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PubMed1977

Calf thymus alkaline phosphatase. II. Interaction with detergents.

1. A number of detergents were used to dissolve calf thymus plasma membranes rich in alkaline phosphatase (orthophosporic-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) activity. 2. The Stokes' radius (r) of alkaline phosphatase in each detergent was measured by gel filtraton. The size of the solubilized enzyme varied from r = 6.2 nm in sodium cholate to r = 8.3 nm in Berol EMU-043. With N-alkylsulphates, the apparent size increased with alkyl chain length, with r = 6.4 nm (C9) and r = 7.3 nm (C12). Tween 20 failed to solubilise the enzyme. 3. The effect of each detergent on the catalytic activity of alkaline phosphatase was determined. The non-ionic detergents Triton X-100, Nonidet P-40, Berol EMU-043, Tween 20 and the zwitterionic detergent Empigen BB increased V by 10--50% without substantially altering the Km for p-nitrophenylphosphate. The bile salts sodium deoxycholate and sodium cholate decreased V and increased the apparent affinity of the enzyme for nitrophenylphosphate. Inhibition was concentration-dependent up to the critical micellar concentration, above which it remained constant (deoxycholate, 33% cholate, 76%). Alkylsulphates (C8-12) had no significant inhibitory effect during 24 h at 23 degrees C. 4. Exchanging one detergent for another altered alkaline phosphatase activity to a state characteristic for the second detergent, e.g. the activity of cholate-inhibited alkaline phosphatase was restored to normal levels by excess of Triton X-100 and vice versa. The inhibitory effect of deoxycholate and cholate therefore result primarily from interactions between detergent and alkaline phosphate, rather than from selective removal of lipids from the enzyme. 5. Pure lecithin, lysolecithin and an ether-deoxylysolecithin each reactivated cholate-inhibited alkaline phosphatase in a concentration-dependent fashion. Cholesterol had no effect. 6. The half-life (t1/2) of membrane-bound alkaline phosphatase at 55 degrees C was 64 min. With the exception of Berol, solubilisation in non-ionic detergents caused no marked change in this sensitivity. The enzyme became more labile in deoxycholate (t1/2) = 31 min), but less labile in cholate (t1/2 = 99 min). Alkylsulphates, which are strong denaturants, markedly increased the sensitivity of the enzyme to heat-inactivation (C8, t1/2 = 13 min; C9--12, t1/2 less than 2 min). 7. It is concluded that membrane-bound alkaline phosphatase is separated from most if not all of its neighbouring lipid moieties by these detergents, which bind to the solubilised enzyme. The number and character of molecules binding to the enzyme influence its size and shape, its susceptibility to inactivation and its catalytic activity.

Alkaline Phosphatase↗

PubMed1988

Evidence for high molecular weight Na-Ca exchange in cardiac sarcolemmal vesicles.

Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na-Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na-Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (Mr) of approximately 226,000 +/- 20,000 SD (n = 6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 1:1 with mobile phase buffer containing 50 mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na-Ca exchange activity. Na-Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na-Ca exchange activity were 91 +/- 7 and 68 +/- 11%, respectively. In the peak fraction, Na-Ca exchange specific activity increased two- to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimum Mr of 224,000 Da. Specific 45Ca2+-binding SL proteins with Mr of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose. These data suggest that native cardiac Na-Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na-Ca exchange protein(s) remains incomplete.

Animals↗

PubMed1985

Effects of cycloartenol on absorption and serum levels of cholesterol in rats.

The possibility that cycloartenol magnifies the hypocholesterolemic effect of beta-sitosterol was studied in two strains of rats fed on cholesterol-enriched (0.5%) diets. Cycloartenol was added to diets containing 1.0% or 0.5% beta-sitosterol at the 0.05% level and to diet free of plant sterol at the 0.5% level. In one experiment, diets included sodium cholate (0.125%). Due to the potent hypocholesterolemic efficacy of beta-sitosterol under the present dietary regimens, no clear additional effect (so-called synergistic effect) of cycloartenol was observed. However, in the experiment using Wistar rats, the decrease in serum apo A-I due to feeding cholesterol was ameliorated more effectively in combination with beta-sitosterol than with beta-sitosterol alone. The hepatic deposition of cholesterol was mitigated by dietary beta-sitosterol, and further, although slightly, by a combination of beta-sitosterol and cycloartenol, except in the experiment with diets containing sodium cholate. Fecal excretion of neutral and acidic steroids was not meaningly magnified by cycloartenol. Cycloartenol itself was not as effective as beta-sitosterol in mitigating lipid disorders due to dietary cholesterol. The rate of appearance of cholesterol in the thoracic duct lymph was not interfered with further by a combination of beta-sitosterol and cycloartenol compared to beta-sitosterol alone. This trimethylsterol was absorbed at a rate approximately 4-fold higher than that of beta-sitosterol, though much lower compared to cholesterol. These results suggest a preference for cycloartenol in cholesterol dynamics. However, cycloartenol seems unlikely to influence cholesterol absorption in the small intestine.

Animals↗

PubMedDate not supplied

Poly(4-vinylpyridine)-coated liposomes: stability studies and release of acetylsalicylic acid.

Liposomes of dimyristoylphosphatidylcholine (DMPC) and dicetylphosphate (DCP) reacted with 4-vinylpyridine (4-VP) to form a salt and, subsequently, autopolymerized for form poly(4-vinylpyridine) (poly(4-VP))-coated liposomes. The conditions for optimization of polymer coating have been determined; also, the effects of polymer coating on liposome stability, the encapsulation of ASA and its release kinetics have been measured. The coating efficiency was maximum at a DMPC:DCP 1:1 mole ratio, at pH 4.0 in acetate buffer, and a polymerization time of 40 min. The polymer-coated liposomes were stable in 2 mM sodium cholate and 4 per cent isopropanol solutions, as determined from turbidity measurements, versus a 20-25% decrease in stability of uncoated liposomes. The encapsulation efficiency of ASA reached a maximum of 9 per cent at DMPC:DCP 1:1 mole ratio. The release of ASA at 37 degrees C, pH 7.0 was characterized by an initial fast release (85 and 63 per cent in 20 min from uncoated and polymer-coated liposomes, respectively) followed by a slow, constant release rate up to 140 min. Thus, autopolymerization of a polymerizable monomer at liposome surfaces represents a potentially feasible stabilization approach for liposomes exposed to sodium cholate solutions with greater retention of solute than uncoated liposomes.

Aspirin↗

PubMed1988

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin.

A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-100-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated.

Bacteriorhodopsins↗

PubMed1992

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.

Animals↗

PubMed1988

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent.

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH-cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemoproteins catalyzed the 16 alpha- and 16 beta-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase*. The activity of cytochrome P-450b and P-450b* increased up to 50% when reconstituted with reductase* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.

Animals↗

PubMed1998

Purification and characterization of a membrane-associated ganglioside sialidase from bovine brain.

A membrane-associated ganglioside-hydrolyzing sialidase was purified to apparent homogeneity from bovine brain. The enzyme was solubilized with Triton X-100 plus sodium cholate from the particulate fraction and purified over 100,000-fold by sequential chromatography on DEAE-cellulose, octyl-Sepharose, heparin-Sepharose, Sephacryl S-200, MonoQ, RCA-agarose, thiol-activated Sepharose, and ganglioside-affinity Sepharose. The final enzyme preparation exhibited a specific activity of 4,851.3 micromol/h/mg protein and an apparent molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme preferentially hydrolyzed gangliosides other than GM1 and GM2 but demonstrated hardly any activity against glycoproteins and oligosaccharides. Gangliosides GD3, GD1a, and GT1b were much better substrates than GM3 and GD1b in the presence of Triton X-100, but the latter became more sensitive to the sialidase with addition of sodium cholate. The enzyme was activated by dithiothreitol, strongly inhibited by 4-hydroxy-mercuribenzoate, and firmly adsorbed to thiol-activated Sepharose, indicating that free sulfhydryl groups are essential for its catalytic activity. Subcellular fractionation experiments revealed that the enzyme is mainly located in the synaptosomal fraction.

Animals↗

PubMed1994

Analysis of dansyl amino acids in feedstuffs and skin by micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MECC) using sodium dodecyl sulphate (SDS) and sodium cholate have been used for analyses of 30 dansylated (Dns) amino acids. The influences of sample preparation, Dns/amino acid ratio, sample solvent composition, and separation conditions including voltage, temperature, pH and buffer composition were investigated. Complete separations of acidic and neutral amino acids were obtained within 45 min in the SDS system. The efficiency expressed as number of theoretical plates for the applied capillary 0.52 m long were between 210,000 and 343,000, and the repeatability was very good with relative standard deviations on relative migration times between 0.09 and 0.70% and on relative normalised peak areas (RNPAs) between 0.85 and 3.41%. The linearity studies gave correlation coefficients between 0.9957 and 0.9993 for RNPAs against concentration. Detection limits were between 3 and 6 fmol or approximately 2 pg of each amino acid. Basic amino acids were separated in a MECC system using sodium cholate. Procedures and problems using Dns derivatisation for amino acids analysed by the MECC methods are described. Finally, examples of analyses of hydrolysates of real complex samples show, that this method can be applied to determine the amino acid composition of proteins in feedstuffs and skin.

Amino Acid Sequence↗

PubMed1987

Detergents as probes of reconstituted rat liver cytochrome P-450 function.

A series of 16 ionic, zwitterionic, and nonionic detergents have been used to perturb the catalytic activities of major cytochrome P-450 (P-450) forms from untreated (UT-A), phenobarbital-treated (PB-B) and beta-naphthoflavone-treated (BNF-B) rats in reconstituted systems with NADPH--P-450 reductase Detergent effects on R warfarin hydroxylase activities were correlated with detergent effects on the quaternary structures of P-450 and reductase, and on their 1:1 complexes as determined by gel exclusion chromatography using sodium cholate as a prototype detergent. The detergent concentrations used did not in most cases affect rates of NADPH-dependent reduction of cytochrome c by the reductase. With P-450 BNF-B, ionic and zwitterionic detergents enhanced warfarin hydroxylase activities at low concentrations and produced marked inhibition at higher concentrations, while nonionic detergents only inhibited. With P-450 UT-A, some nonionic and zwitterionic detergents increased rates at low concentrations and inhibited at higher concentrations. P-450 PB-B was inhibited by detergents of all three classes at low and high concentrations. The concentrations of a detergent required to affect 50% inhibition differed for the three P-450s, suggesting, together with the differential susceptibilities to detergent-mediated rate enhancing effects, that the reductase interacts functionally differently with the three P-450s. Chromatographic studies demonstrated that concentrations of sodium cholate which optimally enhanced metabolic rates with P-450 BNF-B facilitated the uptake of the P-450 into the functional reductase/P-450 complex, and higher concentrations of cholate, which completely inhibited activity, produced profound disruptions of the complex. The data have provided insight into the functional interactions required for monooxygenase activity.

Animals↗

PubMed1987

Tri(n-butyl) phosphate/detergent treatment of licensed therapeutic and experimental blood derivatives.

Incubation of an AHF concentrate with 0.3% tri(n-butyl)phosphate (TNBP) and 0.2% sodium cholate was shown to inactivate at least 10,000 infectious doses of lipid-enveloped viruses, including hepatitis B and non-A, non-B viruses and HTLV-III [Prince et al., Lancet i, pp. 706-710, 1986]. The use of TNBP/detergent combinations for virus sterilization was evaluated further to determine its effect on the structure and function of a wide variety of blood proteins. Vesicular stomatitis and Sindbis viruses were used as markers of virus inactivation. TNBP/detergent treatment did not significantly alter the function of AHF, factor VII, factor IX, factor X, fibrinogen, factor XIII, fibronectin, anti-HBsAg and anti-HA in normal serum globulin, haptoglobin, tumor necrosis factor, alpha-interferon, and both native and chemically polymerized stroma-free hemoglobin. As compared with partially purified derivatives, the extent of virus sterilization of plasma and component cryoprecipitate with 0.3% TNBP and 0.2% sodium cholate at ambient temperature could be improved by raising the TNBP concentration and temperature. Virus sterilization by TNBP/detergent mixtures appears to be generally applicable to blood protein derivatives.

Antiviral Agents↗

PubMed2001

Determination of synthesized alpha-vitamin E by micellar electrokinetic chromatography.

We developed a micellar electrokinetic chromatography method (MEKC) for the direct determination of the content of synthesized alpha-vitamin E. It was found that under the optimum separation conditions 7 mM borate + 14 mM phosphate + 15 mM sodium dodecyl sulfate (SDS) + 10 mM sodium cholate (NaCh) + 8% acetonitrile (pH 9.2) with UV detection wavelength at 214 nm, 16 kV constant voltage, and 26 degrees C constant temperature, alpha-vitamin E and its isomers can be baseline separated and alpha-vitamin E was quantitatively analyzed. In addition, the sample recovery, the limit of detection and the repeatability of the method were investigated. The influence of various parameters on the separation such as SDS concentration, NaCh concentration, buffer pH and acetonitrile percentage were also discussed.

Acetonitriles↗

PubMed2005

Analysis of ecdysteroids by micellar electrokinetic chromatography with on-line preconcentration.

In order to enhance the UV detection sensitivity, an application study of an on-line preconcentration technique for micellar electrokinetic chromatographic (MEKC) was carried out. The simultaneous determination of four test ecdysteroids, 20-hydroxyecdysone, ajugasterone C, polypodine B and ponasterone A has been investigated by using the normal stacking mode in MEKC with UV detection. The effects of anionic surfactant composition and concentration, the applied voltage, the pH buffer, the kind and the amount of organic solvent and the injection time on the analyte resolution were evaluated. The optimised conditions for the separation involved the use of a 50 mM borate as the running buffer containing 50 mM of a mixture of sodium dodecyl sulphate (SDS) and sodium cholate (SC) in the ratio of 1:1 together with a concentration of 10% (v/v) of 2-PrOH at pH 9.0. Hydrodynamic injection of 12 s at 50 mbar and separation voltage of 20 kV at temperature of 20 degrees C were employed. These conditions allowed a repeatability separation within 21 min. Concentration detection limit for the neutral analytes studied improve about an order of magnitude. The method was also applied to the determination of ecdysteroids in a real sample.

Amaranthaceae↗

PubMed1989

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles.

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

Analysis of post-harvest fungicides by micellar electrokinetic chromatography.

A method based on solid-phase extraction (SPE) and micellar electrokinetic chromatography (MEKC) was developed for the simultaneous determination of carbendazim, imazalil, methylthiophanate, O-phenylphenol, prochloraz, procimidone, thiabendazole and triadimefon residues in grape, lettuce, orange and tomato. Selectivity and resolution were studied changing the pH and the concentration of the buffer, the type and concentration of surfactant and the methanol content in the mobile phase. A buffer consisting of 4 mM borate with 75 mM sodium cholate (pH 9.2) gave the best results. The recoveries of the fungicides in spiked fruit and vegetable samples ranged from 30 to 105%, and the limits of detection were between 0.1 and 1 mg kg(-1). The reproducibility and repeatability of the combination of SPE pretreatment and MEKC were good for all the compounds, except for imazalil and O-phenylphenol in oranges, due to some matrix compounds interfering with the separation. The method was applied to post harvest treated samples, and the fungicides were sometimes detected at concentration levels lower than maximum residue limits (MRLs).

Buffers↗

PubMed1982

Regulation of bile salt transport in rat liver. Evidence that increased maximum bile salt secretory capacity is due to increased cholic acid receptors.

Expansion of the bile salt pool size in rats increases maximum excretory capacity for taurocholate. We examined whether increased bile salt transport is due to recruitment of centrolobular transport units or rather to adaptive changes in the hepatocyte. Daily sodium cholate (100 mg/100 g body wt) was administered orally to rats. This treatment was well tolerated for at least 4 d and produced an 8.2-fold expansion of the bile salt pool. This expanded pool consisted predominently (99%) of cholic and deoxycholic acids. Significantly increased bile salt transport was not observed until 16 h after bile acid loading, and maximum elevations of transport capacity to 2.3-fold of control required approximately 2 d. In contrast, maximum sulfobromophthalein excretion rates increased 2.2-fold as early as 4 h and actually fell to 1.5-fold increase at 4 d. We studied the possibility that this adaptive increase in bile salt secretory transport was due to changes in canalicular surface membrane area, lipid composition, or increased number of putative carriers. Canalicular membrane protein recovery and the specific activities of leucine aminopeptidase, Mg(++)-ATPase and 5'-nucleotidase activities were unaltered by bile salt pool expansion. The content of free and esterified cholesterol and total phospholipids was unchanged in liver surface membrane fractions compared with control values. In contrast, sodium cholate administration selectively increased specific [(14)C]cholic acid binding sites twofold in liver surface membrane fractions. Increased numbers of [(14)C]cholic acid receptors (a) was associated with the time-dependent increase in bile salt transport, and (b) was selective for the taurine conjugate of cholate and (c) was reduced by chenodeoxycholate. Changes in bile acid binding sites 16 h following taurocholate and chenodeoxycholate and the lack of change with glycocholate was associated with comparable changes in bile salt transport. In conclusion, selective bile salts increase bile salt transport in the liver through an adaptive increase in the density of putative bile acid carriers in liver surface membrane.

Animals↗

PubMed1997

Activity and stability of a Rhizomucor miehei lipase in hydrophobic media.

The effects of detergents and organic solvents on a commercial lipase (Lipozyme) from Rhizomucor miehei were investigated. It was shown that the detergent sodium cholate is possibly an activator of the enzyme, increasing lipase activity 2.5 times (250% of the control) when the enzyme was preincubated with 7 mM cholate. Lipozyme activity was over twice as high (230% of the control) in the presence of 80 mM Tween 80 or 90 mM Tween 20 (polyoxyethylenesorbitan monolaurate), apparently through an additional emulsifying action on the substrate. Preincubation with Tween 80 (polyoxyethylenesorbitan mono-oleate) did not affect enzyme activity. In contrast, lipase activity was completely inhibited in the presence of an 8.9 mM concentration of another non-ionic detergent, Brij 58, whereas with a 16.4 mM concentration of the cationic detergent cetyltrimethylammonium bromide (CTAB), enzyme activity was reduced by 80%. Preincubation of Lipozyme with the same concentrations of Brij 58 [poly(oxyethylene)20 cetyl ether] and CTAB promoted total inactivation of the enzyme. Organic solvents had different effects on lipase activity and stability. Of the tested solvents, hexane was least deleterious to lipase activity and did not alter enzyme stability on preincubation. These results suggest that Lipozyme can be used in esterification reactions with hexane as solvent or in hydrolysis reactions with Tween 20 or Tween 80 as emulsifying agents, after pretreatment with sodium cholate.

Culture Media↗

PubMed1996

A noninjury, diet-induced swine model of atherosclerosis for cardiovascular-interventional research.

To investigate whether atherosclerotic vascular disease in the microswine model can be induced by atherogenic diet alone and does not require balloon injury or endothelial denudation as widely stated in the literature, 28 female Yucatan microswine were fed a high-fat, high-cholesterol diet, including 2% sodium cholate, for an average of 310 +/- 13 days. Four control swine were placed on a regular diet for an average of 287.2 +/- 7.8 days. Selective coronary arteriography and morphologic and histologic studies were performed at the end of this period. Coronary arteries were fixed in vivo by pressure perfusion of formalin. Angiograms and sequential histologic sections were reviewed by a double-blind team. The angiography did not show apparent disease in all vessels but generally revealed mild irregularity. Quantitatively, there was a 30.5 +/- 3.5% stenosis (mean +/- standard error, P < 0.05 vs. control) in left anterior descending (LAD), 40.7 +/- 4.5% of stenosis in right coronary artery (RCA) (P < 0.01 vs. control), and 24.8 +/- 3.7% of stenosis in left circumflex artery (LCX). The lesions were eccentric in 95% of LCA, 95.8% of RCA, and 75% of LCX, and the remainder were concentric lesions. Typical lesions were characterized by significant intimal proliferation, cholesterol clefts, necrotic cores, heavy extracellular fat deposition, and calcification. Control animals had only occasional, minimal intimal lipid deposition in coronary arteries. These findings suggest that the Yucatan microswine is an ideal coronary atherosclerosis animal model for vascular research. Lesions can be induced by atherogenic diet alone. Cholesterol uptake is increased by adding sodium cholate to the feed. Moreover, balloon injury of the intima or media is not required to induce significant atherosclerotic lesions in coronary arteries.

Animals↗