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PubMed1977

[The effects of 4 detergents on the respiration of mitochondria isolated from rat liver].

The effects of these four detergents (two non-ionic: Titron X100 and Tween 80, two ionic: sodium cholate and sodium dedeoxycholate) upon the respiratory intensities of mitochondria and upon the ADP/0 and respiratory control ratios were observed. At low concentrations and in the absence of exogenous ADP, non ionic as well as ionic detergents provoked a threefold (or fourfold) increase of the respiratory intensities of mitochondria. At higher concentrations, the four detergents were inhibitory for mitochondrial oxidations in the order: Triton X 100 greater than DOC greater than cholate greater than Tween 80. At increasing doses, the four detergents progressively decreased the phosphorylating capacities of mitochondria.

Adenosine Diphosphate↗

PubMed1977

[Membrane lipids and electron transfer. Effects of four detergents on NADH-ferricyanide reductase and NADH-cytochrome c reductase activities of potato tuber microsomes].

The NADH-ferricyanure reductase activity of Potato microsomes is stimulated by non ionic detergents (Triton X100 and Tween80) and is partially inhibited by ionic detergents (sodium-cholate and deoxycholate). All these four detergents progressively decreased the NADH-cytochrome c reductase in the following order: sodium deoxycholate greater than Triton X100 greater than sodium cholate greater than Tween80.

Cytochrome Reductases↗

PubMed1978

[Effect of increasing concentrations of detergents on electron transfer in chloroplasts].

The effects of increasing concentrations of sodium cholate and deoxycholate, "Tween 80" and "Triton X 100" upon O2 evolution by intact or osmotically broken spinach chloroplasts were investigated polarographically. Except for cholate, low concentrations of any detergent increased considerably (up to four times) the rates of oxygen evolution by broken chloroplasts, without interrupting the electron flux between photosystem II and photosystem I.

Chloroplasts↗

PubMed1979

Dissolution of human cholesterol gallstones in vitro with bile salts. A scanning and stereoscanning electron microscopic study.

This report deals with the in vitro dissolution of cholesterol gallstones by bile salts, as observed with the scanning electron microscope. Scanning electron microscopic techniques permit excellent visualization of the substructure of these stones and hence allow insight into the structural basis of gallstone dissolution. The small faceted, mixed cholesterol gallstone was used since this is the most common type clinically. Scanning electron microscopy revealed that the gallstone has four structurally different zones; an outer surface, a cortex of compactly layered cholesterol plates, an inner layer of cholesterol mixed with other components, and a center or nidus. The cortex is of particular importance in terms of gallstone dissolution with bile salts, since the interior of the stone remains unaffected until this zone is removed. The speed and completeness of dissolution of the stone depends mainly upon the thickness of the cortex and in part upon the content of noncholesterol components in the interior of the stone. Chenodeoxycholate and cholate behave similarly in terms of the dissolution process as observed by scanning electron microscopy. The solubilization process is very similar to that observed in vitro with solvents such as ether and ethanol. Stones treated with 400 mM cholate were completely solubilized in 3 weeks. The process described in this paper is probably similar to that which occurs in vivo in spontaneous gallstone dissolution in man and in patients treated with bile salts for gallstone dissolution.

Chenodeoxycholic Acid↗

PubMed1979

[Reconstitution of liver endoplasmic reticulum membranes from solubilized proteins and lipids by removal of detergent].

A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown. A removal of the detergent results in a formation of membranes, which are chemically close to the original ones but ultrastructurally very different from the latter. On the other hand, absorption or dialysis of cholate-solubilized proteins and lipids results in reconstituted membranes with asymmetrically arranged intramembrane particles located on the hydrophobic surfaces of the membrane halves. The number and size of these particles are similar to those of the original microsomal membranes.

Animals↗

PubMedDate not supplied

[Disturbance of the intensity of secretion and chemical composition of bile in liver dystrophy induced by carbon tetrachloride].

Liver dystrophy induced by carbon tetrachloride results in inhibition of intensity of bile secretion and cholate formation and upsets discharge of bilirubin and cholesterol with bile. Inhibition of cholate synthesis might be accounted for by repressed production of deoxycholic and cholic acid from cholesterol as well as by repressed conjugation of cholic acid with taurine and glycine. Cessation of carbon tetrachloride injections is followed by steady restoration of bile formation: first of all is discharge of bilirubin restored, then discharge of cholesterol, and much later intensity of bile discharge and formation of bile acids.

Animals↗

PubMed1984

Lipid-dependent recovery of alpha-bungarotoxin and monoclonal antibody binding to the purified alpha-subunit from Torpedo marmorata acetylcholine receptor. Enhancement by noncompetitive channel blockers.

Recently the purified alpha-subunit from Torpedo marmorata acetylcholine receptor was shown to bind alpha-bungarotoxin with a KD approximately 3 nM in the presence of sodium dodecyl sulfate (Tzartos, S.J., and Changeux, J.P. (1983) EMBO J. 2, 381-387). Here we describe a further significant step toward renaturation of the alpha-subunit as judged by toxin and monoclonal antibody binding. Purified T. marmorata receptor subunits were diluted with 1% lipids (asolectin) plus 0.5% Na+ cholate. An anion-exchange resin eliminated most of the detergents, leaving approximately 0.1% Na+ cholate and the lipids. After this treatment, about 20% of the alpha-subunit recovered (but not the beta-, gamma-, or delta-subunit) exhibited a high affinity for radioiodinated alpha-bungarotoxin with a KD approximately 0.5 nM. The 34,000- and 27,000-dalton proteolytic peptides of the alpha-subunit conserved this lipid-dependent toxin binding. Unlabeled alpha-toxins, hexamethonium, and carbamylcholine competed with alpha-bungarotoxin for the renatured alpha-subunit. Noncompetitive channel blockers doubled the lipid-dependent toxin-binding capacity of the alpha-subunit but had no effect on the 27,000-dalton peptide. The binding of several monoclonal antibodies to the main immunogenic region (which is particularly sensitive to denaturation) significantly increased. In particular, binding of antibody 16 changed from 1% to denatured to 100% to the lipid-renaturated alpha-subunit. The binding of these antibodies was lost with the lipid-renatured 34,000- and 27,000-dalton peptides.

Animals↗

PubMed1983

Modulation of gamma-glutamyl transpeptidase activity by bile acids.

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.

Animals↗

PubMed1982

Phosphatidylcholine-promoted interaction of the catalytic and regulatory proteins of adenylate cyclase.

The catalytic protein of rabbit hepatic adenylate cyclase, after chromatographic separation from the GTP-binding regulatory protein (G/F) (Ross, E. M. (1981) J. Biol. Chem. 256, 1949-1953), is essentially free of endogenous phospholipids. This preparation is active in the presence of Mn2+ and is markedly stimulated by forskolin, but it is stimulated only slightly by the addition of purified G/F plus an activator (GTP gamma S or NaF). The ability of activator-liganded G/F to stimulate the activity of the resolved catalyst is increased up to 8-fold by the addition of either dimyristoylphosphatidylcholine (0.3-1.5 mM optimal concentration) or several other phosphatidylcholines. Phosphatidylcholine stabilizes the catalyst to denaturation but has little effect on its basal activity or on its stimulation by Mn2+ or forskolin. It also had no stimulating effect on the activation of G/F by GTP gamma S. These data are interpreted as showing that phosphatidylcholine promotes or is required for the stimulatory interaction of activator-liganded G/F with the catalytic protein of adenylate cyclase. Lubrol 12A9, Triton X-100, cholate, lysophosphatidylcholine, digitonin, and phosphatidylserine could not substitute for phosphatidylcholine. The detergents inhibited stimulation by liganded G/F even in the presence of phosphatidylcholine. Removal of cholate from a mixture of soluble catalytic protein and phosphatidylcholine by dialysis and sucrose density gradient centrifugation caused the binding of catalytic protein to large unilamellar vesicles. This preparation was further reconstituted with increasing amounts of G/F to yield vesicles with varied G/F:catalyst ratios and similarly varied responses to G/F-mediated activating ligands.

Adenylyl Cyclases↗

PubMed1984

Kinetics and thermodynamics of the formation of mixed micelles of egg phosphatidylcholine and bile salts.

The kinetics of dissolution of dispersions of egg phosphatidylcholine (lecithin) by bile salts was studied by observing the decrease in turbidity as mixed micelles of lecithin and bile salt were formed. The rate of dissolution of lecithin corresponding to the formation of mixed micelles was studied in the presence of dihydroxy bile salts, sodium deoxycholate, sodium chenodeoxycholate, sodium ursodeoxycholate, and one trihydroxy bile salt, sodium cholate. The rate of dissolution of lecithin and mixed micelle formation decreased in the order: chenodeoxycholate greater than deoxycholate greater than cholate greater than ursodeoxycholate. Kinetic solvent isotope studies in D2O, along with measurement of enthalpies of mixed micelle formation both in H2O and D2O, appear to indicate that the formation and stabilization of mixed micelles is related to "hydrophobicity" as estimated by high performance liquid chromatography retention factors.

Bile Acids and Salts↗

PubMed1984

Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases. Purification of the human erythrocyte proteins without the use of activating regulatory ligands.

Methods were developed to adequately extract, separate and, without the use of NaF as stabilizing agent, purify to better than 90% purity human erythrocyte Ns and Ni, the stimulatory and inhibitory guanine nucleotide- and Mg-binding regulatory components of adenylyl cyclases, as well as a protein containing Mr = 35,000 subunits. On the basis of a functional assay for Ns, it was purified about 5,000-fold from starting washed erythrocyte membranes with a yield of about 10%. A typical purification yields from 60 units of outdated human blood, between 500 and 1,000 micrograms of pure Ns, and a similar amount of Ni. Pure Ns and Ni contain each at least one alpha and one beta subunit (Northup, J.K., Sternweis, P.C., Smigel, M.D., Schleifer, L.S., Ross, E.M., and Gilman, A.G. (1980) Proc. Natl. Acad. Sci. U.S.A. 74, 6516-6520; Codina, J., Hildebrandt, J.D., Iyengar, R., Birnbaumer, L., Sekura, R.D., and Manclark, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 77, 4276-4280). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at varying acrylamide concentrations yielded Mr values of 42,000 and 40,000 for the alpha subunits of Ns and Ni, and of 35,000 for the beta subunits of Ns and Ni. Two-dimensional thin layer analysis of tryptic peptides obtained from digesting 125I-labeled subunits of Ns and Ni confirmed the finding of Manning, D., and Gilman, A.G. (1983) J. Biol. Chem. 258, 7059-7063) that while their alpha subunits are clearly different, their beta subunits are the same. Hydrodynamic analysis of the molecular weights of the nondenatured proteins showed behavior consistent with Mr = 95,500 for Ns, the same for Ni, and Mr = 40,000 for the protein containing the Mr = 35,000 beta subunit. Sedimentation coefficients and Stokes radii of the purified Ns were indistinguishable from those of Ns activity present in initial cholate extracts from human erythrocyte membranes. Further, the overall kinetics with which Ns activity in cholate extracts and Ns activity in the purified protein reconstituted the Ns-deficient adenylyl cyclase system of cyc- S49 cells was also indistinguishable. We conclude that we have purified the native unactivated form of Ns, and by serendipity the Ni, as well as a protein containing the 35 kDa beta subunit of Ns and Ni.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗

PubMed1984

One transporter per vesicle: determination of the basis of the insulin effect on glucose transport.

The basis for insulin stimulation of glucose transport in rat adipocytes has been investigated by determining the relative number of functional glucose transporters in the plasma and microsomal membranes from basal and insulin-treated cells. Each fraction was solubilized with cholate and then reconstituted into vesicles of about 500 A in diameter through removal of the cholate by dialysis. This procedure distributed the glucose transporters into the vesicles at a density of either one or none per vesicle. Consequently the fraction of the intravesicular volume that rapidly equilibrated with D-glucose provided an estimate of the relative number of functional transporters. By means of this one-transporter-per-vesicle method, it was found that insulin increased the number of transporters in the plasma membrane by a factor of 2.4 and decreased the number in the microsomes to 68% of the original value. These results provide independent evidence for the hypothesis that insulin causes the translocation of functional transporters from an intracellular location to the plasma membrane.

Adipose Tissue↗

PubMed1984

Chemical dissolution of common bile duct stones.

Dissolution of bile duct calculi is complicated by the facts that about 30-40% of them are pigment stones and the stone cannot be unambiguously identified by radiography before the start of therapy. Thus it does not appear logical only to infuse irrigation media that dissolve cholesterol (cholate, Capmul) but to use solutions that also dissolve calcium bilirubinate. Calcium bilirubinate is the most important compound in primary pigment stones in the bile duct. Thin sections of calcium bilirubinate stones can be dissolved in EDTA 4Na. The rate is determined by the temperature, the pH, and the surface tension of the solution. In vitro experiments showed that cholesterol stones and composition stones can be dissolved more rapidly by alternating therapy with an EDTA solution and a Capmul preparation than by monotherapy with glycerol octanoate, and that bovine pigment stones can also be disaggregated. Since calcium bilirubinate stones consist up to 20-60% of an organic matrix, a mixture of glycerol octanoate and EDTA was prepared containing SH-activated papain. It was possible, by using this mixture, to disaggregate human calcium bilirubinate stones. The process of dissolution is complex and is not yet understood in detail. It is supposed that the important steps are the extraction of calcium, the chemical solution and molecular dispersion of bilirubin and cholesterol, and the disaggregation of the structure of the stone by surface-active substances. The irrigation media have but little effect on black pigment stones. Toxicity studies have shown that cholate, glycerol octanoate, and glycerol octanoate preparations are locally toxic and can lead to cholangitis and cholecystitis in animals. EDTA solutions bring about lesser changes. In humans, it has not been possible to distinguish these inflammatory changes unambiguously from those found in untreated gallstone patients.

Aged↗

PubMed1983

ATP-driven proton fluxes across membranes of secretory organelles.

The ATP-dependent proton uptake by chromaffin granule membranes, lysosomes, and synaptosomes was examined. In synaptosomes the reaction was absolutely dependent on the presence of chloride, while in chromaffin granules chloride had a profound effect and in lysosomes only a minor effect. The presence of chloride markedly increases the rate of collapse of delta pH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in all three organelles. Ascorbate with phenazine methosulfate uncoupled the ATP-dependent proton uptake by chromaffin granules, but had no effect on lysosomes and synaptosomes. Proton uptake by submitochondrial particles was about 50-fold more sensitive to dicyclohexylcarbodiimide than the proton uptake by chromaffin granule membranes. Chromaffin granule membranes were treated with 2 M sodium bromide to inactivate the mitochondrial ATPase. The treatment caused a complete inhibition of the ATP-dependent proton uptake. Solubilization of these membranes by sodium cholate, followed by reconstitution by cholate dilution revealed the ATP-dependent proton uptake of the system. It is concluded that the genuine ATPase enzyme of chromaffin granules is a proton translocator.

Adenosine Triphosphate↗

PubMed1981

[Preservation of the allosteric properties of the protein receptor for acetylcholine in detergent solution without addition of lipids].

Solubilization by Na cholate of acetylcholine receptor-rich membrane fragments from Torpedo marmorata, followed by exchange of NA cholate by the neutral detergent "Tween 80" yields the receptor protein in its 9 S soluble light form; under these conditions, without adding lipids, the receptor protein conserves its characteristic binding properties for the fluorescent agonist Dns-C6-Cho as followed by fast kinetic techniques, and the allosteric regulation by non-competitive blockers.

Allosteric Regulation↗

PubMedDate not supplied

Properties of reconstituted rabbit pulmonary mixed-function oxidase system with cytochrome P-450.

Reconstituted rabbit pulmonary mixed-function oxidase (MFO) system is composed of four components: NADPH cytochrome P-450 reductase, cytochrome P-450, phosphatidylcholine and cholate. Evidence has been presented that phosphatidylcholine and cholate form micelles that take an active part in the reconstitution. Order of addition of MFO system components in the reconstitution has been intensively studied. Several titration curves well describe reconstituted system. Experimental data suggest formation of dissociable complex of reductase, cytochrome P-450 and micelles A model for reconstituted rabbit pulmonary MFO system is discussed.

Animals↗

PubMed1982

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-monooxygenase.

7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.

Animals↗

PubMed1982

Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity.

Proteoliposome vesicles containing apoA-I, lecithin, and cholesterol (including labeled cholesterol) were prepared from various molar ratios of the three components by the cholate dialysis technique. Comparative studies on the sensitivity and efficiency of these proteoliposomes to serve as substrate for lecithin:cholesterol acyltransferase (LCATase) indicated that the proteoliposome with apoA-I:lecithin:cholesterol molar ratio of 0.8:250:12.5 was ideal for assaying LCATase activity of both plasma and purified enzyme. This proteoliposome was shown to be comparable in size by gel filtration (radius, 131.9 +/- 4.8 A, n = 6) and by electron microscopy (radius, 123.4 +/- 5.1 A, n = 100). The proteoliposome preparation was stable as LCATase substrate for at least 3 and 5 weeks, respectively, when stored at 4 degrees C and -20 degrees C, and was a better substrate for the enzyme activity assay than were lecithin-cholesterol liposomes incubated with apoA-I. Under the standardized assay system LCATase activity was a linear function of plasma enzyme added and was independent of the amount of plasma cholesterol added to the proteoliposomes in the range of 3 to 20 microliters of plasma. The mean LCATase activity by this method was 95.1 +/- 14.0 (range 76.5-122.5) nmol/hr per ml of plasma from fifteen normal human subjects. This method of substrate formation using the cholate dialysis technique permits the preparation of large amounts of stable, efficient, homogeneous, and well-defined substrate that is suitable for measuring low levels of enzyme activity, comparative studies, and large scale investigations of plasma LCATase, as well as studies of the mechanism and regulation of LCATase reaction.

Apolipoprotein A-I↗