共查询到20条相似文献,搜索用时 15 毫秒
1.
T F Shevchenko 《Biofizika》1976,21(2):321-323
Changes in the activity of calcium ions in the medium containing outer fragments suspension of bovine eye retina rods have been studied by the method of calcium-selective electrodes. Illumination of the suspension increases calcium ion activity in the incubation medium. Photoinduced yield of calcium ions depends on Ca+2 concentration: it equals 0.11+/-0.015 M Ca2+/1m rodopsin in the medium containing 0.1 mM CaCl2 and 0.046+/-0.002Ca2+/1M rodopsin in the medium containing 0.05 mM CaCl2. In the medium containing more than 10(-4) M CaCl2 both an increase and a decrease of Ca2+ ions have been observed. 相似文献
2.
Formation of giant liposomes promoted by divalent cations: critical role of electrostatic repulsion. 总被引:1,自引:1,他引:0
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Spontaneous formation of giant unilamellar liposomes in a gentle hydration process, as well as the adhesion energy between liposomal membranes, has been found to be dependent on the concentration of divalent alkali cations, Ca2+ or Mg2+, in the medium. With electrically neutral phosphatidylcholine (PC), Ca2+ or Mg2+ at 1-30 mM greatly promoted liposome formation compared to low yields in nonelectrolyte or potassium chloride solutions. When negatively charged phosphatidylglycerol (PG) was mixed at 10%, the yield was high in nonelectrolytes but liposomes did not form at 3-10 mM CaCl2. In the adhesion test with micropipette manipulation, liposomal membranes adhered to each other only in a certain range of CaCl2 concentrations, which agreed with the range where liposome did not form. The adhesion range shifted to higher Ca2+ concentrations as the amount of PG was increased. These results indicate that the divalent cations bind to and add positive charges to the lipids, and that membranes are separated and stabilized in the form of unilamellar liposomes when net charges on the membranes produce large enough electrostatic repulsion. Under the assumption that the maximum of adhesion energy within an adhesive range corresponds to exact charge neutralization by added Ca2+, association constants of PC and PG for Ca2+ were estimated at 7.3 M(-1) and 86 M(-1), respectively, in good agreement with literature values. 相似文献
3.
Effects of perchlorate on the molecules of excitation-contraction coupling of skeletal and cardiac muscle
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《The Journal of general physiology》1993,102(3):423-448
To understand the nature of the transmission process of excitation- contraction (EC) coupling, the effects of the anion perchlorate were investigated on the voltage sensor (dihydropyridine receptor, DHPR) and the Ca release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum (SR). The molecules, from rabbit skeletal muscle, were either separated in membrane vesicular fractions or biochemically purified so that the normal EC coupling interaction was prevented. Additionally, the effect of ClO4- was investigated on L-type Ca2+ channel gating currents of guinea pig ventricular myocytes, as a native DHPR not in the physiological interaction of skeletal muscle. At 20 mM, ClO4- had minor effects on the activation of ionic currents through Ca channels from skeletal muscle transverse tubular (T) membranes fused with planar bilayers: a +7-mV shift in the midpoint voltage, V, with no change in kinetics of activation or deactivation. This is in contrast with the larger, negative shift that ClO4- causes on the distribution of intramembrane charge movement of skeletal muscle. At up to 100 mM it did not affect the binding of the DHP [3H]PN200-110 to triad-enriched membrane fractions (TR). At 8 mM it did not affect the kinetics or the voltage distribution of gating currents of Ca channels in heart myocytes. These negative results were in contrast to the effects of ClO4- on the release channel. At 20 mM it increased several-fold the open probability of channels from purified RyR incorporated in planar bilayers and conducting Ba2+, an effect seen on channels first closed by chelation of Ca2+ or by the presence of Mg2+. It significantly increased the initial rate of efflux of 45Ca2+ from TR vesicles (by a factor of 1.75 at 20 mM and 4.5 at 100 mM). ClO4- also increased the binding of [3H]ryanodine to TR fractions. The relative increase in binding was 50-fold at the lowest [Ca2+] used (1 microM) and then decayed to much lower values as [Ca2+] was increased. The increase was due entirely to an increase in the association rate constant of ryanodine binding. The chaotropic ions SCN- and I- increased the association rate constant to a similar extent. The binding of ryanodine to purified RyR protein reconstituted into liposomes had a greater affinity than to TR fractions but was similarly enhanced by ClO4-. The reducing agent dithiothreitol (5 mM) did not reduce the effect of ClO4- , and 5% polyethylene glycol, with an osmolarity equivalent to 20 mM ClO4-, did not change ryanodine binding.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
4.
Binding of divalent cations of dipalmitoylphosphatidylcholine bilayers and its effect on bilayer interaction 总被引:4,自引:0,他引:4
We have confirmed that CaCl2 swells the multilayer lattice formed by dipalmitolyphosphatidylcholine (DPPC) in an aqueous solution. Specifically, at room temperature 1 mM CaCl2 causes these lipid bilayers to increase their separation, dw, from 19 A in pure water to greater than 90 A. CaCl2 concentrations greater than 4 mM cause less swelling. We have measured the net repulsive force between the bilayers in 30 mM CaCl2 at T = 25 degrees C (below the acyl chain freezing temperature). For interbilayer separations between 30 and 90 A, the dominant repulsion between bilayers is probably electrostatic; Ca2+ binds to DPPc lecithin bilayers, imparting a charge to them. The addition of NaCl to CaCl2 solutions decreases this repulsion. For dw less than 20 A, the bilayer repulsion appears to be dominated by the "hydration forces" observed previously between both neutral and charged phospholipids. From the electrostatic repulsive force, we estimate the extent of Ca2+ binding to the bilayer surface. The desorption and bound Ca2+, apparent when bilayers are pushed together, is more rapid than one would expect if an association constant governed Ca2+ binding. The association affinity does not appear to be a fixed quantity but rather a sensitive function of ionic strength and bilayer separation. 相似文献
5.
X-ray diffraction studies have been made on the effects of cations upon the dipalmitoyl phosphatidylcholine/water system, which originally consists of a lamellar phase with period of 64.5 A and of excess water. Addition of 1 mM CaCl2 destroys the lamellar structure and makes it swell into the excess water. The lamellar phase, however, reappears when the concentration of CaCl2 increases: a partially disordered lamellar phase with the repeat distance of 150-200 A comes out at the concentration of about 10 mM, the lamellar diffraction lines become sharp and the repeat distance decreases with increasing CaCl2 concentration. A small amount of uranyl acetate destroys the lameellar phase in pure water. MgCl2 induces the lamellar phase of large repeat distance, whereas LiCl, NaCl, KCl, SrCl2 and BaCl2 exhibit practically no effect by themselves. Addition of cholesterol to the phosphatidylcholine bilayers tends to stabilize the lamellar phase. The high-angle reflections indicate that molecular arrangements in phosphatidylcholine bilayers change at CaCl2 concentrations around 0.5 M. The bilayers at high CaCl2 concentration seem to consist of two phases of pure phosphatidylcholine and of equimolar mixture of phosphatidylcholine and cholesterol. 相似文献
6.
By use of small-angle X-ray scattering it is shown that addition of alkali halide salts in small amounts (0-200 mM) shifts the repeat spacing in multilamellar DC13PC vesicles and alters the anomalous swelling behaviour close to the main transition. Both effects follow the Hofmeister series of the ions. We suggest that the shift of repeat spacing can be explained by ion effects on the van der Waals attractive forces between the membranes and on the decay length of the repulsive hydration force. The anomalous swelling is explained in terms of a critical unbinding of the membranes. The proximity of the critical temperature of the unbinding to the main transition temperature can be tuned by varying the concentration and type of salt in the sample. 相似文献
7.
In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process. 相似文献
8.
Uhríková D Kucerka N Teixeira J Gordeliy V Balgavý P 《Chemistry and physics of lipids》2008,155(2):80-89
Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+. 相似文献
9.
The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle. 相似文献
10.
The binding of aqueous anions (ClO4-, SCN-, I-, and NO3-) to lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using deuterium (2H) and phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy. The ability of these anions to influence the 2H NMR quadrupole splittings of POPC, specifically labeled at the alpha or beta position of the choline head group, increased in the order NO3- much less than I- less than SCN- less than ClO4-. In the presence of these chaotropic anions, the quadrupole splitting increased for alpha-deuterated POPC and decreased for beta-deuterated POPC, indicating a progressive accumulation of negative charge at the membrane surface. Calibration of the 2H NMR quadrupole splittings with the amount of membrane-bound anion permitted binding isotherms to be generated for perchlorate, thiocyanate, and iodide, up to concentrations of 100 mM. The binding isotherms were analyzed by considering electrostatic contributions, according to the Gouy-Chapman theory, as well as chemical equilibrium contributions. For neutral POPC membranes, we obtained ion association constants of 32, 80, and 115 M-1 for iodide, thiocyanate, and perchlorate, respectively. These values increase in the order expected for a Hofmeister series of anions. We conclude that the factor determining whether a particular anion will bind to lipid bilayers is the ease with which that anion loses its hydration shell.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore. 相似文献
12.
In order to examine the effect of a metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca(2+) binding site as in human alpha-lactalbumin by Escherichia coli expression system (Ser(-1) CaB lysozyme). In the presence of 2 mM CaCl(2), the refolding yield of Ser(-1) CaB lysozyme at a low protein concentration (25 microg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 microg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%). However, the refolding yield of Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) even at a protein concentration of 200 microg/mL was 80% and was higher than that of the wild-type lysozyme. From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser(-1) CaB lysozyme in the presence of various concentrations of Ca(2+), it was suggested that the dissociation constant of Ca(2+)-peptide complex was estimated to be 20-36 mM. Moreover, the solubility of the denatured Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) was higher than that in the presence of 2 mM CaCl(2) whereas the solubility of the denatured Ser(-1) lysozyme in the presence of 100 mM CaCl(2) was not higher than that in the presence of 2 mM CaCl(2). Therefore, it was concluded that the reduced lysozyme possessing the Ca(2+) binding site was efficiently folded in the presence of high concentration of Ca(2+) (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca(2+) to the polypeptide chain in Ser(-1) CaB lysozyme. 相似文献
13.
Mg(ClO4)2 induces the cooperative B-to-Z transition of poly[d(G-C)]; the salt concentration at the midpoint is 0.26 M. A comparison with previous data for NaCl, MgCl2 and NaClO4 (F.M. Pohl and T.M. Jovin, J. Mol. Biol. 67 (1972) 375) indicates that Mg(ClO4)2 is more effective than would be anticipated from the simple additive effects of the Mg2+ and ClO4- ions (the ionic strengths of the respective transition points are: NaCl, 2.4; MgCl2, 2.1; NaClO4, 1.8 and Mg(ClO4)2, 0.78). These results suggest the importance of specific interactions involving ClO4-, particularly in the presence of Mg2+. The B-Z transition of poly[d(G-C)] can be monitored spectroscopically via the large hyperchromic shift at 295 nm and the inversion in the CD spectrum. The reaction is fully reversible and can be fitted by a monoexponential function with half times varying between 8 and 150 min. The observed relaxation times are strongly dependent on the concentration of Mg(ClO4)2 with a distinct maximum at the transition point, in accordance with a concerted mechanism involving only the B and Z states. As the polymer assumes the Z conformation it progressively aggregates into a gel-like precipitate, which, however, redissolves rapidly upon lowering the salt concentration. The natural DNA from Micrococcus lysodeikticus which has a high GC content of 72% is also precipitated by Mg(ClO4)2 but we do not have direct spectroscopic evidence for the involvement of the Z conformation in this phenomenon. Neither calf thymus DNA (41% GC) nor poly[d(A-T)] (0% GC) aggregates under the same conditions. 相似文献
14.
Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells 总被引:2,自引:0,他引:2
Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
Christopher Earl rew Chantry Nazar Mohammad Paul Glynn 《Journal of neurochemistry》1988,51(3):718-724
Myelin basic protein (MBP) dissociated from brain myelin membranes when they were incubated (37 degrees C; pH 7.4) at physiological ionic strength. Zinc ions inhibited, and calcium promoted, this process. Protease activity in the membrane preparations cleaved the dissociated MBP into both small (less than 4 kilodaltons) and large (greater than 8 kilodaltons) fragments. The latter were detected, together with intact MBP, by gel electrophoresis of incubation media. Zinc ions appeared to act in two distinct processes. In the presence or absence of added CaCl2, zinc ions in the range 0.1-1 mM inhibited MBP-membrane dissociation. This process was relatively insensitive to heat and Zn2+ could be substituted by either copper (II) or cobalt (II) ions. A second effect was evident only in the presence of added calcium ions, when lower concentrations of Zn2+ (less than 0.1 mM) inhibited MBP-membrane dissociation and the accumulation of intact MBP in incubation media. This process was heat sensitive and only copper (II), but not cobalt (II), ions could replace Zn2+. To determine whether endogenous zinc in myelin membranes is bound to MBP, preparations were solubilised in buffers containing Triton X-100/2 mM CaCl2 and subjected to gel filtration. Endogenous zinc, as indicated by a dithizone-binding method, eluted with fractions containing both MBP and proteolipid protein (PLP). Thus, one means whereby zinc stabilises association of MBP with brain myelin membranes may be by promoting its binding to PLP. 相似文献
16.
I G Safonova D D Sviridov G B Men'shikov V S Repin 《Biulleten' eksperimental'no? biologii i meditsiny》1989,108(12):666-668
The effect of temperature and Ca2+ ions on the interaction of high-density lipoprotein (HDL3) with human enterocytes was studied. It was shown that Kd measured at 4 degrees C is similar to that at 37 degrees C. Maximal number of binding sites at 37 degrees C is 15-fold times higher than that at 4 degrees C. EDTA (10 mM) and CaCl2 (0.5-5 mM) did not affect binding and uptake of HDL3 by human enterocytes. The obtained results indicate that HDL3-binding with these cells depends on temperature and does not depend on Ca2+ ions. 相似文献
17.
1. Absorption of ingested calcium (2 ml of a 10mM CaCl2 solution + 45Ca) by the adult rat was shown to be facilitated by the simultaneous ingestion of an active carbohydrate, L-arabinose. As the carbohydrate concentration is increased from 10 to 200 mM, the adsorption of calcium is maximized at a level corresponding to about twice the control adsorption level. 2. A similar doubling of calcium adsorption is obtained when a 100 mM concentration of any one of a number of other carbohydrates (gluconic acid, mannose, glucosamine, sorbitol, lactose, raffinose, stachyose) is ingested simultaneously with a 10 mM CaCl2 solution. 3. Conversely, the simultaneous ingestion of increasing doses (10 to 100 mM) of phosphate (NaH2PO4) with a 10 mM CaCl2 solution results in decreased 45Ca absorption and retention by the adult rat. 4. The maximum inhibition of calcium adsorption by phosphate is independent of the concentration of the ingested calcium solution (from 5 to 50 mM CaCl2). 5. The simultaneous ingestion of CaCl2 (10 mM) with lactose and sodium phosphate (50 and 10 mM, respectively) shows that the activating effect of lactose upon 45Ca adsorption may be partly dissimulated by the presence of phosphate. 6. These various observations indicate that, within a large concentration range (2 to 50 mM CaCl2), calcium adsorption appears to be a precisely modulated diffusion process. Calcium absorption varies (between minimum and maximum levels) as a function of the state of saturation by the activators (carbohydrates) and inhibitors (phosphate) of the calcium transport system. 相似文献
18.
Membrane vesicles were prepared from Artemia nauplii (San Francisco Bay variety) 45 h after hydration of the dry cysts. Na+-loaded vesicles accumulated up to 10 nmol Ca2+/mg protein when diluted 50-fold into 160 mM KCl containing 15 microM CaCl2. Practically no accumulation of Ca2+ was observed if the vesicles were diluted into 160 mM NaCl instead of KCl, or if they were treated with monensin, a Na+ ionophore, for 30 s prior to addition of CaCl2 to the KCl medium. These observations indicate that the Artemia vesicles exhibit Na-Ca exchange activity. The velocity of Ca2+ accumulation by the vesicles in KCl was stimulated 2.6-fold by the K+ ionophore valinomycin, suggesting that the exchange system is electrogenic, with a stoichiometry greater than 2Na+ per Ca2+. Km,Ca and Vmax values were 15 microM and 7.5 nmol/mg protein.s, respectively. Exchange activity in the Artemia vesicles was inhibited by benzamil (IC50 approximately equal to 100 microM) and by quinacrine (IC50 approximately equal to 250 microM), agents that also inhibit exchange activity in cardiac sarcolemmal vesicles. Unlike cardiac vesicles, however, exchange activity in Artemia was not stimulated by limited proteolysis, redox reagents, or intravesicular Ca2+. This indicates that the two exchange systems are regulated by different mechanisms. Vesicles were prepared from Artemia at various times after hydration of the dry cysts and examined for exchange activity. Activity was first observed at approximately 10 h after hydration and increased to a maximal value by 30-40 h; hatching of the free swimming nauplii occurred at 18-24 h. The results suggest that hatching Artemia nauplii might be a particularly rich source of mRNA coding for the Na+-Ca2+ exchange carrier. 相似文献
19.
The one-way fluxes of 14CO2 and a series of 22Na (Cl, Br, HCO3, ClO4, I) salts across n-hexadecane-impregnated solid-support liquid membranes have been measured in water and low dielectric media (50-90 vol% dioxane/water). One-way fluxes for 14CO2 (J14CO2) were 0.84 and 1.03 x 10(-9) mol cm-2 s-1 in 75% dioxane (aq.) and water, respectively, across both impregnated cellulose and teflon membranes. 22Na fluxes across impregnated cellulose membranes in 75% dioxane (aq.) ranged from 1.8 to 11.4 x 10(-10) mol cm-2 s-1 and had the order NaCl less than NaBr less than NaHCO3 less than NaClO4 less than Nal. 22Na fluxes across impregnated teflon membranes were slightly smaller, 1.5-7.1 x 10(-10) mol cm-2 s-1, but had the same order for the anions tested. No measurable 22Na fluxes were observed in aqueous media. For NaI and NaClO4 there was a 3-6-fold enhancement of fluxes in 90% dioxane (aq.) compared to 75% dioxane (aq.). The corresponding enhancement for fluxes of NaHCO3, NaBr and NaCl was 1.5-fold. The results are discussed in terms of ion-paired salt transport in low dielectric media. 相似文献
20.
The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case 总被引:13,自引:0,他引:13
M W Pantoliano M Whitlow J F Wood M L Rollence B C Finzel G L Gilliland T L Poulos P N Bryan 《Biochemistry》1988,27(22):8311-8317
A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' (EC 3.4.21.14) was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65 degrees C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log Ka = 1.49. A series of refined X-ray crystal structures (R less than or equal to 0.15, 1.7 A) of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein (ionic strength = 0.10). X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 A, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献