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PubMed1988

Characterization of complexes of egg yolk phosphatidylcholine and apolipoprotein A-II prepared in the absence and presence of sodium cholate.

Complexes of apolipoprotein A-II and egg yolk phosphatidylcholine were prepared in mixtures of different composition in the absence and presence of sodium cholate. By gradient gel electrophoresis, complex preparations were polydisperse and particle size distributions were influenced by the composition of the reconstitution mixture. Complexes generally exhibited a discoidal morphology by electron microscopy, but showed increased formation of vesicular complexes at elevated levels of egg yolk PC in the mixtures. By chemical crosslinking, complexes formed in the absence of cholate were shown to consist primarily of discoidal species with three apolipoprotein A-II molecules per particle in the mixtures investigated; complexes formed in the presence of cholate included species ranging from three to five apolipoprotein A-II per particle. The number of apolipoprotein A-II per particle and the sizes of the complexes, prepared in cholate, increased with increase of egg yolk PC in the reconstitution mixture. Relative to the particle size distribution of discoidal complexes formed in the absence of cholate, those prepared in cholate showed a distribution shifted to larger particle sizes. Complexes of similar particle size distribution formed in the presence or absence of cholate showed similar physical-chemical properties. Discoidal complexes with the same number of apolipoprotein A-II per particle but of different size and composition were observed, suggesting the possibility of some conformational adaptation of apolipoprotein A-II leading to stabilization of egg yolk PC bilayers of different diameter. Properties of particle size distributions of discoidal complexes prepared in cholate of apolipoprotein A-II and egg yolk PC were compared with those of complexes of apolipoprotein A-I previously reported (Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) Biochim. Biophys. Acta 750, 353-364).

Apolipoprotein A-II↗
PubMed1990

Identification of cholate as a shared substrate for the unidirectional efflux systems for methotrexate in L1210 mouse cells.

The bidirectional transport properties of cholate have been examined in leukemic L1210 mouse cells and compared with the transport of methotrexate. The cell entry of [3H]cholate was Na(+)-independent, linear with increasing concentrations of substrate, enhanced by decreasing pH, and uneffected by excess unlabeled cholate or by various anion-transport inhibitors and hence had the characteristics of passive diffusion or a pH-dependent mediated process with a high Kt for cholate. The efflux of [3H]cholate, however, could be attributed to carrier-mediated and energy-dependent transport. Efflux was rapid (t1/2 = 1.5 min) and could be increased with glucose and decreased with metabolic inhibitors, and it was inhibited by various compounds including bromosulfophthalein, probenecid, prostaglandin A1, reserpine, verapamil, quinidine, diamide, 1-methyl-3-isobutylxanthine and vincristine. The most potent inhibitor was prostaglandin A1, which reduced efflux by 50% at a concentration of 0.10 microM. Half-maximal inhibition by vincristine occurred at 4.8 microM. The maximum extent of inhibition with most of the inhibitors was 95%, although a lower value was observed with bromosulfophthalein (85%). When cholate efflux was compared with the efflux of methotrexate, both processes responded similarly to changes in the metabolic state of the cell. Moreover, the various inhibitors of cholate efflux also inhibited the efflux of methotrexate and the same concentration of each inhibitor was required for half-maximal inhibition of both processes. The efflux of folate and urate also proceeded via outwardly directed, unidirectional processes which were sensitive to bromosulfophthalein and probenecid. The results suggest that L1210 cells have the capacity for the unidirectional extrusion of cholate, methotrexate and probably other large, structurally dissimilar organic anions and that this efflux occurs via two or more very similar transport systems with a broad anion specificity. The function of an organic anion efflux system in vivo may be to facilitate the extrusion of cytotoxic metabolic anions which are too large to exit via the general anion-exchange carrier of these cells. Similarities in inhibitor specificity were also apparent between unidirectional anion efflux in L1210 cells and the drug efflux pump which is over-produced in cells with multidrug resistance.

Animals↗
PubMed2003

Temperature effect on formation of sodium cholate micelles.

The micellization of sodium cholate (NaC) at 293.2, 298.2, 303.2, 308.2, and 313.2 K by cholate anion concentration was studied over the pH range from 6.0 to 7.2. Using a stepwise association model of cholate anions without bound sodium counterions, the aggregation number (nmacr;) of the cholate micelles was evaluated and found to increase with the total concentration, indicating that the stepwise association model is applicable. The nmacr; values go up and down with increasing temperature; 17 at 298.2 and 12 at 313.2 K and at 60 mM of the sodium cholate. The fluorescence of pyrene was measured in sodium cholate solution to determine the critical micelle concentration (CMC), indicating a narrow concentration range for CMC. A sodium-ion-specific electrode was used to determine a relatively low degree of counterion binding to micelles, supporting the validity of the present association model of cholate anions. The aggregation numbers evaluated at a constant ionic strength of 0.15 and at lower but variable ionic strengths were similar except for higher cholate concentrations.

Journal Article↗
PubMed1987

Multiple equilibria binding treatment of lipid and detergent interactions with membrane proteins. Application to cytochrome c oxidase solubilized in cholate.

A modified multiple binding equilibria treatment is presented that allows determination of thermodynamic parameters of the interaction of phospholipids with integral membrane proteins solubilized in excess detergent. Lipid binding is modeled as a series of exchange reactions between lipid molecules and detergent molecules at the hydrophobic protein surface. A general equation is derived which expresses a relative association constant (K) and the total number of contact sites at the lipid-protein interface (N) in terms of experimentally measurable variables. A useful simplification of the general equation occurs when the amount of detergent is high relative to the total number of lipid binding sites in the sample. Computer simulations show that in cases we have examined there appears to be an experimentally accessible range of detergent to protein molar ratios where the approximation at high detergent is useful for analyzing experimental data. This model is used to examine the competition between cholate and spin-labeled phospholipids for the hydrophobic surfaces of bovine heart cytochrome c oxidase. We find, for example, that K = 12 +/- 2 for phosphatidylcholine relative to cholate (i.e., the cholate molecules are relatively easily displaced by membrane lipids). This helps to explain the experimental observation that cholate is an effective detergent both for solubilizing cytochrome c oxidase and for reconstituting this protein into a defined lipid bilayer environment. An excess of cholate readily displaces almost all of the native phospholipids, and the protein is dispersed in cholate micelles. However, when phospholipids are added back, the cholate molecules at the protein surface are replaced because of the higher relative binding of the phospholipids. Observed differences between the behavior of phosphatidylcholine and phosphatidylglycerol suggest that reconstitution in cholate is a selective process in which detergent molecules in localized areas on the protein surface are more readily displaced by certain phospholipids.

Animals↗
PubMed1995

Simultaneous dietary supplementation of sodium cholate and beta-carotene markedly enhances accumulation of beta-carotene in mice.

This study evaluated whether simultaneous supplementation of sodium cholate and beta-carotene to a diet enhanced the accumulation of beta-carotene in mice. For 2 wk, male ICR mice were fed either a basal diet or a diet containing Dunaliella-bardawil beta-carotene 50 mg/100g that was or was not supplemented with sodium cholate (0.25 g/100 g). The concentrations of beta-carotene in liver and plasma were approximately 5 and 10 times higher, respectively. In the mice fed the beta-carotene diet with sodium cholate than in those fed the beta-carotene diet without sodium cholate. Beta-carotene was not detectable in the liver or plasma of mice fed either basal diet. The concentrations of vitamin E in the plasma and liver of mice fed either beta-carotene diet or the basal diet with sodium cholate were significantly lower than in those fed the basal diet. In a second study, mice were fed a diet containing 50 mg/100 g synthetic beta-carotene supplemented with various concentrations of sodium cholate (0, 0.05, 0.1, 0.25, 0.5 g/100 g) for 2 wk. The concentrations of beta-carotene and vitamin E in plasma, liver and bone marrow cells were higher in mice fed the beta-carotene diet supplemented with 0.05 g/100 g of sodium cholate than in those fed the unsupplemented diet. These findings show that simultaneous supplementation of sodium cholate and beta-carotene to a diet markedly enhances the accumulation of beta-carotene. This dietary protocol may be useful to introduce a high amount of beta-carotene in the tissue of mice in a short period of time.

Animals↗
PubMed1991

Evidence for cAMP and cholate extrusion in C6 rat glioma cells by a common anion efflux pump.

C6 rat glioma cells were investigated for a shared unidirectional efflux system for cAMP and cholate. [3H]Cholate was accumulated (at pH 7.3) by scraped C6 cell monolayers via a process which was rapid initially and then slowed to a steady state after 10 min at 37 degrees C. Release of the accumulated label was also rapid (t1/2 = 2 min), was essentially complete within 15 min, and exhibited energy dependence since it could be blocked by antimycin A. Half-maximal inhibition by antimycin A occurred at 0.87 microM, and maximal inhibition exceeded 90%. Various other compounds also inhibited [3H]cholate efflux. The most effective was prostaglandin A1, which reduced efflux half-maximally at a concentration of 0.14 microM. Other inhibitors, prostaglandin B1, verapamil, probenecid, and bromosulfophathalein, produced half-maximal inhibition at 5.3, 42, 78, and 110 microM, respectively. Cholate efflux was also blocked by 40 microM vincristine. Initial influx of [3H]cholate was not affected by antimycin A, prostaglandin A1, or vincristine and hence was attributed to a process separate from efflux. C6 rat glioma cells also have the ability to produce high intracellular levels of cAMP in response to isoproterenol and to release cAMP into the medium via a carrier-mediated efflux system. When measured under the same conditions employed for cholate efflux, the efflux of cAMP was found to be sensitive to each of the inhibitors of cholate efflux. Moreover, plots of cAMP efflux versus varying concentrations of prostaglandin A1, antimycin A, prostaglandin B1, verapamil, and probenecid showed similar response curves and comparable values for half-maximal These results indicate that C6 rat glioma cells contain a unidirectional efflux pump for cholate and that this same system also appears to mediate the unidirectional efflux of cAMP. These findings support the hypothesis that various cells contain efflux pumps which exhibit a broad specificity for large organic anions of diverse structure and that the function of these efflux pumps resides primarily in cellular anion detoxification. Analogous efflux pumps for hydrophobic drugs are overproduced in tumor cells exhibiting multidrug resistance.

Animals↗
PubMed1988

Hydrolysis of phosphatidylcholine in phosphatidylcholine-cholate mixtures by porcine pancreatic phospholipase A2.

Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1974

Alteration of the kinetics of thrombin-catalyzed hydrolysis of amino acid ester substrates by sodium cholate and other steroids.

Thrombin-catalyzed hydrolysis of TAME proceeds by an initial zero-order phase which later falls off into an apparent first order reaction as substrate becomes limiting. Optimum amounts of sodium cholate not only accelerated TAME hydrolysis but also altered its kinetics to apparent zero-order to complete substrate hydrolysis. As this implies, the rate of hydrolysis in the presence of cholate was found to be independent of substrate concentration, provided concentrations TAME and cholate were low enough to prevent precipitation of some TAME-cholate as an insoluble complex. The formation of a soluble complex composed of polymeric molecules of TAME and cholate may explain both the acceleration and the change in reaction order. Although the pH and temperature optima for TAME hydrolysis by thrombin rising pH and temperature on the ascending protion of the curves. This is believed to be due to the greater solubility of the TAME-cholate complex. The effects of cholate on thrombin-catalyzed hydrolysis of other arginine esters as well as esters of lysine, histidine and phenylalanine were also studied. Solutions of sodium desoxycholate and androsterone-3-sulfate accelerated TAME hydrolysis as did supensions of testosterone, etiocholanolone, androsterone, androsterone-3-hemisuccinate and pregnandiol-3-glucuronidate. However, isoandrosterone, progesterone, pregnandiol, estradiol, estrone, estriol, estrone-3-sulfate, cholesterol, corticosterone, hydrocortisone and hydrocortisone-3-phosphate had no significant effect on TAME hydrolysis by thrombin. The ability of the andorogenic hormones to accelerate hydrolysis appeared to depend to some extent on the configuration of the substituent group at C3 and the hydrogen at C5. Androsterone-3-hemisuccinate was, like cholate, able to accelerate the hydrolysis of TAME at apparent zero-order kinetics to complete substrate hydrolysis.

Adrenal Cortex Hormones↗
PubMed2005

Cholate and deoxycholate counteract the calcium-induced lowering of fat digestion in rats.

The objective of the present experiment was to investigate whether deoxycholate and cholate would differ in their effectiveness of counteracting the inhibitory effect of calcium on fat digestibility in rats. Rats were fed one of four experimental diets, a diet low in calcium, high in calcium or high in calcium with either 0.5% sodium cholate or 0.5% sodium deoxycholate. Both deoxycholate and cholate supplementation of the high-calcium diet reduced feed intake and body-weight gain. Low-calcium intake increased fat digestibility. Supplemental bile acids partially counteracted the calcium-induced inhibition of fat digestion, cholate being more effective than deoxycholate. The outcome is explained by the suggestion that cholate is bound to the calcium phosphate sediment in the small intestinal lumen with less affinity than deoxycholate. As a result, more cholate than deoxycholate would be available to support the process of fat digestion. Rats fed cholate had higher liver and serum cholesterol concentrations than did the rats fed deoxycholate.

Animals↗
PubMed2000

Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter.

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L. lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.

Adenosine Triphosphate↗
PubMed2003

Evaluation of efficiency of insulin suppository formulations containing sodium salicylate or sodium cholate in insulin dependent diabetic patients.

Two formulations of insulin suppositories were prepared to contain different amounts of sodium salicylate and sodium cholate as absorption promoters and also of insulin with the purpose of obtaining the most effective formulation in reducing plasma glucose levels after rectal administration to diabetic patients. The results show that insulin suppositories containing 100 mg sodium salicylate and 100 or 200 U of crystalline insulin showed no significant difference in AUC, Cmax and Tmax and both formulations showed significant reduction in plasma glucose level compared to initial values within 1.5-2 h. The results from experiments carried out in health volunteers showed that 100 mg sodium salicylate is the optimum amount to be included in insulin suppositories producing significantly higher Cmax and AUC compared to those produced after rectal administration of insulin suppositories containing 50 or 200 mg sodium salicylate. The results also show that using sodium cholate in 50 mg amount did not produce any significant reduction in plasma glucose levels of insulin dependent diabetic patients given suppositories containing 100 U of insulin, but this amount in suppositories containing 200 U of insulin was able to produce significant (p < 0.05) reduction in plasma glucose level within 1 h which lasted till end of experiment producing Cmax of 29.7 +/- 6.61% at Tmax of 1.5 +/- 0.61 h. On increasing the amount of sodium cholate to 100 mg in the suppositories, a marked (p < 0.01) reduction in plasma glucose level took place and the Cmax increased to 47.7 +/- 12.24% at Tmax of 1.5 +/- 0.63 h. This resulted in AUC of 86.7 +/- 22.4 mg%h which was non significantly higher from that produced after administration of suppositories containing 50 mg sodium cholate and 200 U insulin (62.5 +/- 17.6 mg%h). The results also show that insulin suppositories containing 100 mg sodium cholate and 200 U insulin resulted in a non significant differences in Cmax and AUC from those produced by S.C. injection of insulin (20 U) but significantly (p < 0.001) shorter Tmax. This formulation also shows non significant differences in Tmax and AUC and significantly (p < 0.05) higher Cmax than from those produced after rectal administration of suppositories containing 100 mg of sodium salicylate and same amount of insulin. Further more this formulation produced severe hypoglycemia in control healthy volunteers within 1 h of administration producing Cmax of 57.0 +/- 18.8% at Tmax of 0.75 +/- 0.35 h. The results of this study showed that the formulation containing 100 mg of sodium cholate and 200 U of insulin tested in fasted insulin dependent diabetic patients produced a maximum % reduction in plasma glucose levels (Cmax) of 47.7 +/- 12.24% at tmax of 1.5 +/- 0.63 h compared to Cmax of 50.56 +/- 6.8% at tmax of 2.93 +/- 0.19 h resulted after subcutaneous injection of 20 U insulin. These suppositories produced an area under the curve (AUC) of 87 +/- 22.4 mg%h compared to an AUC of 81 +/- 13.4 mg%h obtained after subcutaneous injection. This formulation of suppositories studied in 7 insulin dependent diabetic patients was found to abolish the 2-h post-prandial significant rise in plasma glucose levels after meal. These results show that these insulin suppositories containing 100 mg of sodium cholate and 200 U of insulin can serve as effective buffer against meal related hyperglycemia. The suppositories were safe, effective, accepted and well tolerated by the tested individuals.

Adult↗
PubMed1998

Equilibrium Penetration of DMPC Monolayers by Sodium Cholate.

The interaction of dimyristoyl phosphatidylcholine (DMPC) with sodium cholate was studied by equilibrium penetration measurements. Surface pressure-area isotherms for DMPC on substrates containing various concentrations of sodium cholate are presented. From these isotherms, the saturation adsorptions of the bile salt for various surface concentrations of DMPC were calculated. It is shown that the equilibrium penetration of DMPC monolayers by sodium cholate can be satisfactorily described by the accessible-area theory of McGregor and Barnes. A plot of the surface excess concentration of cholate against the inverse of the area per DMPC molecule is linear. This plot yields a value for the adsorption into the accessible areas of the monolayer-covered surface that corresponds to the adsorption of cholate in a monolayer-free surface. The cholate molecules lie flat on the air-water interface. The effective cross-sectional area of the DMPC in the mixed monolayer is similar to the area occupied by a DMPC molecule in a close-packed liquid expanded monolayer. The composition of the penetrated monolayer at different areas per DMPC molecule was calculated. The penetration of cholate into DMPC monolayers is correlated with different results taken from the literature dealing with the incorporation of bile salts into lecithin bilayers. Copyright 1998 Academic Press.

Journal Article↗
PubMed1983

Common properties of hepatocellular uptake of cholate, iodipamide and antamanide, as distinct from the uptake of bromosulfophthalein.

The uptake of iodipamide and of the cyclopeptide antamanide by isolated hepatocytes was reduced reversibly in the absence of oxygen as recently shown for the transport of cholate. Oligomycin, antimycin A and carbonylcyano-chlorophenylhydrazone (CCCP) completely blocked the uptake of iodipamide and antamanide whereas the uptake of cholate was only partially decreased. Reduction of ATP in hepatocytes following replacement of glucose by fructose inhibited the uptake of iodipamide, of antamanide, and also of cholate. In contrast, the penetration of bromosulfophthalein remained unaffected under the above conditions. Arrhenius paralysis yielded high apparent activation energies for the uptake of cholate, iodipamide, and antamanide being 89, 77 and 55 kJ/mol respectively but only 22 kJ/mol for bromosulfophthalein. Mutual transport inhibition was found for iodipamide, antamanide and cholate as well as for bromosulfophthalein. Cholate inhibited the uptake of iodipamide and antamanide competitively. In contrast, bromosulfophthalein inhibited iodipamide uptake in a mixed order fashion. The results suggest a common uptake mechanism for cholate, iodipamide and antamanide different from that of bromosulfophthalein.

Absorption↗
PubMed1995

The effect of albumin on the state of aggregation and phase transformations in phosphatidylcholine-sodium cholate mixtures.

Dilution of phospholipid-detergent mixed micellar systems results in an increase of the fraction of monomeric detergent and, consequently, in a decrease of the effective ratio Re between non-monomeric detergent and phospholipid. The value of Re has been previously shown to be the main determinant of the state of aggregation in such mixtures: at Re values below a critical value ReSAT, the mixture is vesicular; at Re values higher than ReSOL, the mixture is micellar, whereas within the range of ReSAT-ReSOL, vesicles and micelles co-exist. Albumin binds bile salts. Therefore, in PC-cholate mixtures, Re is reduced by the presence of albumin in the system. Within the range of PC concentrations of 2-23 mM, cholate concentrations of 2-15 mM and BSA concentrations of 0-100 mg/ml, binding of cholate to BSA results in reduction of the effective cholate concentration to the extent of 0.11 mM cholate per 1 mg/ml BSA, namely up to 7 cholate molecules bind to each BSA molecule. Yet, the values of ReSAT and ReSOL are essentially independent of BSA. In addition, at any given Re value, the size of vesicles made by dilution of mixed micelles is a complex function of albumin and PC concentrations. Possible mechanistic details which may cause this effect are discussed. These effects of albumin on the state of aggregation of PC-cholate mixtures must be taken into account in studies of such mixtures in the presence of albumin when other effects of albumin (e.g., on phospholipolysis) are investigated. Practical conclusions are reached with respect to the procedures that can be used to prepare vesicles of identical composition and size in the presence of different concentrations of albumin.

Cholic Acid↗
PubMed1988

Effect of calcium on kinetic and structural aspects of dilution-induced micellar to lamellar phase transformation in phosphatidylcholine-cholate mixtures.

Previously, we have shown [Almog, S., Kushnir, T., Nir, S., & Lichtenberg, D. (1986) Biochemistry 25, 2597-2605] that the distribution of cholate between phosphatidylcholine (PC) vesicles and aqueous media apparently obeys a single distribution coefficient, K. In PC-cholate mixed micellar systems, the monomer concentration does not rise much above the cholate's critical micelle concentration (cmc). Consequently, for vesicular systems, the cholate:PC molar ratio in the mixed aggregates (Re) is given by Re = [cholate]/([PC] + 1/K) whereas for mixed micellar systems Re = ([cholate] - cmc)/[PC]. Dilution of mixed micellar systems results in a decrease of Re, due to an increase in the fraction of monomeric PC. If the decrease in Re is to values lower than 0.3, micellar to lamellar transformation occurs. This process involves a sequence of three steps, namely, micellar equilibration followed by vesiculation and subsequent vesicle size growth via a lipid transfer mechanism. The ultimate size of the resultant vesicles is an increasing function of Re. This work is devoted to the effect of calcium on the dilution-induced vesicle formation. Its major findings and conclusions are as follows: (i) Calcium reduces the cmc of the detergent and raises its distribution coefficient between PC vesicles and the aqueous medium. Thus, for any given cholate and PC concentrations, calcium causes an increase of Re. (ii) The rate of all the steps which ultimately lead to an apparent equilibrium vesicle size distribution increases dramatically with increasing calcium concentration. Thus, equilibration is attained in seconds to minutes rather than many hours required in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1997

In situ aggregational state of M13 bacteriophage major coat protein in sodium cholate and lipid bilayers.

The in situ aggregational behavior of the bacteriophage M13 major coat protein was determined for the protein isolated in sodium cholate and reconstituted into DOPC lipid bilayers. For this purpose, the cysteine mutants A49C and T36C of the major coat protein were labeled with either a maleimido spin-label or a fluorescence label (IAEDANS). The steric restrictions sensed by the spin-label were used to evaluate the local protein conformation and the extent of protein-protein interactions at the position of the labeled residue. In addition, fluorescent labels covalently attached to the protein were used to determine the polarity of the local environment. The labeled coat protein mutants were examined under different conditions of protein association (amphiphile environment, ionic strength, temperature, and pH). The aggregational state of the major coat protein solubilized from the phage particle in sodium cholate was not dependent on the ionic strength, but was strongly dependent on cholate concentration and pH during sample preparation. At pH 7.0 and high sodium cholate concentration, the protein was in a dimeric form. The unusually strong association properties of the protein dimer in sodium cholate at pH 7.0 were attributed to the inability of sodium cholate to disrupt the strong hydrophobic forces between neighboring protein subunits in the phage particle. Such a "structural protein dimer" was, however, completely and irreversibly disrupted at pH 10.0. Qualitatively the same aggregational tendency was found upon changing the pH for the coat protein reconstituted in DOPC lipid bilayers. This reveals that the dimer disruption process is primarily a protein property, because there are no titratable groups on DOPC in the experimental pH range. The results are interpreted in terms of a model relating the protein aggregational state in the assembled phage to the protein aggregational behavior in sodium cholate and lipid bilayers.

Bacteriophage M13↗
PubMed1978

Protein-lipid interactions in cytochrome oxidase from Saccharomyces cerevisiae. Effects of detergents and reconstitution of enzyme activity by phospholipids by using cholate-mediated exchange.

Cytochrome oxidase, purified from the yeast Saccharomyces cerevisiae, was shown to have associated phospholipid, cholate or detergent, which could be varied by dialysis or (NH4)2SO4 precipitation of the protein. Cholate and the detergents Triton X-100 and Tween 80 were shown to differ in their ability to support enzyme activity. Changes in the Vmax, but not the Km, for ferrocytochrome c as the cholate concentration was varied indicate that cholate increases the number of exposed active sites of the enzyme. Cholate was used to introduce chosen phospholipids into the lipid environment of yeast cytochrome oxidase. Kinetic studies clearly showed that cholate can mediate exchange of exogenous for endogenous phospholipid. All phospholipids screened supported activity up to the basal value for the unsubstituted enzyme, whereas mitochondrial phosphatidylethanolamine and various phosphatidlycholines (except 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) produced enhanced activity. A detailed kinetic examination revealed that the major effect of phosphatidylethanolamine is to increase k+1, whereas the major effect of phosphatidylcholine is to increase K+2 in the minimal kinetic scheme E + S k+1 in equilibrium k-1 ES k+2 leads to E + P Cardiolipin, although supporting activity, does not give any enhancement of k+1 or k+2 over the values for the cholate control. The relevance of these observations to protein-lipid interactions in cytochrome oxidase is discussed.

Cholic Acids↗
PubMed1994

Cholate extracts of mitochondrial outer membranes increase inhibition by malonyl-CoA of carnitine palmitoyltransferase-I by a mechanism involving phospholipids.

It has been reported that sodium cholate can separate the catalytic component of carnitine palmitoyltransferase-I (CPT-I) from a putative malonyl-CoA-binding regulatory protein capable of conferring sensitivity to malonyl-CoA on CPT-II. We found that cholate preferentially extracted a contaminating malonyl-CoA-sensitive CPT from mitochondrial inner membranes. When cholate extracts of outer membranes were incubated either with cholate extracts of inner membranes or with osmotically swollen mitochondria, inhibition of CPT by malonyl-CoA was increased. Treatment of intact mitochondria with subtilisin abolished the increased inhibition by malonyl-CoA, suggesting that the outer-membrane CPT-I was responsible for the increased inhibition. Incubation of cholate extracts with proteinase K did not prevent the increased inhibition. Fractionation of the cholate extract indicated the presence of phospholipids. Addition of cardiolipin or phosphatidylglycerol to osmotically swollen mitochondria increased sensitivity of CPT to malonyl-CoA, but several other phospholipids did not. When cardiolipin was added to intact mitochondria from either starved or fed rats, there were large increases in inhibition by malonyl-CoA; sensitivity in mitochondria from starved rats increased to that normally observed with mitochondria from fed rats. These results suggest that phospholipids are responsible for the increased inhibition of CPT by malonyl-CoA with added cholate extracts and that changes in membrane composition may be involved in the physiological regulation of CPT-I.

Animals↗