) and Babior (Biochem. Biophys. Res. Comm. 91, 222–226). The paradoxical results previously presented can be explained by recognizing that shortly after activation of resting cells an O2 diffusion layer is established at or near the outer surface of these cells. The presence of this diffusion layer can markedly alter the anticipated stochiometric relationship between trapped and apparent O2 consumed by these cells when they are exposed to trapping agents. 相似文献
20.
(1) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of 3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F1) was studied in the absence of photoirradiation, and compared to that of . The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by upon photoirradiation. (3) Photoirradiation of F1 by resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol . Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound was localized on the α- and β-subunits of F1. At low concentrations (less than 10 μM), bound was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F1. 相似文献