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At least 451 records · Page 25Linked to original sources
PubMed1989

Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography.

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1984

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes.

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.

Animals↗
PubMed1984

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution. I. A 56 kilodalton managnese-containing protein, a probable component of the coupling factor enzyme.

The binding of endogenous manganese (Mn) to proteins released from spinach grana-thylakoid membranes by 2% cholate detergent or by osmotic shock is investigated. A mixture of 15-20 proteins is released by cholate and has been separated by isoelectric focusing in a sucrose gradient or by chromatofocusing. Mn coelutes with several proteins, but is lost upon dialysis. A dramatic redistribution of this Mn occurs in proteins released by osmotic shock in the presence of hydrophobic and hydrophilic oxidants. Maintaining an oxidizing solution potential during extraction apparently precludes reduction of the higher oxidation states of Mn to the labile Mn(II) state by reducing agents released from the membranes during lysing. This allows proteins to be separated which bind non-labile Mn ions. Under these extraction conditions, a protein is isolated which has an apparent molecular weight (Mr) of 65000 or 56000 on SDS-polyacrylamide gel electrophoresis depending on the sample buffer system used. The nondissociated protein occurs as a monomer of 58 kDa (90%) and an apparent dimer of 112 kDa (10%) by gel filtration. This protein binds little Mn if extracted by cholate and separated by isoelectric focusing. However, extraction by osmotic shock in the presence of oxidants and separation by chromatofocusing results in the retention of 1.9 +/- 0.3 Mn ions per monomer. This protein is identical to that reported by Spector and Winget (Spector, M., and Winget, G.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 957-959). Contrary to their result, this protein does not reconstitute O2 evolution when added to depleted membranes. Rabbit antibody to this purified protein inhibits O2 evolution by 20% when incubated with intact grana-thylakoid membranes or 10-20% with partially inverted, French-pressed thylakoids. This inhibition is completely removed by 10(-3)M NH3Cl as an uncoupler of photophosphorylation. These results support a role in Phosphorylation and a location on the outer surface of the thylakoids. This antibody also selectively binds purified coupling factor, CF1, the multisubunit phosphorylation enzyme which is located on the outer thylakoid surface and which is known to bind two Mn ions tightly (Hochman, Y. and Carmeli, C. (1981) Biochemistry 20, 6293-6297). Thus the beta-subunit of CF1, which has a molecular weight of 56 kDa, can be identified as the locus of Mn binding in CF1 and as the Mn protein isolated by Spector and Winget. This protein plays no role on O2 evolution.

Cytoplasmic Granules↗
PubMed1987

The effect of detergents on bovine cytochrome c oxidase: a kinetic approach.

(1) Investigation of the relationship between the detergent concentration and steady-state and pre-steady-state kinetics of cytochrome c oxidase proved to be a valid approach in the study of protein-detergent interaction. (2) Laurylmaltoside, sodium cholate and Triton X-100 influenced the kinetics of cytochrome c oxidase cooperatively at detergent concentrations near their critical micelle concentration. This mode of interaction reflects disaggregation of the oxidase as a result of cooperative binding of the detergent. (3) Addition of increasing concentrations of Tween-80 to the aggregated enzyme caused a more gradual decrease in aggregation of the oxidase, which did not result in a change in activity of the enzyme. This suggests that aggregation of cytochrome c oxidase occurs in a highly regular manner in which no catalytic sites are shielded off. (4) Oxidase aggregates present at detergent concentrations below the critical micelle concentration of laurylmaltoside and Triton X-100 showed considerable activity. Their kinetics were equal to those of the oxidase in Tween-80, suggesting that the protein molecules are aligned in a similar way in all oligomers. Aggregates present in low concentrations of sodium cholate showed turnover rates that were twice as low as those observed with other aggregates. (5) Solubilisation of the oxidase by sodium cholate or Triton X-100 resulted in almost complete inhibition of enzymic activity, whereas the association rate of ferrocytochrome c was almost equal to that found for monomeric oxidase in laurylmaltoside. These results are in agreement with a mixed-type inhibition.

Animals↗
PubMed1975

A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo.

The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques. In the closed-loops, 5 mM deoxycholate selectively inactivated (Na+ + K+)-ATPase, and net secretion of Na+ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-0-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5 plus or minus 2.9% per g wet st of gut) greatly exceeded those of taurocholate (4.3 plus or minus 1.0). In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na+ and C1-, and inhibited active glucose transport; concomitantly "total" ATPase, (Na+ + K+)-ATPase, and Mg-2+-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg-2+-ATPase activity. Taurocholate and cholate at 1mM had no effect on transport of (Na+ + K+)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate. These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na+ + K+)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.

Adenosine Triphosphatases↗
PubMed1981

Rapid transmembrane movement of phosphatidylcholine in small unilamellar lipid vesicles formed by detergent removal.

Small unilamellar phosphatidylcholine vesicles, formed by solubilizing phosphatidylcholine with sodium cholate and removing the detergent by gel filtration, have been studied in their interaction with phospholipid exchange protein. The exchange of phosphatidylcholine between the vesicles and erythrocyte ghosts was greatly stimulated by the phosphatidylcholine-specific exchange protein from bovine liver. It was found that 95% of the phosphatidylcholine was readily available for exchange within 3 h at 37 degrees C. In similar vesicles prepared by sonication only 70% of the phosphatidylcholine was rapidly exchangeable. Our results indicate that the transmembrane movement of phosphatidylcholine across the bilayer of vesicles prepared by the cholate technique is a relatively fast process. The results are discussed with respect to the presence of trace amounts of lipid-associated cholate which may facilitate the transbilayer exchange of phosphatidylcholine.

Animals↗
PubMed1982

Solubilization and reconstitution of membranes containing the Na+ -Ca2+ exchange carrier from rat brain.

The Na+ -Ca2+ exchange carrier from brain plasmalemma was solubilized in cholate and reconstituted into asolectin vesicles by the cholate dilution method. Optimal solubilization and reconstitution required the presence of high NaCl (greater than or equal to 1.3 M). The reconstituted vesicles rapidly accumulated 45Ca2+ in the presence of an outward directed Na+ gradient. Other monovalent ion gradients (K+, Li+ or cholate+) did not drive transport. Further, Mg2+ X ATP did not drive Ca2+ uptake in the reconstituted vesicles. Uptake was temperature dependent with highest uptake occurring at 37 degrees C. Intravesicular Ca2+ accumulated by the Na+ -dependent process could be released by the Ca2+ ionophore A23187 or by extravesicular Na+ but not by external EGTA. Ca2+ uptake was inhibited by extravesicular Li+ or Na+. The Ki for Na+ inhibition was 35 mM for both the original membrane vesicles from brain plasmalemma and for the reconstituted vesicles. Ca2+ uptake was saturable with respect to extravesicular Ca2+ (Km(Ca2+) = 27 microM).

Animals↗
PubMed1988

Effects of detergent on the binding of solubilized sodium channels to immobilized wheat germ agglutinin: structural implications.

The binding of the solubilized voltage-dependent sodium channel from rat brain to immobilized wheat germ agglutinin (WGA) is detergent-dependent. When solubilized in sodium cholate, only 11% of total recovered channels bound to a WGA-Sepharose column. When solubilized in Triton X-100 or CHAPS, however, 80% and 60% could bind, respectively. The effect of cholate on sodium channel binding is relatively specific: the major rat brain glycoproteins which bind to immobilized WGA are roughly the same in either Triton or cholate, as analyzed by SDS gel electrophoresis. The structural implications for the channel are discussed.

Animals↗
PubMed1991

Influence of the 53 kDa glycoprotein on the cooperativity of the Ca(2+)-ATPase of the sarcoplasmic reticulum.

Previous results from this laboratory suggest that the 53 kDa glycoprotein (GP-53) of rabbit skeletal muscle sarcoplasmic reticulum membrane (SR) may influence coupling between Ca2+ transport and ATP hydrolysis by the Ca(2+)-ATPase. Here we report evidence that GP-53 may influence the cooperative behavior of the Ca(2+)-ATPase. The ATPase activity of the Ca(2+)-ATPase displays negative cooperative dependence (Hill coefficient n less than 1) on [MgATP] and has positive cooperative dependence (n greater than 1) on [Ca2+]free. We have determined the degree of cooperativity for native SR vesicles, SR preincubated with antiserum against GP-53 or preimmune serum, and SR partially extracted with KCl-cholate. Our results show that SR preincubated with preimmune serum or SR treated with cholate in 50 mM KCl (yielding membranes rich in GP-53) demonstrate a cooperative dependence of Ca(2+)-ATPase activity on both [ATP] and [Ca2+] similar to that of untreated SR. SR preincubated with anti-GP-53 antiserum (which causes an uncoupling of Ca2+ transport from ATP hydrolysis) or SR extracted with cholate in 1 M KCl (yielding membranes depleted of GP-53) displays decreased positive cooperative dependence on [Ca2+] and decreased negative cooperative dependence on [ATP]. The results are consistent with the interpretation that GP-53 may influence the cooperative behavior of the Ca(2+)-ATPase.

Adenosine Triphosphate↗
PubMed1979

Conversion of deoxycorticosterone to 3-keto-4-etienic acid, catalyzed by a partially purified preparation from bovine adrenocortical mitochondria.

Bovine adrenocortical mitochondria were sonicated and subjected to extraction with sodium cholate. The extract contained not only cytochrome P-450 activities, but also an activity which catalyzed the conversion of deoxycorticosterone to an unknown steroid (designated X). The latter activity was concentrated by (NH4)2SO4 fractionation in the presence of sodium cholate, and separated from P-450 by taking advantage of their different solubilities in phosphate buffer without sodium cholate. The specific activity of the partially purified enzyme fraction was 70 times higher than that of sonicated mitochondria. The conversion of deoxycorticosterone to steroid X required NAD or NADP. The conversion rate was dependent on the concentration of deoxycorticosterone. The major product, steroid X, was isolated from the reaction mixture by means of silicic acid and Iatrobeads column chromatography. The steroid was characterized as 3-keto-4-etienic acid (3-oxoandrost-4-ene-17beta-carboxylic acid). This result suggests that an enzyme system for the conversion of deoxycorticosterone to 3-keto-4-etienic acid exists in adrenocortical mitochondria.

Adrenal Cortex↗
PubMed1983

Further studies on the bile salt induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridium absonum.

Optimal induction of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase in 100-ml cultures grown to stationary phase was achieved by the addition of metabolizable bile salt inducers: chenodeoxycholate, 7-ketolithocholate or cholate at 2.5-3 h after inoculation. Bile salt addition prior to or after this period markedly reduced the enzyme levels induced. However, when the non-metabolizable inducers deoxycholate and 12-ketolithocholate were similarly added, no significant differences in enzyme levels were observed between addition at 2.5-3 h or at earlier times. The ability of both metabolizable and non-metabolizable bile salts to induce the enzymes fell markedly when additions were made later than approximately 3.5 h. Kinetic studies using 1-l cultures suggest that in a larger culture a somewhat earlier inducer addition period is optimal. When ranked according to the level of enzymes induced the order in decreasing induction power was: chenodeoxycholate, 7-ketolithocholate, deoxycholate, 12-ketolithocholate and cholate. Mixtures of cholate and suboptimal concentrations of deoxycholate induced the culture better than the sum of the two concentrations individually. The end product, ursodeoxycholate, was very effective in blocking the induction by chenodeoxycholate or deoxycholate. Ursocholate (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoate) was less effective. Cultures when grown for 3 h with various bile salts or none, then centrifuged and recultured for a further 3 h in fresh medium containing chenodeoxycholate, all yielded identical enzyme levels within experimental error. We conclude that exposure of the organism to bile salt inducer in the last 3 h of culture was important, while the history of the culture prior to this time was unimportant in the induction process.

Bile Acids and Salts↗
PubMed1987

Hydrolysis of galactosylsphingosine and lactosylsphingosine by beta-galactosidases in human brain and cultured fibroblasts.

Enzymatic properties of beta-galactosidases with galactosylsphingosine (psychosine) and lactosylsphingosine as the substrates were examined. Although bile salts were stimulatory on the hydrolysis of the glycolipids in normal brain and cultured fibroblasts, the hydrolytic activities could be readily assayed, without detergents. The in vitro hydrolysis of lactosylsphingosine in cultured fibroblast homogenates was catalyzed by two enzymes, as is the case with the hydrolysis of galactosylceramide and lactosylceramide. Lactosylsphingosine beta-galactosidase activities assayed in the absence and the presence of taurocholate (probably lactosylceramidase I) were deficient in fibroblasts from patients with globoid cell leukodystrophy, while the activity assayed with sodium cholate (probably lactosylceramidase II) was deficient in GM1 gangliosidosis fibroblasts. In contrast, galactosylsphingosine beta-galactosidase was not activated by cholate and the enzyme activities assayed with the no-additive and taurocholate systems were deficient in brain and fibroblasts from patients with globoid cell leukodystrophy, thereby indicating that the hydrolysis of galactosylsphingosine is catalyzed by one enzyme, galactosylceramidase I. Exogenous lipids and an activator protein purified from normal spleen activated galactosylsphingosine beta-galactosidase but they were inhibitory to lactosylsphingosine beta-galactosidase. Because the Km values of lactosylsphingosine beta-galactosidase assayed with cholate were several magnitude higher than those obtained with the no-additive system and because lactosylsphingosine is readily hydrolyzed with the no-additive system in vitro, it is likely that the in vivo hydrolysis of the lipid is catalyzed by only one enzyme, lactosylceramidase I.

Bile Acids and Salts↗
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↗
PubMed1984

Reconstitution of [3H]3-cyanoimipramine ([3H]Ro 11-2465) sites solubilised from human platelets.

[3H]3-Cyanoimipramine has been characterised as a pseudoirreversible ligand at imipramine binding sites making it useful for molecular characterisation studies. Using this ligand, I now report that it is possible to solubilise the binding site molecule with cholate. Gel permeation chromatography on a Sepharose 4B column equilibrated in 0.1% cholate revealed a micellar size of 4.1 nm. Subsequent removal of cholate (from an initial concentration of 2% to less than 0.02%) in the presence of Azolectin (soybean lipid extract) led to reconstituted material that could be retained on glass fibre filters. Electron microscopy revealed apparent micelles of approximately 1 micron diameter. Over 16% of the original, membrane-bound, binding sites were recovered in the reconstituted material along with a similar percentage of the protein. This reconstituted material was moderately stable for up to 3 days after its preparation.

Binding Sites↗
PubMed1987

Giardia lamblia: the role of conjugated and unconjugated bile salts in killing by human milk.

Killing of Giardia lamblia trophozoites by nonimmune human milk in vitro is dependent upon the presence of cholate which activates the milk bile salt-stimulated lipase to cleave fatty acids from milk triglycerides. In the present studies, conjugated bile salts, which predominate in vivo, displayed striking differences from unconjugated bile salts in ability to support killing by milk. Human milk killed greater than 99% of the parasites in the presence of cholate, but not glycocholate or taurocholate. In contrast, after brief sonication which disrupts milk fat globules, milk killed G. lamblia after addition of either conjugated or unconjugated bile salts. Whereas cholate stimulated milk lipase to cleave triglycerides of either unsonicated or sonicated human milk, glycocholate or taurocholate stimulated lipolysis only in sonicated milk. Since the concentration of bile salts in the small intestine fluctuates, the effect of this variable on killing was examined. Each bile salt at and above its critical micellar concentration increased Giardia survival of human milk probably because it sequestered released fatty acids in micelles. This partial protection could be overcome by increasing the milk concentration. Human hepatic and gall bladder bile and artificial bile also activated human milk to kill at low concentrations but partly protected the parasite at higher concentrations. These studies show that conjugated bile salts can activate the bile salt-stimulated lipase of sonicated human milk to release fatty acids; and kill G. lamblia. Conversely, bile salts in concentrations above their critical micellar concentration sequester fatty acids and interfere with killing. Thus, nonimmune host secretions such as milk and bile may affect the course of infection by G. lamblia.

Animals↗
PubMed1989

Effects of bile salts on energy metabolism and acid secretion by the isolated toad gastric mucosa.

The effects of bile salts on energy metabolism and acid secretion were investigated in the amphibian gastric mucosa in vitro. Serosal exposure to deoxycholate (0.2-2 mM), cholate (2-8 mM), or taurocholate (4-80 mM), at pH 7.4, decreased acid secretion depending on the concentration and time of exposure. Mucosal application of cholate and taurocholate at pH 5.6 caused a more pronounced reduction in the apparent rate of acid secretion. Oxygen uptake and substrate oxidations were significantly inhibited by bile salts in a dose-dependent manner. At pH 7.4, 1 mM deoxycholate inhibited the respiration by 49% and the rates of oxidation by 51%, 52%, 78%, 74%, and 54% of control values, for glucose, pyruvate, succinate, acetate, and butyrate, respectively. Cholate and taurocholate were found to be less potent than deoxycholate. Tissue adenosine triphosphate concentration was decreased by 13%, 57%, and 67% with 0.5, 1, and 2 mM deoxycholate, respectively. We believe the impairment of energy metabolism could be involved in the mechanism of bile salt injury to the gastric mucosa.

Animals↗
PubMed1992

Bile acid transport in the anhepatic rat.

Hepatocyte dysfunction eventually results in the loss of canalicular bile formation. Without canalicular flow, intestinal bile acid may originate from plasma by reverse transport. Anhepatic rats with preserved intestinal function permit evaluation of such transport. In the present study, plasma taurocholate clearance was markedly decreased in anhepatic rats. The relative proportion of free cholate increased with time. Peripheral tissues contained virtually only cleared taurocholate, but the intestinal contents were mainly free cholate. This indicates the intestinal contents as the source of the plasma cholate and shows an equilibrium between intestinal and plasma bile acid even without bile flow. The enteral administration of an anion exchange resin to anhepatic rats increased intestinal bile acid recovery and decreased the bile acid recovery in tissue. Plasma bile acid concentration was decreased and fractional loss increased threefold, confirming the anhepatic plasma-intestine bile acid equilibrium. However, the enhanced plasma clearance produced by the resin was less than 1% of the fractional loss found in the intact rat. These data show a very limited bile acid flux between intestine and plasma without bile flow, which could be modestly influenced by an intestinal bile acid sequestrant.

Animals↗
PubMed1996

Separation of pharmaceutically important estrogens by micellar electrokinetic chromatography.

Baseline separation of ten estrogens of the type found in conjugated estrogens tablets from pregnant mares' urine were separated by micellar electrokinetic chromatography with a 20 mM sodium borate-sodium phosphate buffer (pH 8) containing either 75 mM sodium cholate and 15 mM beta-cyclodextrin or 50 mM sodium dodecyl sulfate and 20 mM gamma-cyclodextrin. The system containing sodium cholate was used to identify estrogens in a pharmaceutical tablet formulation of conjugated estrogens and to determine the amounts and ratio of sodium equilin sulfate to sodium estrone sulfate to confirm tablet conformity with regulatory requirements. Baseline separation of the seven ethynyl steroids registered as oral contraceptives in the USA was obtained with the system 20 mM sodium borate-sodium phosphate buffer (pH 8) containing 50 mM sodium cholate and 10% (v/v) methanol. Factors that affect selectivity and reproducibility of the above separation systems are discussed.

Animals↗