共查询到20条相似文献,搜索用时 81 毫秒
1.
Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin. 相似文献
2.
D Lichtenberg N Younis A Bor T Kushnir M Shefi S Almog S Nir 《Chemistry and physics of lipids》1988,46(4):279-291
In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder. 相似文献
3.
The rate of peroxidation of unconjugated bilirubin (UCB), catalyzed by horseradish peroxidase (HRP), has been employed by Jacobsen (1969. FEBS Lett. 5: 112-114) to assess the fraction of unbound UCB in the presence of serum albumin. We used this method to examine the interactions of UCB with taurocholate (TC) at pH 8.2, assuming solubilization of UCB by TC is due to pigment binding and/or to partitioning into the micelle, thus rendering UCB unavailable for peroxidation. Inhibition of UCB peroxidation conformed with predictions based on these assumptions and demonstrated significant interaction of UCB with both monomeric and micellar TC. Although significant inhibition of UCB peroxidation was seen with TC monomer, inhibition was even greater with TC micelles. In contrast, pyrogallol, another substrate of HRP, acted very differently in the presence of TC. Inhibition of pyrogallol peroxidation by TC was much less than with UCB and occurred primarily with monomeric TC, with little further inhibition in the micellar range. The results of this study suggest that at taurocholate concentrations above 50 mM, similar to the physiologic bile salt concentrations in human bile, at least 99% of UCB is bound to bile salt, dramatically decreasing the concentration of unbound UCB. Since bile salts also bind Ca2+, they play a dual role in protection against the precipitation of calcium bilirubinate from bile. Therefore, bile salts are a major factor in the prevention of the formation and growth of pigment gallstones. 相似文献
4.
Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions. 总被引:3,自引:0,他引:3
The principles governing the in vitro solubility of the common natural conjugated and unconjugated bile acids and salts in relation to pH, micelle formation, and Ca2+ concentration are considered from a theoretical standpoint and then correlated first with experimental observations on model systems and second with the formation of precipitates containing bile acids in health and disease. In vitro, taurine-conjugated bile acids are soluble at strongly acidic pH; glycine-conjugated bile acids are poorly soluble at moderately acidic pH; and many of the common, natural unconjugated bile acids are insoluble at neutral pH. For both glycine-conjugated and unconjugated bile acids, solubility rises exponentially, with increasing pH, until the concentration of the anion reaches the critical micellization concentration (CMC) when micelle formation occurs and solubility becomes practically unlimited. In vivo, in health, conjugated bile acids are present in micellar form in the biliary and intestinal tract. Unconjugated bile acids formed in the large intestine remain at low monomeric concentrations because of the acidic pH of the proximal colon, binding to bacteria, and absorption across the intestinal mucosa. In diseases in which proximal small intestinal content is abnormally acidic, precipitation of glycine-conjugated bile acids (in protonated form) occurs. Increased bacterial formation of unconjugated bile acids occurs with stasis in the biliary tract and small intestine; in the intestine, unconjugated bile acids precipitate in the protonated form. If the precipitates aggregate, an enterolith may be formed. In vitro, the calcium salts of taurine conjugates are highly water soluble, whereas the calcium salts of glycine conjugates and unconjugated bile acids possess limited aqueous solubility that is strongly influenced by bile acid structure. Precipitation occurs extremely slowly from supersaturated solutions of glycine-conjugated bile acids because of metastability, whereas super-saturated solutions of unconjugated bile acids rapidly form precipitates of the calcium salt. In systems containing Ca2+ ions and unconjugated bile acids, pH is important, since it is the key determinant of the anion concentration. For bile acids with relatively soluble calcium salts (or with a low CMC), the concentration of the anion will reach the CMC and micelles will form, thus precluding formation of the insoluble calcium salt. For bile acids, with relatively insoluble calcium salts (or with a high CMC), the effect of increasing pH is to cause the anion to reach the solubility product of the calcium salt before reaching the CMC so that precipitation of the calcium salt occurs instead of micelle formation.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
5.
【目的】随着抗生素生长促进剂(AGPs)在动物饲料中逐步禁止使用,AGPs替代物的研究成为热点。由于胆盐水解酶(BSH)在脂类代谢中的关键作用,成为AGPs替代物研究的一个重要方向。在原核表达和纯化的基础上鉴定鸡源和猪源乳杆菌BSH在酶学性质方面的差异性。【方法】分别对鸡源胆盐水解酶(BSHc)和猪源胆盐水解酶(BSHp)基因进行原核表达和蛋白纯化,通过测定对6种甘氨结合胆盐和牛磺结合胆盐的水解效率获得两种酶的酶学动力学性质,进而测定了温度、pH和金属离子对酶活力的影响。【结果】BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc对甘氨结合胆盐的水解效率较BSHp稍高;BSHc和BSHp的最适酶解温度分别为45°C和42°C;BSHc和BSHp的最适反应pH分别为6.0和5.4;含有Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)的金属盐对BSHc和BSHp的酶活力均具有不同程度的抑制作用,特别是Cu~(2+)和Fe~(3+)抑制作用比较强;含有Na~+、K~+、Mg~(2+)和Ca2+的金属盐对BSHc和BSHp酶活力的抑制作用相对较弱或无抑制作用,但KIO3对BSHc和BSHp酶活力具有强抑制作用,KI和CaCl_2对BSHp酶活力也具有较强的抑制作用。【结论】原核表达和纯化的BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc的最适酶解温度和pH稍高于BSHp,Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)等金属离子对BSHc和BSHp酶活力具有明显抑制作用,试验结果为鉴定BSH抑制物进而研制AGPs替代物奠定了基础。 相似文献
6.
Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100. 总被引:5,自引:1,他引:4
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100. 相似文献
7.
J J Gu A F Hofmann H T Ton-Nu C D Schteingart K J Mysels 《Journal of lipid research》1992,33(5):635-646
The approximate solubility products of the calcium salts of ten unconjugated bile acids and several taurine conjugated bile acids were determined. The formation of micelles, gels, and/or precipitates in relation to Ca2+,Na+, and bile salt concentration was summarized by "phase maps." Because the ratio of Ca2+ to bile salt in the precipitates was ca. 1:2, and the activity of Ca2+ but not that of bile salt (BA-) could be measured, the ion product of aCa2+ [BA-]2 was calculated. The ion product (= Ksp) ranged over nine orders of magnitude and the solubility thus ranged over three orders of magnitude; its value depended on the number and orientation of the hydroxyl groups in the bile acid. Ion products (in units of 10(-9) mol/l)3 were as follows: cholic (3 alpha OH,7 alpha OH,12 alpha OH) 640; ursocholic (3 alpha OH,7 beta OH,12 alpha OH) 2300; hyocholic (3 alpha OH,6 alpha OH,7 alpha OH) 11; ursodeoxycholic (3 alpha OH,7 beta OH) 91; chenodeoxycholic (3 alpha OH,7 alpha OH) 10; deoxycholic (3 alpha OH,12 alpha OH) 1.5; 12-epideoxycholic (lagodeoxycholic, 3 alpha OH,12 beta OH) 2.2; hyodeoxycholic (3 alpha OH,6 alpha OH) 0.7; and lithocholic (3 alpha OH) 0.00005. The critical micellization temperature of the sodium salt of murideoxycholic acid (3 alpha OH,6 beta OH) was greater than 100 degrees C, and its Ca2+ salt was likely to be very insoluble. Taurine conjugates were much more soluble than their corresponding unconjugated derivatives: chenodeoxycholyltaurine, 384; deoxycholyltaurine, 117; and cholyltaurine, greater than 10,000. Calcium salts of unconjugated bile acids precipitated rapidly in contrast to those of glycine conjugates which were metastable for months. Thus, hepatic conjugation of bile acids with taurine or glycine not only enhances solubility at acidic pH, but also at Ca2+ ion concentrations present in bile and intestinal content. 相似文献
8.
Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions. 相似文献
9.
The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site. 相似文献
10.
11.
William C. Duane 《Biochemical and biophysical research communications》1977,74(1):223-229
The equilibrium of bile salt between aqueous phase and mixed micelle was studied in solutions of pure bile salt and lecithin comparing taurocholate and taurochenodeoxycholate. The relationship between bile salt concentration in the aqueous phase and the ratio of bile salt/lecithin in the mixed micelle was determined by equilibrium dialysis on serial dilutions of these solutions. Extrapolation of this relationship to zero mixed-micellar bile salt permitted calculation of the critical micelle concentration (CMC) of the mixed micelle. For taurocholate, taurochenodeoxycholate, and an equimolar mix of these two bile salts, the mixed micelle CMC's were 3.1 mM, 0.47 mM, and 0.89 mM respectively. In the most concentrated solutions, aqueous phase bile salt concentration surpassed the CMC of the simple bile salt micelle by more than four-fold indicating the presence of simple micelles as well as mixed micelles. At all dilutions taurochenodeoxycholate had a much greater affinity for the mixed micelle than did taurocholate. This last finding may be the reason for the superior cholesterol solubilizing capacity of taurochenodeoxycholate-lecithin solutions compared to taurocholate-lecithin solutions. 相似文献
12.
Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding 总被引:20,自引:0,他引:20
The preferential interactions of proteins with solvent components were studied in concentrated aqueous solutions of the sulfate, acetate, and chloride salts of Mg2+, Ba2+, Ca2+, Mn2+ and Ni2+ [except for CaSO4, BaSO4, Mn-(OAc)2, and Ni(OAc)2], and results were compared with those of the Na+ salts. It was found that, for all the salts, the preferential hydration increased in the order of Cl- less than CH3-COO- less than SO42- regardless of the cationic species used, in agreement with the anionic lyotropic series, and that the same parameter exhibited a tendency to increase in the order of Mn2+, Ni2+ less than Ca2+, Ba2+ less than Mg2+ less than Na+. The salting-out and stabilizing or salting-in and destabilizing effectiveness of the salts were interpreted in terms of the observed preferential interactions. The surface tension increment of salts, which is a major factor responsible for the preferential interactions of the Na+ salts, had no correlation with those of the divalent cation salts. It was shown that the binding of divalent cations to the proteins overcomes the salt exclusion due to the surface tension increase, leading to a decrease in the preferential hydration. In conformity with this mechanism, the preferential interaction of MgCl2 was strongly pH dependent, because of the protein charge-dependent affinity of Mg2+ for proteins, while NaCl showed no pH dependence of the preferential interaction. The proposed mechanism was supported by a strong correlation between the preferential interaction results and the interaction of these salts with the model peptide compound acetyltetraglycine ethyl ester, described by Robinson and Jencks. 相似文献
13.
Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Selective inhibition of [3H]nitrendipine binding to brain and cardiac membranes by bacitracin 总被引:1,自引:0,他引:1
The effects of bacitracin were investigated on [3H]nitrendipine binding to rat brain and cardiac membranes in a low ionic strength (5 mM Tris-HCl) buffer. Bacitracin inhibited [3H]nitrendipine binding to rat brain and cardiac membranes with IC50 values of 400 +/- 100 and 4600 +/- 400 micrograms/mL, respectively. Scatchard analysis in brain membranes revealed that bacitracin inhibited [3H]nitrendipine binding primarily by reducing the Bmax but also by producing a small increase in the Kd. In brain membranes, Na+ (100 mM) and Ca2+ (2 mM) reduced the potency of bacitracin to inhibit [3H]nitrendipine binding by approximately sixfold with IC50 values of 2600 +/- 300 and 2100 +/- 400 micrograms/mL observed for bacitracin in the presence of 100 mM Na+ and 2 mM Ca2+, respectively. The EC50 values for the effects of Na+ and Ca2+ were 800 +/- 200 microM and 25 +/- 5 mM. K+, Mg2+, choline, and increasing the assay buffer of Tris-HCl to 50 mM also decreased the inhibition of [3H]nitrendipine binding by bacitracin. These results suggest that bacitracin specifically modulates [3H]nitrendipine binding in a cation-dependent manner and that brain and cardiac dihydropyridine binding sites are either biochemically different or exist in a different membrane environment. 相似文献
15.
The effect of K+ and Na+ on the Ca2+ binding site in the dense core of monoaminergic vesicles of pineal nerves was investigated in the rat. Rat pineal glands, bisected immediately after decapitation, were incubated at room temperature in solutions containing high K+ or high Na+ in the presence or absence of Ca2+. Fixation was performed in glutaraldehyde-osmium tetroxide in collidine buffer, with and without CaCl2. It was confirmed that, after fixation in Ca2+-containing solutions, an electron-dense particle, located in the vesicle core, which can be considered a calcium deposit, appears within the synaptic vesicles. It was observed that this Ca2+ deposit may be modified by incubation in a high K+ or high Na+ milieu before fixation in Ca2+ containing solutions. When the incubation was carried out with high K+ and high Ca2+ simultaneously, Ca2+ deposits were considerably increased. With K+ alone, no Ca2+ deposits were apparent, as when electrical stimulation is applied before fixation. This effect was not observed when the incubation was done in high Na+. Consecutive incubations in high K+ and high Na+, respectively, restored the capability of the vesicle cores to bind Ca2+. Prolonged incubation in high Na+ before fixation increased Ca2+ deposits within the vesicles. These findings are in line with data on the effect of these ions upon the storage and release of biogenic amines and suggest that these ions modify the capability of synaptic vesicles to bind Ca2+. 相似文献
16.
Electrophoresis was used to study effects of salinity on the characteristics of Ca2+ binding to the outer surface of plasma membrane (PM) of protoplasts isolated from two types of tobacco (Nicotiana tabacum L., cv. Bright Yellow) cultured cells that were adapted (tolerant) and unadapted (sensitive) to 50 mM NaCl stress. Electrophoretic analysis of salt-sensitive NaCl-unadapted cells shows that Na+ induced an appreciably higher degree of reduction in the amount of Ca2+ bound to PM compared with K+ with increasing concentration from 0.1 to 30 mM. In salt-tolerant NaCl-adapted cells, however, both Na+ and K+ ions induced almost the same degree of reduction in the amount of Ca2+ bound to PM in the physiological concentration range of Ca2+ in the medium between 2 and 4 mM. These results suggest that, under the physiological conditions, PM of salt-sensitive NaCl-unadapted cells has an appreciable amount of PM-bound Ca2+ that is desorbed much easier by Na+ than K+, whereas PM of salt-tolerant NaCl-adapted cells has the PM-bound Ca2+ that can be equally desorbed by Na+ and K+. 相似文献
17.
A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements. 相似文献
18.
L G Lelievre J D Potter M Piascik E T Wallick A Schwartz D Charlemagne B Geny 《European journal of biochemistry》1985,148(1):13-19
The Kd for ouabain for inhibition of Na+,K+-ATPase isolated from murine plasmocytoma MOPC 173 cells is 120 microM, but when isolated in the presence of EDTA, it is 100-fold lower (1.2 microM). Simultaneous addition of muscle tropomyosin and calcium to sensitive membranes restored the original insensitivity (tropomyosin bound to the membranes in an irreversible and saturable manner). For comparison 86Rb influx into intact cells, mediated by the Na+,K+-pump, is half-maximally inhibited at 50 microM ouabain. Calcium converts the enzyme to an insensitive form. This appeared to involve calmodulin because after extraction of calmodulin with EDTA and EGTA from sensitive membranes, they could not be made insensitive by the addition of tropomyosin and Ca2+. Addition of exogenous calmodulin to these calmodulin-depleted membranes was required, in addition to tropomyosin and Ca2+, to decrease the ouabain sensitivity. The involvement of calmodulin was further assessed by measuring the range of Ca2+ concentrations required to convert to the insensitive form. At saturating concentrations of tropomyosin, increasing free [Ca2+] up to 3 microM led to an heterogeneous population of Na+,K+-ATPase forms. The calcium dependency was a saturable process. The shift to the insensitive form was half maximal at 0.65 + 0.11 microM free Ca2+ and was abolished by the addition of troponin I or trifluoroperazine (0.1 mM). These results suggest that, in murine plasmocytoma cells, the intrinsic sensitivity of Na+,K+-ATPase to ouabain might be regulated by a calmodulin-dependent process within a submembrane contractile-like environment. 相似文献
19.
Competition of the trivalent cation, Co(NH3)(3+)(6), with K+ and Na+ ions in binding to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH), containing different combinations and concentrations of KCl and NaCl and constant concentration (0.8 mM) of Co(NH3)(6)Cl(3). The degree of Co(NH3)(3+)(6) binding to DNA does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na+ or K+). The ion exchange selectivity coefficient of monovalent-trivalent ion competition, D(1)(c3), increases with the concentration of Me+, C(o)(+), and the monotonic dependence of log D(1)(c3) vs log C(o)(+) has an inflection between 100 and 300 mM that is caused by a structural transformation of DNA from A- to B-form. The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, discretely charged polyions with density and spatial distribution of the charged groups modeling B- and A-forms of DNA. The GCMC method for discretely charged models of the DNA polyion produces a quantitative agreement with experimental data on trivalent-monovalent ion competition in dependence on DNA structural state and salt concentration. Based on this and previous studies it is concluded that the affinity of DNA for the cations decreases in the order Co(NH3)(3+)(6) > Ca2+ > Mg2+ > Na+ approximately K+ > Li+. DNA does not exhibit selectivity for Na+ or K+ in ethanol/water solutions either in the absence or in the presence of Co(NH3)(3+)(6), Ca2+, and Mg2+. 相似文献
20.
A von Kieckebusch U Fritsche V Vogel V Witzemann V P Whittaker 《Biochimica et biophysica acta》1988,957(1):131-137
The Ca2+-regulated lipid-binding properties of the H- and L-forms of calelectrin present in the electric organ of Torpedo marmorata have been compared. Binding of H-calelectrin required Ca2+ in millimolar concentrations, whereas that of L-calelectrin occurred in the micromolar range. Dissociation of H-calelectrin previously bound to lipids in the presence of 2 mM Ca2+ took place only when the Ca2+ concentration was reduced to micromolar concentrations. Binding was most effective to acidic phospholipids such as phosphatidylserine. Both forms of calelectrin promoted the aggregation of membrane vesicles in the presence of Ca2+.Mg2+, Na+ and K+ inhibited the Ca2+-induced binding to phospholipid, decreasing in effectiveness in that order. Binding was also inhibited by high pH. The surface activity and hydrophobicity index showed that H-calelectrin is a hydrophilic molecule. It may represent a less active, more highly phosphorylated "down-regulated" form of L-calelectrin. The role of calcium in H-calelectrin binding to lipid appeared to be consistent with the formation of a ternary complex of the protein, an acidic lipid and Ca2+, rather than with a direct interaction of lipid with hydrophobic sequences in H-calelectrin whose accessibility is Ca2+-regulated. 相似文献