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PubMed1987

Purification of NADPH:cytochrome c (cytochrome P-450) reductase from hamster liver microsomes by detergent extraction and affinity chromatography.

NADPH:cytochrome c (cytochrome P-450) reductase (Fp) from hamster liver microsomes has been purified to near homogeneity using a simple and rapid method. Microsomes were treated with the detergent Chaps (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid) in combination with 0.07% protamine sulfate and then centrifuged to pellet insoluble material. While over 60% of the total microsomal protein was solubilized, all Fp activity remained in the pellet. Fp was extracted from the Chaps-insoluble material using a combination of the detergents sodium cholate and Lubrol PX. This treatment resulted in a fivefold increase in Fp specific activity and allowed direct processing of the enriched Fp fraction by 2',5'-ADP agarose affinity chromatography. The purified Fp had a total flavin content of 23 nmol/mg protein (flavin adenine dinucleotide:flavin mononucleotide ratio = 1:1), a specific activity of 26,000 units/mg protein at 22 degrees C using cytochrome c as electron acceptor, and migrated as a single band on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis with a relative molecular weight of 76,000. The purity, specific activity, and yield were nearly identical to results obtained when the flavoprotein was purified by conventional methods. This procedure eliminates the need for anion-exchange chromatography and allows for the rapid purification of large amounts of Fp suitable for use in studies concerning cytochrome P-450-mediated drug metabolism. Importantly, this method is equally effective when used to purify Fp from rat liver microsomes.

Animals↗

PubMed1991

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification.

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.

Angiotensin II↗

PubMed1987

Purification of acetylcholine receptor-like protein from fetal calf thymus.

A cobrotoxin binding protein from the fetal calf thymus was isolated by affinity chromatography after solubilization with sodium cholate. The specific activity as a nicotinic acetylcholine receptor (AChR) was determined by assessing the binding to [3H]-alpha-bungarotoxin (BuTx), using the high-pressure liquid chromatography. An AChR-like protein was detected in the amount of 1.39-2.14 nmol per g protein. The first peak of 420k-protein from gel filtration of the eluate of affinity chromatography on a Sephacryl column showed one major polypeptide band with an Mr of 40k, by polyacrylamide gel electrophoresis in sodium dodecylsulfate, two major protein bands with pI 5.4-5.6 and 9.2 by isoelectric focusing, and reacted with sera from patients with myasthenia gravis.

Animals↗

PubMed2000

Analysis of phospholipid molecular species in brain by (31)P NMR spectroscopy.

Techniques are described for the (31)P NMR analysis of glycerophospholipid (PL) headgroup and molecular species in brain. The (31)P NMR spectrum of PLs from human temporal cortex, solubilized in aqueous Na cholate, typically showed 3 major resonances, assigned to phosphatidylcholine (PC) molecular species containing 0, 1, or 2 fully saturated acyl chains. Less species resolution was obtained for the other PL headgroups under these conditions. Alkylacyl- and alkenylacyl-PC were readily discerned using the CHCl(3)-CH(3)OH-H(2)O solvent method. The chain-length, temperature, and species dependences of the (31)P NMR chemical shifts were explored in model PLs. Assignments of signals from phosphatidylethanolamine (PE) subclasses were confirmed in the sodium-cholate system by lipase-mediated selective hydrolysis of bovine-brain PE. The utility of (31)P NMR to monitor enzymatic PL oxidation was further demonstrated. Possible changes in PL composition with postmortem interval (PMI) in rat brain were examined. No significant changes were seen in PL headgroup or PC species composition with PMI at up to 18 hours. Where comparable, the Na-cholate-solubilization and solvent methods gave similar quantitative results for headgroup analysis on the same samples. The present work demonstrates the feasibility and utility of the dual system for analysis of PLs in brain. Magn Reson Med 44:215-223, 2000.

Aged↗

PubMed1986

Purification and properties of human placental NADPH-cytochrome P-450 reductase.

Human placental NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 microM 4-androstene-3,17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35-60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2',5'-ADP-Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolysis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 +/- 0.7 mumol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.

Amino Acids↗

PubMed1977

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.

Animals↗

PubMed1983

Solubilization and partial purification of n,n'-dicyclohexylcarbodiimide-sensitive ATPase from pea cotyledon mitochondria.

The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F(0)F(1) preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.

Journal Article↗

PubMed1989

Solubilization and reconstitution of the sodium-and-chloride-coupled glycine transporter from rat spinal cord.

Synaptic membranes from rat spinal cord were solubilized in the presence of 2% sodium cholate, phospholipids and 15% ammonium sulphate. The soluble extract was incorporated into liposomes consisting of asolectin and crude rat brain lipids. Reconstitution of the functional transporter protein was achieved by removal of detergent by gel filtration. Several parameters proved to be important for optimal reconstitution efficiency: (a) the lipid composition of the liposomes, (b) the type of detergent, and (c) the phospholipid/protein and detergent/protein ratio during reconstitution. In the reconstituted system, the transport of glycine showed a specific activity about twice that of native vesicles. The ionic dependence of the transport, the inhibitory effect of nigericin in the presence of external sodium and the stimulatory effect of valinomycin in the presence of internal potassium on glycine transport were preserved and more clearly observed in the reconstituted system. These results indicate that, in this preparation, the glycine transporter protein retains the same features displayed in the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity and inhibitor sensitivity.

Amino Acid Transport Systems, Neutral↗

PubMed1984

Detergent solubilization and affinity purification of a local anesthetic binding protein from mammalian axonal membranes.

Previously, we identified a local anesthetic receptor in the membranes of nerve cells, especially enriched in axonal membranes, by the binding assay of a fluorescence local anesthetic probe, quinacrine (Greenberg, M., and Tsong, T.Y. (1982) J. Biol. Chem. 257, 8964-8971). We report here affinity chromatography purification of the quinacrine binding protein from axonal membranes of bovine brain. One per cent sodium cholate solubilized more than 90% of the membrane proteins, and the quinacrine binding activity was preserved in the extract; KD remained at 1.0 microM. An affinity column of quinacrine mustard covalently linked to Affi-Gel 10 (Bio-Rad) retained about 5% of the total solubilized proteins, and the subsequent elution with 1.0 M lidocaine revealed a protein peak which coeluted with the lidocaine peak. After removal of lidocaine, the protein peak retained the high affinity to quinacrine (KD = 1.5 microM). Furthermore, bound quinacrine could be displaced with dibucaine in the range of its nerve-blocking concentration, around 10 microM. Sodium dodecyl sulfate-gel electrophoresis of the peak protein fractions gave a predominant band of approximately 16 kDa. A major protein band with the same molecular weight was also seen in the sodium dodecyl sulfate gel of the total axonal membrane proteins. The receptor concentration in the axonal membranes was estimated to be 2-5 nmol/mg protein. With a molecular weight of 16 kDa, the receptor should constitute 3-8% of total proteins, consistent with the finding that it is a major protein constituent of the axonal membranes. The purity of the binding protein after the affinity chromatography step was about 90%. The purified protein contained roughly 60 nmol of quinacrine binding sites/mg.

Animals↗

PubMed1980

Molecular characterization, reconstitution, and "transport-specific fractionation" of the saxitoxin binding protein/Na+ gate of mammalian brain.

The saxitoxin (STX) binding protein has been solubilized by sodium cholate, both from axolemma and from synaptosomal membranes of mammalian brain. On the basis of agarose gel filtration and sedimentation properties in H2O and 2H2O, the solubilized particle has the following molecular properties: Stokes radius, 120 A; partial specific volume, 0.85 cm3/g; mass, 1,020,000 daltons; frictional ratio f/fo, 1.6. The solubilized STX binding protein was incorporated into unilamellar (approximately 550-A) artificial phosphatidylcholine vesicles. Based on the expectation that the STX binding protein contains functional monovalent cation gating activity ("action potential Na+ gate") that can be activated by veratridine and inhibited by tetrodotoxin, a strategy was devised for partial purification of the reconstituted sodium gate/STX binding protein by "transport-specific fractionation." When the entire vesicle population was preloaded with 0.4 M cesium ion, addition of veratridine allowed Cs+ efflux from specifically those vesicles containing the ion gate; the concomitant reduction in intravesicular density permitted the ion gate/STX binding protein to be fractionated on density gradients. These observations demonstrate functional reconstitution and partial (30- to 50-fold) purification of the STX binding protein/Na+ gate of mammalian brain.

Amphibian Proteins↗

PubMed1984

Purification and characterization of epimeric estradiol dehydrogenases (17 alpha and 17 beta) from equine placenta.

Estradiol 17 alpha-dehydrogenase (EC 1.1.1.148) and estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from horse placenta have been purified to homogeneity. Both enzymes are localized in the microsomal fraction and are solubilized in 1.5% sodium cholate. The 17 alpha- and 17 beta-dehydrogenases are separated by selective elution from hydroxylapatite with 0.5 and 1.0 M potassium phosphate, respectively. Subsequent purification is achieved by two affinity-absorption steps using reactive blue 2-agarose and estriol hemisuccinate-Sepharose. Homogeneous estradiol 17 alpha-dehydrogenase has a specific activity of 10 IU/mg and has been purified 5900-fold with an 87% recovery. Homogeneous estradiol 17 beta-dehydrogenase has a specific activity of 10.6 IU/mg and has been purified 15 000-fold with an apparent recovery of 100%. Each enzyme exhibits a single band on polyacrylamide disc gel electrophoresis and on sodium dodecyl sulfate (SDS) gel electrophoresis. The mobilities of the two on SDS gels are identical and correspond to subunit molecular weights of 33000. The apparent molecular weight of the undenatured, active enzyme, as determined by gel filtration on Sephacryl S-300, is 52000 in the case of the 17 alpha-dehydrogenase and 68500 for the 17 beta-dehydrogenase. Both enzymes exhibit pH optima at 9.0-9.5; both prefer NAD+ over NADP+ but utilize both cofactors. Both are highly specific for their respective epimers of estradiol with apparent Km values of 1.7 microM at pH 9.5.

17-Hydroxysteroid Dehydrogenases↗

PubMed1980

The isolation and purification of cytochrome c1 from bovine heart.

A large-scale isolation method for cytochrome c1 from beef heart is presented, based in principle on the procedure of Yu et al. (Yu, C.A., Yu, L. and King, T.E. (1972) J. Biol. Chem. 247, 1012--1019). Optimal solubilization of cytochrome c1 from succinate-cytochrome c oxidoreductase was achieved with 15% beta-mercaptoethanol, 1.5% cholate, 0.5% deoxycholate in 8% saturated ammoniun sulphate. The protein is purfied to a higher degree by chromatography on DEAE-cellulose and Ultrogel AcA 44. The method is reproducible and gives highly purified cytochrome c1 with a yield from succinate-cytochrome c oxidoreductase of 40%. The purified cytochrome c1 contains 32 nmol of heme/mg protein and has a spectral heme-to-protein ratio (Ared417nm/Ax276nm) of 2.7. Reduced cytochrome c1 is oxidized very rapidly by ferricytochrome c (k = 3 . 10(7) M-1 . S-1 at 10 degrees C, 100 mM potassium phosphate (pH 7.0) and 1% Tween 20). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate shows that the isolated protein consists of one peptide, with a molecular weight of 31 000, carrying the chromophore. In the presence of 1% sodium cholate or 1% Tween 80, cytochrome c1 is in the monomeric state, whereas at lower concentrations of detergent the protein aggregates. The aggregation of cytochrome c1 is found to be reversible.

Ammonium Sulfate↗

PubMed1990

Separation and determination of lipophilic corticosteroids and benzothiazepin analogues by micellar electrokinetic chromatography using bile salts.

The separation of corticosteroids and benzothiazepin analogues by micellar electrokinetic chromatography (micellar EKC) was studied in comparison with capillary zone electrophoresis. The separation of these substances was not successful under neutral and alkaline conditions because they migrated with the same velocity as that of the electroosmotic flow. Micellar EKC with sodium dodecyl sulphate (SDS) solutions was also not successful because these substances migrated with almost the same velocity as that of the SDS micelle, owing to their high lipophilicity. The use of bile salts, which have a similar skeleton to corticosteroids, as the micellar phase permitted the separation of these substances with high theoretical plate numbers (150,000-350,000) within a short time (ca. 15 min). Sodium cholate was particularly useful. The effects of bile salt concentration, pH and the addition of methanol were investigated. Micellar EKC was also applied to the determination of the drug substances in tablets and cream using the internal standard method and to purity testing of drug substances and tablets.

Adrenal Cortex Hormones↗

PubMed1983

Solubilization and partial purification of two forms of cytochrome P-450 from trout liver microsomes.

Liver microsomal cytochrome P-450 content, NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethylmorphine N-demethylase activities of rainbow trout, Salmo gairdneri, were found to be 0.16 (n = 10) nmol P-450/mg protein, 38 (n = 5) units/mg protein, 0.04 (n = 4) nmol p-aminophenol/min/mg protein and 0.174 (n = 3) nmol formaldehyde/min/mg protein, respectively. Trout liver cytochrome P-450 was solubilized by treatment of microsomes with sodium cholate. Chromatography on DEAE-cellulose column yielded two distinct cytochrome P-450 fractions from solubilized microsomes. Cytochrome P-450-I was eluted with Emulgen 913-containing buffer. Application of 0.08 M KCl in Emulgen 913-containing buffer to the DEAE-cellulose column eluted cytochrome P-450-II fraction. Cytochrome P-450-I was further purified on hydroxylapatite column. CO-difference spectrum of dithionite-reduced cytochrome P-450-I gave a peak at 449 nm while the similar spectrum of cytochrome P-450-II showed a maximum absorbance at 451 nm. Monomer molecular weights of cytochrome P-450-I and cytochrome P-450-II were determined by sodium dodecyl sulfate gel electrophoresis and found to be 56,000 and 48,500, respectively.

Animals↗

PubMed1978

The interaction of detergents with bilayer lipid membranes.

Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025--0.15 mM; dodecyl sulphate 0.82 in the range 0.01--1 mM; sodium deoxycholate 1.03 in the range 0.01--1 mM and sodium cholate 1.37 in the range 0.1--10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.

Cholic Acids↗

PubMed1996

Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique.

A novel method using fluorescence depolarization to determine the critical micelle concentrations (CMC) of surfactants was developed. Fluorescence anisotropies of Triton X-100, sodium dodecyl sulfate, and sodium cholate were measured using 1,6-diphenyl-1,3, 5-hexatriene as a fluorescence probe. Fluorescence anisotropy decreased with increasing surfactant concentrations below the CMC and leveled off above the CMC. The depolarization method does not depend on the concentration of DPH and is largely immune to light-scattering problems encountered in turbid aqueous systems.

Cholic Acid↗

PubMed1987

Purification of the alpha 2-adrenergic receptor from porcine brain using a yohimbine-agarose affinity matrix.

A procedure has been developed for purification of the porcine brain alpha 2-adrenergic receptor to homogeneity. alpha 2-Adrenergic receptors were solubilized from porcine brain particulate preparations using sequential extraction into sodium cholate- and digitonin-containing buffers. The alpha 2-adrenergic receptors in the digitonin extract were identified using the alpha 2-adrenergic selective antagonist, [3H]yohimbine, and demonstrated the same specificity for interaction with adrenergic ligands as did the receptors in particulate preparations. Extraction into digitonin-containing buffers eliminated the modulation of receptor-agonist interactions by guanine nucleotides, but not by monovalent cations. A novel affinity resin, yohimbine-agarose, was synthesized and used for purification of alpha 2-adrenergic receptors. Using two sequential yohimbine-agarose affinity chromatography steps, digitonin-solubilized alpha 2-adrenergic receptors from porcine brain cortex were purified to homogeneity as assessed by radioiodination and silver stain analysis of these preparations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified alpha 2-adrenergic receptor has an approximate Mr = 65,000, as determined by photolabeling of the adrenergic ligand-binding subunit. The yohimbine-agarose affinity resin should be useful for purifying quantities of receptor sufficient for studies of receptor structure and function.

Animals↗

PubMed1981

Transglutaminase activity in normal and hereditary cataractous rat lens and its partial purification.

Hereditary cataractous rat lenses showed significantly higher specific activity for transglutaminase than the normal lenses of comparable age. Transglutaminase activity of normal lenses was distributed predominantly in the buffer-soluble fraction. The buffer-insoluble fraction showed 14% of total activity. The cortical and nuclear fractions of the normal lens showed 43% and 57% distribution of total activity, respectively. Protein solubilizing agents enhanced the activity of the enzyme in the lens homogenate in the following order: Triton X-100 greater than Tween 20 greater than Sodium dodecyl sulfate greater than Sodium deoxycholate greater than Sodium cholate greater than NaSCN greater than KI. Transglutaminase was purified 15 fold by hydrophobic affinity chromatography employing omega-amine octylagarose matrix. The purified enzyme was activated by calcium and inactivated by iodoacetamide and upon freeze-drying. Lens crystallins served as exogenous substrate for transglutaminase, with gamma-crystallin as the most effective amine acceptor.

Animals↗