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1.
lumisomes produce a bioluminescent flash when the vesicles are disrupted with hypotonic solutions containing Ca2+. A flash is also observed in the presence of Ca2+ using isotonic solutions of monovalent cations under the following conditions: When the ratio inside the lumisomal membrane is high and when this ratio outside the membrane is low. We suggest that Na+ may be the counter ion for Ca2+ transport. Na+, when outside the membrane, inhibits Ca2+-triggered luminescence suggesting that Na+ blocks Ca2+ channels. Ca2+ uptake into the lumisomal membrane, as measured by bioluminescence, is very rapid in the presence of the ionophore A23187. X537A is much less effective. The Ca2+ triggered bioluminescence flash observed with lumisomes provides a rapid and sensitive assay for ionophores that are specific for divalent cations such as Ca2+. 相似文献
2.
The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na+. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na+. The extent of transport inhibition by Na+ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na+ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the values for Na+ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order . Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na+, in addition to the competitive inhibition of transport Na+ induces an increase in membrane permeability for amino acids. 相似文献
3.
Showdomycin inhibited pig brain ( with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1), (2) , , and none, (3) , and , (4) and , , , and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition () were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i.e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of ( proceeds via at least four kinds of enzyme form (E1, E2, E1 · nucleotide and EP), which all have different conformations. 相似文献
4.
Single potential-dependent K+ channels were studied using the patch-voltage-clamp method. Two types of channel with identical, but oppositely directed, potential dependences were found. The channels of the first type (slow channels) are assumed to be responsible for the outward rectification. The properties of the channels of the second type (fast channels) are similar to those of the K+ channels in neurone soma which create the fast transient currents. The kinetic characteristics of both types of channel are presented. The conductances of slow and fast K+ channels are approx. 30 and 40 pS, respectively, at zero membrane potential and a K+ concentration of 50 mmol/l at the inner side of the membrane. The following sequence of channel selectivity with respect to monovalent cations was found: . The probability of the channel open state monotonically decreases with free Ca2+ concentration at the inside membrane surface for both types of channel. It was found that the channels have discrete and multiple conductance substrates. It is supposed that a unitary K+ channel consists of approx. 16 elementary ones with conductances of approx. 2 pS (slow channels) and approx. 2.5 pS (fast channels) at zero potential. At + 100 mV the elementary conductances are equal to approx. 4 and 5.5. pS, respectively. Thus, according to this assumption, the unitary channel is a cluster of elementary channels. 相似文献
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6.
A potent inhibitor of activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma with an of 1 μM in the presence of 1 mM acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate in increased by free Mg2+. The inhibition is half maximally reversed by 250 μM epinephrine. Equine muscle ATP was also found to contain a second inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 μM epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and . 相似文献
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8.
Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca2+ transport in this preparation were characterized and compared to those of (Ca2+ + Mg2+)-ATPase activity. The time course of Ca2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca2+/mg protein after 3–4 min of incubation. The rate of Ca2+ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca2+/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent values for Mg2+ and Ca2+ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ()-ATPase. The presence of potassium was required for maximum Ca2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca2+ transport and ( activity was transport and ( activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM sulfonate. The results indicate that Ca2+ transport in adipocyte endoplasmic reticulum is mediated by a and suggest an important role for endoplasmic reticulum in control of intracellular Ca2+ distribution. 相似文献
9.
At least ten distinct ATP-hydrolyzing activities are associated with mitochondria, endoplasmic reticulum-, Golgi-, and plasma membrane-enriched fractions from the marine diatom, Nitzschia alba. These activities are divided into four groups: Ca2+-dependent, Mg2+-dependent monovalent cation-stimulated, Mg2+-anion-stimulated ATPases, and Mg2+-dependent nucleotidases.The Mg2+-dependent activities hydrolyze nucleoside triphosphates and, in some membranes, nucleoside diphosphates. Molar ratios of 1:2 are preferred. However, their divalent cation requirements are not specific, and they can effectively utilize Ca2+, Mn2+, Mg2+, or Zn2+. The most effective inhibitors of the Mg2+-dependent activities are oligomycin, NaN3, and NaF.Optimal activity of the Mg2+-dependent monovalent cation-stimulated ATPase is obtained at Na+, or Na+ plus K+ concentrations of 100–300 mm. Under these high salt conditions, ATP is hydrolyzed almost exclusively, and Mg2+ is specifically required for activation. Preference is for a molar ratio of , and the sulfhydryl-blocking agents, p-chloromecuribenzoate, N-ethylmaleimide, and iodoacetamide strongly or completely inhibit ATP hyrolysis. 相似文献
10.
(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献
11.
Treatment by EDTA of purified plasma membranes from MF2S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of to ouabain. When added back with Ca2+ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca2+ it was able to restore the original sensitivity to ouabain of from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca2+ the same quantitative biological activity on ouabain sensitivity of the . A possible interaction between β-actinin, calmodulin and membrane-bound is discussed. 相似文献
12.
A Ziegelhoffer M B Anand-Srivastava R L Khandelwal N S Dhalla 《Biochemical and biophysical research communications》1979,89(4):1073-1081
The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate 32P from [γ-32P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca2+ ATPase and Mg2+ ATPase activities without any changes in Na+K+ ATPase activity. The observed increase in ATPase activity was found to be associated with an increase in Vmax value of the reaction whereas Ka value for was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect. 相似文献
13.
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
14.
The high pH-maintaining capacity of yeast suspension after glucose-induced acidification, measured as its ability to neutralize added alkali, was found to be due mainly to actively extruded acidity (H+). The buffering action of passively excreted metabolites (CO2, organic acids) and cell surface polyelectrolytes contributed only 15–40% to the overall pH-maintaining capacity which was 10 mmol NaOH/l per pH unit between pH 3 and 4 and 3.5 mmol NaOH/l per pH unit between pH 4 and 7. The buffering capacity of yeast cell-free extract was still higher (up to 4.5-times) than that of glucose-supplied cell suspension; addition of glucose to the extract thus produced considerable titratable acidity but negligible net acidity. The glucose-induced acidification of yeast suspension was stimulated by univalent cations in the sequence . The processes participating in the acidification and probably also in the creation of extracellular buffering capacity include excretion of CO2 and organic acids, net extrusion of H+ and K+ (in K+-free media; in K+-containing media this is preceded by an initial rapid K+ uptake), and movements of some anions (phosphate, chlorides). The overall process appears to be electrically silent. 相似文献
15.
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
16.
Dennis W. Jung Guey-Yueh Shi Gerald P. Brierley 《Archives of biochemistry and biophysics》1981,209(2):356-361
Depletion of mitochondrial divalent cations by addition of the ionophore A23187 results in a marked increase in passive exchange activity. The exchange is activated by increasing pH and temperature and inhibited by added divalent cations. The reaction is independent of the amount of A23187 present, but depends on the concentration of external K+ (Km = 25 mm). Intramitochondrial 42K+ in cation-depleted mitochondria exchanges passively with external Na+ and Li+, but not with choline+. The evidence suggests that removal of mitochondrial divalent cations by A23187 activates the endogenous exchange component of the mitochondrion and that the activated exchanger promotes cation/cation exchange in the absence of a metabolic pH gradient. 相似文献
17.
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
18.
(1) Eosin bound to the in the presence of K+ has practically the same fluorescence as eosin without enzyme while in the presence of Na+ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K+ concentration but with a low affinity. With equal concentrations of Na+ and K+ more is bound in the presence of Na+, and the difference between 150 mM Na+ and 150 mM K+ shows one high-affinity eosin binding site per 32P-labelling site ( 0.45 μM). With lower concentrations of the cations there are between one and two Na+-dependent high-affinity eosin binding sites per 32P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na+-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na+ (+Mg2+). (4) Eosin, like ATP, increases the Na+ relative to K+ affinity ) for Na+ activation of hydrolysis and for Na+ protection against inactivation by . (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na+ opens a high-affinity site for ATP while K+ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na+ relative to K+ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of the system reacts with two different ATP molecules. 相似文献
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20.
A possible mechanism for the Na,K-ATPase 总被引:2,自引:0,他引:2
P M Wiggins 《Journal of theoretical biology》1982,99(4):665-679
A model previously described for the Ca2+ pump of sarcoplasmic reticulum has been modified in a thought experiment so that it has the properties of a Na,K-adenosinetriphosphatase (ATPase). When the two Ca2+-specific sites are changed into three Na+-specific sites, and the channel which opens in the actively transporting conformation made univalent- instead of divalent-cation-selective, the model has the properties of the Na-ATPase which is observed on red cell membranes in the absence of both Na+ and K+ externally. As in the model for the Ca-ATPase the driving force for transport is generated by a change in solvent structure so that a preformed ionic equilibrium is displaced in favour of less-highly hydrated species; in this case highly hydrated Mg2+ ions displace the less highly hydrated Na+ ions from binding sites; and Na+ diffuses out through a simultaneously opened channel. With the addition of three external K+-selective sites per α-polypeptide chain, and the constraint that pump units with their external sites occupied by any univalent cation cannot be phosphorylated by ATP, the model turns out to have the properties of a Na,K-ATPase. It operates in the exchange, exchange, exchange, K+-dependent phosphatase, uncoupled Na+ efflux and pump reversal modes. It is concluded that if the modified water in the cleft of the phospho-enzymes has properties similar to those of water at 5°C the pump is competent to exchange three intracellular Na+ ions for two extracellular K+ ions, and one intracellular Na+ ion but it is incapable of exchanging three Na+ ions for three K+ ions. 相似文献