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Results for “Sodium Cholate”

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At least 613 records · Page 34Linked to original sources

PubMed1988

Determination of cytochromes in human liver contaminated with hemoglobin.

A spectrophotometric method was developed to determine cytochromes in human liver mitochondria contaminated with hemoglobin. The influence of hemoglobin on the measurement was canceled by keeping hemoglobin in the carbon monoxide bound form throughout determination. Mitochondria were solubilized by 2% sodium cholate, and cytochromes were reduced enzymatically with glutamate and succinate as substrates to the maximal extent. The amount of cytochromes determined spectrophotometrically was linearly correlated with mitochondrial protein at least up to the extent of 8 mg/ml.

Aged↗

PubMed1987

Enzymatic determination of bilirubin fractions in serum.

Novel enzymatic methods using bilirubin oxidase from Myrothecium verrucaria are described for the determination of bilirubin fractions in serum. These fractions include an unconjugated form (Bu), a conjugated form (Bc), and a delta fraction of bilirubin that reacts with direct diazo reagent and is irreversibly bound to serum albumin (B delta). The determination is based on the different reactivities of the enzyme to bilirubin fractions at different pH in the presence or absence of anionic detergent such as sodium dodecyl sulfate (SDS) or sodium cholate. Thus, in the absence of detergents, Bc and B delta are oxidized at acidic pH, while Bc is oxidized at alkaline pH; Bu is not oxidized at either acidic or alkaline pH. The first approach is based on measuring the decreased absorbance of bilirubin colour at 450 nm caused by the enzymatic oxidation. Total bilirubin is measured at pH 8.0 in the presence of SDS. Direct bilirubin is measured at pH 3.7 and Bc is measured at pH 10.0 in the absence of SDS, respectively. The second approach is made by coupling the diazo reaction with the enzyme reaction. Total and direct bilirubin are determined by a conventional diazo method without prior treatment by the enzyme. B delta is determined with a direct diazo method after the serum sample is treated at pH 10.0 by the enzyme to oxidize the Bc fraction. The precision and the accuracy of these methods were compared with a diazo method, an HPLC method and a slide method, and good results were obtained.

Bilirubin↗

PubMed1985

Pulmonary vascular injury in pancreatitis: evidence for a major role played by pancreatic elastase.

Using an experimental model of pancreatitis in the rat, the role of trypsin and elastase in mediating lung vascular injury in this condition was examined. The induction of pancreatitis by injection of sodium cholate in the pancreas resulted in a significant decrease in serum trypsin inhibitory capacity, and in a complete saturation of serum elastase inhibitory capacity matched by the appearance of endothelial injury of pulmonary capillaries and edema formation. The complete lack of serum elastase inhibitory capacity was associated with the presence of elastase activity in serum and bronchoalveolar lavage (BAL) fluids. The pretreatment of animals with N-furoyl saccharin (a potent inhibitor of many serine proteinases) prevented lung capillary injury and the imbalance of serum proteinase-anti-proteinase activities as well as the appearance of any elastolytic activity in serum and BAL fluids. These findings which clearly demonstrate the protease dependence of the pulmonary vascular injury in our experimental model, strongly suggested a major role for elastase(s). The suppression, in the experimental model, of the serum elastase inhibitory capacity by using chloramine-T resulted in an earlier onset of lung vascular damage, a marked worsening of pulmonary lesions, and an increase of elastolytic levels in serum and BAL fluids. Furthermore the physical properties of the protein molecule with enzyme activity detected in BAL fluids were consistent with those of rat pancreatic elastase. The reported data strongly support the hypothesis that pancreatic elastase plays a major role in the development of pulmonary vascular injury after acute pancreatitis.

Animals↗

PubMed1970

Separation of two forms of cytochrome P450 adrenal cortex mitochondria.

The separation of two distinct forms of cytochrome P450 from adrenal cortex mitochondria has been achieved by the following steps; (1) lyophilisation (2) iso-octane extraction, (3) (NH(4))(2)SO(4) fractionation in the presence of sodium cholate. The fraction precipitating between 25-35 percent (NH(4))(2)SO(4) gave a difference spectrum with 11-deoxycorticosterone (11-DOC) but not with 20alpha-hydroxycholesterol (20alpha-HOC). This fraction showed high 11beta-hydroxylase activity but low activity for side chain cleavage of cholesterol (S.C.C.). The fraction precipitating between 45-60 percent (NH(4))(2)SO(4) gave a difference spectrum with 20alpha-HOC but not with 11-DOC and exhibited high S.C.C. activity but low 11beta-hydroxylase activity. The absorption spectrum of the 45-60 percent fraction indicated a preponderance of high spin hemoprotein (lambda(max) 395 nm).

Journal Article↗

PubMed1984

Some properties of a purinergic receptor solubilized from human uterus membranes.

A purinergic receptor was identified in human myometrium membranes using 5'-N-[3H]ethylcarboxamide-adenosine [( 3H] NECA) as radioligand. Scatchard analysis of the binding data gave a Kd of 123 nM with 2.3 pmol ligand bound/mg protein. Displacement studies indicated that the binding site had the characteristics of the A2 adenosine receptors and some of those of the P2 purinoceptors since it was inhibited by two slowly degradable ATP derivatives with IC50 values comparable to that of NECA. The receptor was solubilized with sodium cholate and its binding properties were the same as those of the membrane-bound form. No -SH group appeared to be essential for the binding activity. By density gradient centrifugation the purinergic receptor-detergent complex was estimated to have an apparent molecular mass of 95 kDa.

Adenosine↗

PubMed1987

Identification and purification of a novel G protein from neutrophils.

A novel G protein which appears to couple chemotactic peptide receptors to a polyphosphoinositide phospholipase C has been purified from rabbit neutrophils. Neutrophil membranes were solubilized with sodium cholate and fractionated by successive anion exchange, gel filtration and hydrophobic chromatography. Guanosine-5'-(3-O-thio)triphosphate binding activity was purified 170-fold from the soluble extract. The alpha-subunit of the purified G protein was identified by pertussis toxin-catalyzed ADP-ribosylation, and found to have an Mr of 40,000. The beta-subunit (Mr 36,000) comigrated on SDS-polyacrylamide gel electrophoresis with the beta-subunits of bovine brain Gi and Go. The neutrophil pertussis toxin substrate is highly unstable in cholate solution unless 30% ethylene glycol is added. Structural and functional analysis of this novel G protein will advance our understanding of the molecular mechanisms of coupling of receptors to phospholipase C.

Animals↗

PubMed1988

Purification and characterization of two GTP-binding proteins of 22 kDa from human platelet membranes.

Two GTP-binding proteins (G-proteins) of 22 kDa were purified to near homogeneity from a sodium cholate extract of human platelet membranes by successive chromatographies on DEAE-Sephacel, Ultrogel AcA-44, phenyl-Sepharose CL-4B, Mono Q HR5/5 and hydroxyapatite columns. They bound maximally 0.89 mol of [35S]guanosine 5'-(3-O-thio)triphosphate per mol of both purified proteins, and this binding was inhibited by GTP and GDP but not by ATP and AppNHp. Their molecular masses were somewhat lower than that of ras p21 and they were not recognized by an anti-v-Ki-ras p21 antibody. These results indicate that human platelet membranes contain at least two low-molecular-mass G-proteins distinct from ras p21, in addition to the heterotrimeric G-proteins, the alpha-subunits of which possess molecular mass values of about 40 kDa.

Blood Platelets↗

PubMed1988

A model for the molecular mechanism of interfacial activation of phospholipase A2 supporting the substrate theory.

Changes occurring in the activity of porcine pancreatic phospholipase A2 upon formation of mixed micelles of sodium cholate and the fluorescent phosphocholines 1,2-di[6-(pyren-1-yl)butanoyl]-sn-glycero-3-phosphocholine or 1-[6-(pyren-1-yl)butanoyl]-2-[6-(pyren-1-yl)hexanoyl]- sn-glycero-3- phosphocholine were studied. A 2-fold enhancement was observed in the activity of phospholipase A2 towards both pyrene phospholipids upon exceeding the critical micellar concentration of the system. Changes in the pyrene excimer/monomer fluorescence emission intensity ratio coincide with the enhancement of phospholipase A2 activity at the critical micellar concentration. Due to the different effects of micellization on the alignment of the pyrene in the two fluorescent probes conformational changes could be assessed. A model describing possible conformations of these pyrene phospholipid molecules below and above the critical micellar concentration is presented and correlated with the interfacial activation of phospholipase A2.

Animals↗

PubMed1989

Functional reconstitution of rat ovarian LH/hCG receptor into proteoliposomes.

Rat ovarian membrane LH/hCG receptor was solubilized in various detergents and reconstituted into proteoliposomes. Upon removal of sodium cholate by active absorption on Bio-Beads SM-2, the functional interaction between receptor and adenylate cyclase was restored. Adenylate cyclase was stimulated by hCG, hCG + GTP or GppNHp and NaF. Reconstituted proteoliposomes bound more 125I-hCG (528 fmol/mg protein) than membrane-bound receptors (384 fmol/mg protein). There was no difference, however, in the relative affinity of reconstituted receptor preparations for hCG.

Adenylyl Cyclases↗

PubMed1989

Photoactivation of rhodopsin and interaction with transducin in detergent micelles. Effect of 'doping' with steroid molecules.

On detergent-solubilised bovine rhodopsin, we have studied the formation of the active photoproduct, metarhodopsin II (MII), and its interaction with the rod G-protein, transducin (G1). The measured rate of flash-induced MII formation decreases by a factor of 300 from n-dodecyl-beta-D-maltoside (k = 4 x 10(3) at 18 degrees C, pH 6.5), over (3-(lauroyloxy)propyl)-phosphorylcholine (deoxylysolecithin), n-octyl-beta-D-glucopyranoside, sodium cholate to Chapso. For the two last agents, MII formation is similarly slow as in the native disc membrane; however, the micellar system does not display the very large decrease of the rate with lowering temperature, as is characteristic for the membrane. This points to entropic factors determining the rate in the micellar systems. An admixture of rigid steroid molecules (11-deoxycorticosterone) to lysolecithin micelles ('doped micelles') slows MII formation and shifts the MI/MII equilibrium to values typical for detergents of rigid structure. The observation gives further support to the surface free energy concept of MII formation outlined in previous studies. The free adjustment of the MI/MII equilibrium in these doped micelles allows Gt-induced formation of extra-MII to be measured, providing a convenient monitor of rhodopsin-Gt interaction in solution.

Animals↗

PubMed1995

Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells.

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.

Blotting, Western↗

PubMed1986

Hydrophobic properties of alkaline phosphatases.

The butanol extraction method of Morton (1950), a routine step in enzyme purification, is discussed with special reference to a hydrophobic form of alkaline phosphatase from human liver tissue. This form slowly precipitates from butanol-extracted liver tissue homogenates stored at 4 degrees C. Furthermore, it is lost when acetone precipitation is applied as a purification procedure. The soluble form in liver tissue is shown to have a higher relative hydrophobicity than the serum liver/bone isoenzyme. The use of sodium cholate in the isolation of the hydrophobic form produces an artefact in isoelectric focusing, which can be abolished by dialysis prior to focusing.

Alkaline Phosphatase↗

PubMed1994

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme.

1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage. 2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT. 3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA. 4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the Km for glycerol phosphate. 5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

Animals↗

PubMed1977

Induction of aortic lipid deposition in a high-response (ExHC) rat fed a diet containing cholesterol and cholic acid.

Male ExHC and Sprague--Dawley rats were fed a diet containing 3% cholesterol, 0.6% sodium cholate and 15% olive oil for 16 weeks. The ExHC rat is highly susceptible to dietary hypercholesterolemia. Aortas of the ExHC rats showed Sudan-stained deposits and cholesterol accumulation in the intima and media, whereas no deposited lipids were seen in those of the Sprague--Dawley rats. In male and female ExHC rats fed diets containing the above supplements at 3 dose levels, plasma cholesterol and aortic lipid deposition were found to be dose-dependent. Lipid deposition was more prominent in female than in males, but aortic intimal proliferation was absent.

Animals↗

PubMed1980

Effect of KC-9432, a new hypolipidemic compound, on high density lipoprotein cholesterol in rats.

KC-9432 (a new hypolipidemic compound, alpha-[p-(p-chlorobenzoyl) phenoxy-alpha-cyclohexyl acetic acid ethyl ester) was studied in rats for its effect on plasma lipid and lipoprotein levels. KC-9432 was particularly effective in hyperlipidemia in rats fed with a diet containing 2% cholesterol and 1% sodium cholate. The hypocholesterolemic activity of KC-9432 was far more potent than that of clofibrate. KC-9432 markedly increased the reduced HDL cholesterol level in dietary-induced hyperlipidemia in rats.

Animals↗

PubMed1989

Age-associated ultrastructural changes in the aortic intima of rats with diet-induced hypercholesterolemia.

The ultrastructure of the aortic intima and serum lipid levels in Fischer 344 rats were examined at the ages of 12, 18 and 24 months after feeding the animals an atherogenic diet (2% cholesterol, 0.25% sodium cholate, 5% beef fat) for 6 months. Structural changes in the intima were noticeable only at the age of 24 months. In control rats, the endothelial cells were irregular in shape and each had a well-developed Golgi complex and a few lipid droplets. Simultaneously, reticular, basal lamina-like material and electron-dense granules of extracellular liposomes accumulated in the subendothelium. In fat-fed rats, these structural changes were more conspicuous in association with hypercholesterolemia, and numerous monocytes with lipid droplets were attached to the endothelium, occasionally invading into the subendothelium. Slight foam cell lesions were evident in the intima. The finding that older rats were more susceptible to the atherogenic diet suggests that atherogenesis in the rat is promoted by intrinsic age-associated changes in the aortic intima.

Aging↗

PubMed1993

Separation of cardiac glycosides by micellar electrokinetic capillary electrophoresis.

The separation of mixtures of primary and secondary cardiac glycosides by micellar electrokinetic capillary electrophoresis modified by cyclodextrins, urea and sodium cholate proved to be suitable for the determination of these hydrophobic compounds. It was possible to distinguish the two anomeric cardenolides glucodigifucoside and glucodigiglucomethyloside with all three buffer systems. Electropherograms of crude plant cell extracts from Digitalis lanata were obtained with this method.

Carbohydrate Sequence↗

PubMed1995

Linear solvation energy relationships in micellar liquid chromatography and micellar electrokinetic capillary chromatography.

Linear solvation energy relationships (LSERs) were used to evaluate and characterize chemical interactions that influence retention behavior in micellar liquid chromatography (MLC) and micellar electrokinetic capillary chromatography (MEKC). High correlations were found between solutes' capacity factors in MLC and in MEKC, as well as binding constants to micelles and their solvatochromic parameters using two anionic surfactants, sodium dodecyl sulfate (SDS) and sodium cholate (SC), and one cationic surfactant, tetradecyltrimethylammonium bromide (C14TAB). Surprisingly, in the C14TAB MLC system capacity factor (k') vs. solvatochromic parameters gives better correlation than log k' vs. solvatochromic parameters, which is an opposite behavior to that observed in the SDS MLC system. The capacity factors in the C14TAB MLC system were characterized using LSERs with and without organic modifiers. It was found that the addition of a small amount of short-chain alcohols (e.g., 7% 2-propanol or 5% butanol) does not significantly change the high correlations between k' vs. solvatochromic parameters. The changes in the coefficients with the volume fraction of organic solvents were explained by comparing the differences in chemical natures between mobile phase and stationary phase. Stationary phase shows a significant effect on the chemical interactions in MLC through LSER study using a diphenyl column and a C8 column. LSERs were also used to characterize retention behavior in MEKC. High correlations between the logarithm of solutes' capacity factors and their solvatochromic parameters were observed for a group of 25 uncharged substituted aromatic compounds and polycyclic aromatic hydrocarbons with SDS and SC micelles. It was found that solutes' size and basicity are the two dominant factors that influence the migration behavior in MEKC.

Chromatography, Liquid↗