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1.
The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with p-mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of 203Hg-labelled PMBS. After pretreatment with N-ethylmaleimide to block readily reactive sulphydryl groups, incubation with [203Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal pKa value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups. 相似文献
2.
Charge-pulse relaxation experiments with the negatively charged lipophilic ions, dipicrylamine and tetraphenylborate, (as well as with the positively charged carrier system Rb+-valinomycin) have been carried out in order to study the influence of sterols on the ion transport through the lipid bilayer membrane. The mol fraction of the sterols (cholesterol, epicholesterol, ergosterol, stigmasterol, dihydrocholsterol, epicoprostanol and cholesterololeate) as referred to total lipid was varied in a wide range (mol fractions 0–0.8).The monoolein/sterol or dioleoylphosphatidylcholine/sterol mixtures were dissolved in -hexadecane in order to minimize effects of the sterol on the membrane thickness.Cholesterol had a strong influence on the transport of the lipophilic ions. Its incorporation into monoolein membranes increased the rate constant of translocation up to 8-fold, but incorporation into phosphatidylcholine membranes had virtually no influence on . The other sterols with one hydroxy group and cholesterololeate had no influence on the rate constant or the partition coefficient β. The results are discussed on the basis of a possible change of dipole potential of the membrane caused by cholesterol and its derivatives.In the case of valinomycin-mediated Rb+ transport only cholesterol had a strong influence on transport properties. The rate constants of association () as well as the rate constants of translocation of the complex () and of the free carrier () were reduced by incorporation of cholesterol up to eight-fold. The decrease of and are possibly caused by a decrease of membrane fluidity, whereas the decrease of may be due to an increase of surface potential. The different action of cholesterol on the two transport systems is discussed under the assumption that the adsorption plane of the lipophilic ion is located more towards the aqueous side and that of the ion-carrier complexes more towards the hydrocarbon side of the dipole layer. 相似文献
3.
Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous from methanol to caused an inhibition of calcium uptake and an enhancement of ATPase activity. The treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of vesicles was about twice that of control vesicles. With increasing number () of carbon atoms of the , the maximum increment of ATPase activity increased, and both the alcohol concentration () required to inhibit calcium uptake by 50% and the alcohol concentration () required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows. The ratio, to , was constant for all values. The apparent free energy of binding of the methylene groups of to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of in octanol and water (?670 cal/mole). The effects of on membrane vesicles are discussed on the basis of these data. 相似文献
4.
The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na+,+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a ; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of while ATP binds with an apparent for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is . In the presence of ATP a reduction in size is observed. 相似文献
5.
Quercetin inhibited a dog kidney ( preparation without affecting for ATP or for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the ( activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the for ATP and the for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on , for substrate, and for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations. 相似文献
6.
John A. Jacquez 《生物化学与生物物理学报:生物膜》1973,318(3):411-425
The Michaelis-Menten parameters, and of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na+ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, , decreased with decrease in extracellular Na+ for both α-aminoisobutyric acid and phenylalanine but the for α-aminoisobutyric acid increased markedly as the Na+ concentration fell whereas the for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na+-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na+-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na+-independent but it has a high and is not additive with the Na+-dependent flux. 相似文献
7.
José Remacle 《生物化学与生物物理学报:生物膜》1980,597(3):564-576
The in vitro incorporation of cytochrome into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome than did microsomal preparations; 60% of this cytochrome could not be reduced by the NADH-cytochrome reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome . These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell. 相似文献
8.
9.
Cytochrome was extracted and purified from beef liver by a detergent method (cytochrome ). The hydrophilic moiety which carries the heme group (cytochrome ) was prepared by trypsin action upon pure cytochrome .Single-shelled lecithin liposomes form complexes with cytochromes up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [3H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes.Cytochrome does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents.Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature . i.e. when the aliphatic chains are in a crystalline state, and above , when the alipathic chain are in a fluid state.1H NMR spectra show that even at the maximum cytochrome concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex. 相似文献
10.
The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant () of 13 nM was found in the presence of () and (). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg2+, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K+ and its congeners, by Na+ in the presence of (), and by ATP analogs (ADP-C-P, ATP-OCH3). Ca2+ antagonized the action of K+ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (Rα) and a high affinity conformational state (Rβ). The equilibrium between both states is influenced by ligands of . With 3 mM Mg2+ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot). 相似文献
11.
Chana Vinkler 《Biochemical and biophysical research communications》1981,99(4):1095-1100
The of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K+) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold in the of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K+ are employed, the rate of photophosphorylation is decreased but a several-fold in the of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient. 相似文献
12.
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
13.
An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl? and HCO3?, inhibited by SCN?.Biochemical characterization shows that HCO3? stimulation () is specifically inhibited in a competitive fashion by SCN? (). The residual Mg2+-dependent activity is weakly is weakly affected by SCN?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO3? (); no stimulation is observed in the absence of HCO3?. Thiocyanate exhibits a mixed type of inhibition () towards the Cl? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl?, but this enzyme has a relatively weak affinity for this substrate (). 相似文献
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15.
In the presence of the Na+-channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX–XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value () decreased while the corner frequency () increased as the mucosal amiloride concentration was increased from 0.5 to 24 μM. From the linear relationship between the values and the amiloride concentrations it was possible to determine the binding () and unbinding () constants for amiloride to its receptor on the Na+ channel. With these parameters as well as short-circuit current and values, the current through the individual Na+ channels () was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na+ channels () in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na+ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX–XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in with the amiloride concentration did not occur in these animals. 相似文献
16.
(1) Na+ currents and Na+-current fluctuations were measured in myelinated frog nerve fibres at 15°C during 7.7 ms depolarizations to . (2) The conductance γ of a single Na+ channel and the number of channels per node were calculated from ensemble average values of the mean Na+ current and the variance of Na+-current fluctuations. (3) For a hyperpolarizing holding potential of the mean values of the channel conductance and number were and . (4) After changing the holding potential to the resting potential () the conductance γ increased by a factor of 1.37 whereas the number decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of increased γ by a factor of 1.55 and reduced by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na+ channels in the nodal membrane. 相似文献
17.
G.E. Breitwieser 《生物化学与生物物理学报:生物膜》1982,689(3):457-463
(1) A membrane fraction enriched in (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an specific activity of approx. 2 units/mg and was ouabain-sensitive. (2) The had a for ATP of and a pH optimum of 7.0. It was inhibited by ouabain with a of . (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with . (4) The Mg2+ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the for Mg2+ was , and at 6 mM ATP, the was . High levels of Mg2+ caused inhibition of hydrolysis. (5) The interactions of Na+ and K+ were examined over a range of conditions. K+ levels caused modulations in the Na+ dependence in the range of 1–150 mM. (6) The prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves. 相似文献
18.
Purified cytochrome from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, was less stable than in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal . Liposomal required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal was subjected to treatment. This reagent destroyed the liposomal . These results suggest that the heme is located in the proximity of the reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane. 相似文献
19.
The ionization of fatty acids, fatty amines and acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with 13C and titrated by using NMR spectroscopy. The apparent p of fatty acids in phosphatidylcholine bilayers is 7.2–7.4 and those of fatty amines are approx. 9.5. These p values depend on many different parameters related to the structure of the lipid/ solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the p value whereas a negative one increases it, the total range of variation being 2.5–3 units. In a qualitative macroscopic interpretation, it is proposed that p is essentially determined by the low polarity of the lipidic matrix. 相似文献