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PubMed1990

Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins.

Proteoliposomes were reconstituted by detergent dialysis of a sodium cholate extract of inverted vesicles derived from Escherichia coli plasma membrane. The translocation of precursor proteins into reconstituted vesicles occurred at high efficiency and was SecB dependent. The protein composition of the reconstituted vesicles differed markedly from that of native vesicles. Immunoblot analysis of the sodium cholate extract and of the reconstituted vesicles indicated that PrlA (SecY) protein remained largely unsolubilized under the described conditions and was virtually absent from the reconstituted vesicles, suggesting that PrlA may not be required for in vitro translocation.

ATP-Binding Cassette Transporters↗

PubMed1981

Different states of aggregation for unbleached and bleached rhodopsin after isolation in two different detergents.

Phospholipid-free rhodopsin has been purified in the detergents sodium cholate and octaethylene glycol n-dodecyl ether (C12E8). In both detergents, the native absorption spectrum of the unbleached protein is maintained; however, upon photolysis, the preparation in C12E8 loses its ability to recombine with 11-cis-retinal, whereas the preparation in cholate does not. The circular dichroic spectra of the protein in the two detergents are nearly identical, indicating that the secondary structure of the protein is the same in the two detergents. The state of association of the protein in the two detergents is different. In sodium cholate, the smallest species present was found to be a trimer of the rhodopsin polypeptide chain, and this association was unaffected by exposure to light. On the other hand, in C12E8, the protein is monomeric and undergoes a nonspecific aggregation process on exposure to light. These results suggest that protein--protein interactions may play an important role in the stabilization of the native structure of rhodopsin.

Animals↗

PubMed2005

O-palmitoylcurdlan sulfate (OPCurS)-coated liposomes for oral drug delivery.

O-Palmitoylcurdlan sulfate (OPCurS) was applied to the liposomal surface to improve the stability of liposomes. To synthesize OPCurS, curdlan was chemically sulfated and then modified with a palmitoyl derivative. The synthesized OPCurS was characterized by Fourier transform-infrared spectroscopy (FT-IR). OPCurS-coated liposomes prepared by the solvent evaporation method were characterized for size, shape, surface charge, and stability in simulated gastric fluid (SGF) and sodium cholate solution. The sizes of OPCurS-coated liposomes increased with the OPCurS content of liposomes and zeta potential decreased when OPCurS was applied to the liposomal surface. With the increase in the content of OPCurS attached to the liposomal surface, the stability of liposomes in SGF and sodium cholate solution was gradually induced and the stability was most improved at a lipid/OPCurS weight ratio of 1.5. Liposomes not coated with OPCurS released 99.5+/-2.3% of the initial 5-carboxyfluorescein (5-CF) content, whereas OPCurS-coated liposomes released 53.7+/-3.7%. OPCurS on the surface of liposomes suppressed the release of 5-CF. Theses results indicate that OPCurS-coated liposomes can be effectively used as a drug delivery carrier via oral administration.

Administration, Oral↗

PubMed1988

Constitutive testosterone 6 beta-hydroxylase in rat liver.

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

In vivo regulation of hepatic lipase activity and mRNA levels by diets which modify cholesterol influx to the liver.

The aim of this study was to assess whether diets enriched in cholesterol, sodium cholate and drugs known to modify liver cholesterol biosynthesis can modulate hepatic lipase (H-TGL) expression and activity in vivo. Female lean Zucker rats, known to be good responders to cholesterol, were fed for 7 days with a control C diet or the C diet supplemented (w/w) with either 2% cholesterol, 0.5% sodium cholate, 2% cholestyramine or simvastatin (0.1%) added to the cholestyramine diet or given by gavage (10 mg/rat) for 3 days. H-TGL activity decreased by 34% with cholesterol, and by 27% when both cholesterol and cholate were administered to the rats. Under these conditions, H-TGL mRNA decreased by 34% and 87%, respectively. The sharp decrease in H-TGL expression was associated with a strong increase in cholesteryl ester in total liver and in the liver microsome fraction. H-TGL activity decreased by 33% with cholestyramine and the mRNA level decreased by 47%. Simvastatin lowered H-TGL activity by 55% when added to the cholestyramine diet, probably because of a reduction in food intake. When administrated by gavage, simvastatin increased both the H-TGL activity (by 28%) and mRNA (by 23%). These variations may be linked to the availability of mevalonate-derived sterol and non-sterol products.

Animals↗

PubMed1989

Anti-coccidial activity of human milk.

Sporozoites of Eimeria tenella, an important pathogen of poultry, were killed in vitro in a time- and concentration-dependent manner by exposure to diluted concentrations of normal human milk. Sodium cholate (bile salt stimulator) potentiated the anti-coccidial activity. The anti-coccidial activity was not found in the milk of lower mammals (cow, sheep, goat, dog). The component in human milk showing the activity was initially hypothesized to be a bile salt-stimulated lipase. However, testing of purified lipase (with or without sodium cholate) indicated no anti-coccidial activity. Consequently, we theorize that the active component may be a free fatty acid.

Animals↗

PubMed1980

Reconstitution of a functional acetylcholine receptor. Conservation of the conformational and allosteric transitions and recovery of the permeability response; role of lipids.

The 'functional' state of the acetylcholine receptor protein has been followed during reconstitution with the fluorescent agonist [1-(5-dimethylaminonaphthalene)-sulfonamidol]-n-hexanoic acid-beta-N-trimetylammonium bromide ethyl ester (Dns-C6-Cho) and rapid-mixing techniques. Under appropriate conditions, a majority of the acetylcholine receptor sites can be recovered in a low-affinity state(s) for Dns-C6-Cho, similar to that found with the native membrane-bound receptor. This state can be slowly interconverted to a high-affinity state after rapid mixing with the agonist, and the non-competitive channel blockers, like the local anesthetics, still regulate this transition in an allosteric manner. Several experimental conditions commonly used for the solubilization of the receptor and for its purification in the presence of sodium cholate result in the failure of reconstitution: the soluble receptor protein is stabilized in a low-affinity state which can no longer be interconverted to a high-affinity state in the presence of agonists or local anesthetics. On the other hand, it is demonstrated that if the concentration of lipids remains elevated in the presence of sodium cholate, a soluble (9-S) low-affinity form of the receptor protein can be obtained which shows most of the characteristic properties of the membrane-bound receptor and in particular the slow interconversion to the high-affinity state and the effect of local anesthetics on this transition; furthermore, in these conditions the soluble protein can be manipulated ad libitum and submitted, for instance, to column filtrations and sucrose gradient centrifugations in the presence of detergent, without losing its characteristic conformational and allosteric transitions. After elimination of the detergent this form yields a reconstituted receptor which presents binding properties identical to those of the native membrane-bound receptor and leads to the formation of vesicles which exhibit carbamylcholine-sensitive ion fluxes. A necessary and sufficient condition for functional reconstitution is therefore the conservation, in the presence of lipids, of the allosteric properties of the receptor protein in its detergent-soluble form.

Acetylcholine↗

PubMed1999

Analysis of spiramycin by capillary electrophoresis.

The development and validation of an analytical method for the determination of spiramycin I in the presence of its related substances by capillary electrophoresis is shown. The separation, performed in a phosphate buffer (80 mM, pH 7.5) containing 12 mM cetyltrimethylammonium bromide (CTAB) and 20 mM sodium cholate, with a 50 microm ID and 44 cm long fused-silica capillary (36 cm effective length), applying a voltage of 12 kV (l approximately 80 microA), at 25 degrees C, is achieved in 15 min. Good selectivity among spiramycin I and its related substances was obtained. The influence of the buffer pH, and of the CTAB and sodium cholate concentrations was investigated. The method robustness, examined by means of a full-fraction factorial design, shows that it can be used within the limits set for the three parameters that were investigated. The method is linear (r = 0.9992) and precise (day-to-day corrected peak area repeatability, n = 18, relative standard deviation = 1.3%). The limits of detection and quantitation are 7 pg (0.025%) and 22 pg (0.08%), respectively, relative to a 2 mg/mL solution.

Aminoglycosides↗

PubMed1977

In vitro studies of gallstone dissolution using bile salt solutions and heparinized saline.

A controlled in vitro study of gallstone dissolution has been carried out using a model designed to simulate the in vivo situation in the bile ducts. Sodium taurocholate, sodium cholate, heparinized saline and physiological saline were infused for 10 days over 81 stones from 32 patients and changes in weight and structure were recorded. The bile salt solutions caused weight loss in 81-5 and 85-7 per cent respectively of the stones treated, but the two saline solutions caused weight gains in 74-5 and 88 per cent respectively. Fragmentation occurred in 18-5% of stones treated with sodium taurocholate, in 25% of those treated with sodium cholate and in 11-8 per cent of those treated with heparinized saline. Small stones lost a lower absolute amount of weight than large stones but this represented a greater proportion of their initial weight. These investigations confirm the advantages of a dynamic in vitro model to study gallstone dissolution. Bile salt solutions infused into the bile ducts may clear retained stones by causing reduction in stone weight or fragmentation or both, but heparinized saline appears to be unsuitable for gallstone dissolution. Larger stones may require longer periods of infusion.

Bile Acids and Salts↗

PubMed1965

EFFECT OF HIGH FAT DIETS ON INTESTINAL MICROFLORA AND SERUM CHOLESTEROL IN RATS.

Graber, C. D. (Baylor University College of Medicine, Houston, Tex.), R. M. O'Neal, and E. R. Rabin. Effect of high fat diets on intestinal microflora and serum cholesterol in rats. J. Bacteriol. 89:47-51. 1965.-Differential bacterial counts of feces and total plasma cholesterol determinations were performed on 60 Wistar rats fed several high lipid diets for a period of approximately 6 months. Fecal flora remained relatively stable irrespective of diet, but cholesterol levels rose in animals fed butter and sodium cholate. The six most commonly cultured organisms in all diets were enterococci, Proteus, lactobacilli, Escherichia coli, Staphylococcus aureus, and other micrococci. No enteric pathogens which did not ferment lactose or fermented it slowly were grown. Fungi and yeasts were rare. Aerobes generally outnumbered anaerobes in proportions which were sometimes as high as 300:1. Clostridium perfringens isolations were consistently high in animals given the butter diets, particularly when sodium cholate was added. Evidence is reviewed which indicates that this organism may play a role in bile salt and cholesterol metabolism. This experiment would seem to demonstrate that differences in plasma-cholesterol levels among the various dietary groups of rats were the result of dietary factors rather than alteration in intestinal flora.

Animals↗

PubMed2003

Lecithin vesicles for topical delivery of diclofenac.

Skin penetration of topically applied diclofenac is important for the treatment of rheumatic diseases and actinic keratoses. We have studied the permeation of diclofenac across human cadaver epidermis in-vitro from four lecithin vesicle formulations and a few marketed semi-solid preparations. The lecithin vesicle formulations were prepared by dissolving the lipid contents (lecithin and sodium cholate) in a 1:1 mixture of methanol-chloroform, evaporating the solvents under vacuum, and hydrating the lipid layer with the drug solution in water or 10% ethanol. The vesicles were sonicated for 5 min to reduce the vesicle size and their size and Zeta potential were characterized. The cumulative amount and maximum flux of diclofenac was 69.7+/-40.3 micrograms and 4.77+/-3.16 micrograms/hcm(2) from lecithin vesicles containing sodium cholate and 10% ethanol, and is the highest of all formulations studied. The cumulative amount and mean maximum flux obtained from other formulations were in the range of 2.46+/-1.98-29.9+/-10.1 micrograms and 0.53+/-0.46-3.61+/-0.86 micrograms/hcm(2). Based on the results, lecithin vesicles of diclofenac appear to be advantageous for the topical delivery of diclofenac.

Administration, Topical↗

PubMedDate not supplied

Radioimmunoassay of apolipoprotein a-ii.

A double antibody radioimmunoassay was developed for apolipoprotein A-II (apo A-II), one of the two major apoproteins of human high density lipoproteins (HDL). Apo A-II contains two identical polypeptide chains linked by a disulfide bond. A specific antiserum was raised in sheep. Tracer apo A-II was radiolabeled with 125I by the Bolton-Hunter technique. Reduction and carboxymethylation of apo A-II approximately doubled its immunoreactivity. Since normal sera were found to contain small amounts of monomeric apo A-II, this represented one potential source of error in the radioimmunoassay. Inclusion of 0.1 M sodium cholate in the assay system, however, led to identical immunoreactivity of dimeric apo A-II and the reduced and carboxymethylated protein. Radioimmunoassay using sheep anti-apo A-II detected 75% to 85% of the apo A-II contained in serum or HDL. Masked antigenic sites could be exposed by organic solvent extraction or, more simply, by dilution of serum samples in the buffer containing 0.1 M sodium cholate. Serum levels of apo A-II were measured in a population of consecutively and prospectively selected free-living subjects between 30 to 69 years of age. Levels were significantly (p less than 0.005) higher in females (n = 201; 42.0 +/- 10.3 mg/dl) than in males (n = 189; 39.0 +/- 8.4 mg/dl). Serum apo A-II levels correlated significantly with HDL-cholesterol levels but less strongly than apo A-I, the other major HDL apoprotein.

Adult↗

PubMed1998

Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations.

Bcl-2, Bcl-XL, and Bax are members of the Bcl-2 family that play important roles in apoptosis regulation. These proteins are believed to be membrane-bound and to regulate apoptosis through formation of homo- and heterodimers. However, we recently found by subcellular fractionation that whereas Bcl-2 is predominantly a membrane protein as previously reported, Bax and a significant fraction of Bcl-XL are soluble in thymocyte and splenocyte extracts. In addition, we have demonstrated that the ability of Bax to form dimers appears to be a detergent-induced phenomenon that coincides with a detergent-induced conformational change. We have further investigated the tertiary and quaternary states of Bax in the presence of various detergents. Detergents such as Triton X-100 and Triton X-114 readily enable Bax hetero- and homodimerization. However, other detergents such as polydocanol, W-1, octyl glucoside, dodecyl maltoside, Tween 20, and sodium cholate allow varying degrees of Bax hetero- and homodimerization. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Brij 35 allow neither hetero- nor homodimer formation. Immunoprecipitation analysis with the conformation-sensitive antibody uBax 6A7 revealed that whereas Triton X-100 readily exposes the N-terminal Bax epitope (amino acid 13-19), only limited exposure of the epitope occurs in Triton X-114, polydocanol, dodecyl maltoside, and sodium cholate, and no exposure of this epitope was observed in W-1, Chaps, octyl glucoside, Tween 20, and Brij 35. Moreover, we could not detect any proteins associated with the cytosolic form of Bax based on immunopurification of this protein. Sephacryl S-100 gel filtration chromatography analysis of the cytosolic Bax indicated that this protein is monomeric and displays an apparent molecular mass of 25 kDa. Induction of apo-ptosis which causes the insertion of the soluble form of Bax into membranes did not result in appreciable Bax/Bcl-XL, Bax/Bcl-2 or Bax/Bax dimer formation as determined by cross-linking studies. Further analysis of Bax after apoptosis induction by immunoprecipitation in the presence of Chaps also revealed no significant heterodimer formation. In conclusion, Bax displays several distinct states in different detergents that expose defined regions of the protein. In addition, these results suggest that mechanisms other than the simple dimerization among members of the Bcl-2 family may be required for the regulation of apoptosis.

Amino Acid Sequence↗

PubMed1975

Interconversion between different states of affinity for acetylcholine of the cholinergic receptor protein from Torpedo marmorata.

In receptor-rich membrane fragments from Torpedo, acetylcholine binds, in the presence of 70 muM Tetram, to a homogeneous population of high-affinity sites with Kd = (3.4 +/- 0.8) x 10(08) M. Dissolution of these membrane fragments by sodium cholate causes a decrease of affinity associated with the appearance of medium-affinity (Kd approximately 10(-7) M) and low-affinity (Kd greater than or equal to 10(-6) M) sites. Dissolution by neutral detergents Triton X-100 or Emulphogene preserves the high affinity of the acetylcholine binding sites. In all the soluble states of the receptor protein, Ca2+ ions and local anaesthetics no longer enhance the affinity for acetylcholine. Elimination of sodium cholate by dilution leads to the reassociation of the receptor protein, the recovery of high-affinity sites and the control by Ca2+ ions and local anaesthetics. Purification by affinity chromatography of the receptor protein in Triton X-100 is accompanied by a conversion of a majority of the acetylcholine sites into their state of low affinity. High-affinity sites can no longer be recovered by detergent dilution from these low-affinity ones.

Acetylcholine↗

PubMed1999

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis.

In this work the suitability of micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary electrophoresis (CE) to the analysis of the primary oxidation products of linoleic acid was studied with uncoated fused-silica capillaries. The primary autoxidation products of linoleic acid are the four hydroperoxide isomers 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid, 13-hydroperoxy-trans-9, trans-11-octadecadienoic acid, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid, 9-hydroperoxy-trans-10, trans-12-octadecadienoic acid. Addition of a surfactant such as sodium dodecyl sulfate (SDS) or sodium cholate (SC) into the running buffer (20-30 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or ammonium acetate, pH 9.5-11) was required to enhance the water solubility of the sample and selectivity of the separation. MEKC proved to be a promising new technique for the separation of the primary oxidation products of lipids giving results comparable to high performance liquid chromatography (HPLC). Partial separation of hydroperoxide isomers was also achieved using nonaqueous CE with methanol-acetonitrile-sodium cholate as running buffer.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed1985

Identification of a warfarin-sensitive protein component in a 200S rat liver microsomal fraction catalyzing vitamin K and vitamin K 2,3-epoxide reduction.

A partially purified, 200S submicrosomal fraction exhibiting thiol-dependent vitamin K1 (vitamin K) and epoxide reductase activities has been isolated by partial solubilization of rat hepatic microsomes with sodium cholate and separation by centrifugation at 105 000 g into a discontinuous sucrose gradient. At pH 7.4, the rates of vitamin K and vitamin K 2,3-epoxide reduction per milligram of 200S fraction protein were equivalent and were 2.5-3.0 times faster than in microsomes. Reduction of vitamin K 2,3-epoxide occurred in a tightly coupled, two-step reaction initially to vitamin K and subsequently to vitamin K hydroquinone (vitamin KH2). Incorporation of glycerol or sucrose and of sodium cholate into reaction mixtures equivalently affected the rates of both vitamin K and vitamin K 2,3-epoxide reduction, but in the case of epoxide metabolism, the ratios of vitamin KH2/vitamin K were much lower, suggesting that the second reaction has been partially uncoupled from the first. A 14 000-17 000-dalton warfarin-sensitive protein (WSP) that participates in vitamin K and vitamin K 2,3-epoxide reduction in the 200S fraction was identified by incorporation of N-[3H]ethylmaleimide ([3H]NEM) into the catalytically active reduced form of one or more attached disulfides. Reduction of WSP with dithiothreitol was required for reaction with [3H]NEM, and the substrates vitamin K and vitamin K 2,3-epoxide and the inhibitor warfarin all effectively blocked the reaction. 2-Mercaptoethanol could not substitute for dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Separation of membrane proteins solubilized with a nondenaturing detergent and a high salt concentration by hydroxyapatite high-performance liquid chromatography.

Ceramic hydroxyapatite high-performance liquid chromatography with a solvent containing high concentrations of salts was used for the separation of membrane proteins solubilized under nondenaturing and high salt conditions. The chromatographic conditions were optimized using sodium cholate as the detergent. By this method, most membrane proteins, prepared from rat liver rough microsomes, were effectively resolved from each other with a protein recovery of more than 90%. The method also allowed the single-step purification of the ribosome-binding protein, p34, from a microsomal membrane protein fraction. The good resolution with this method should be applicable to the isolation and characterization of a variety of membrane proteins on the analytical and semipreparative scales. With only the substitution of sodium cholate with other nondenaturing detergents, this method may also be applicable to the purification of membrane proteins requiring such nondenaturing detergents with retention of their biological activities.

Cholic Acid↗

PubMed1990

Partial purification and characterization of phosphatidylinositol kinases from human platelets.

Most of human platelet phosphatidylinositol (PI) kinase activity (approx. 80%) was associated with the membrane fraction and its majority was released by the extraction with Triton X-100 after KCl treatment. Two major activity peaks (mPIK-I and mPIK-III) were obtained by Mono Q column chromatography. They were distinct from each other with regard to Mr (76,000 and 80,000 as determined by gel-filtration chromatography), apparent Km values for ATP, effect of arachidonic acid and phosphatidylserine and detergent requirement. Triton X-100 inhibited the activity of mPIK-I but rather weakly enhanced the mPIK-III activity, and sodium cholate remarkably inhibited both mPIK-I and mPIK-III activities. Their products were identified to be phosphatidylinositol 4-phosphate. On the other hand, about 20% of PI kinase activity was recovered from the cytosolic fraction and two activity peaks (cPIK-I and cPIK-II) were resolved on Mono Q column chromatography. There were no significant differences in biochemical properties between cPIK-I and cPIK-II. Both of them had Mr approx. 550,000 as determined by gel-filtration chromatography and were activated by sodium cholate to a greater extent than by Triton X-100. The results suggest that the major PI kinases (mPIK-I and mPIK-III) are PI 4-kinase and mPIK-I is distinct from PI 4-kinases in other sources especially with regard to the effect of Triton X-100.

1-Phosphatidylinositol 4-Kinase↗