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PubMed1996

Brucine as a potent inducer of CYP2B3, the third member of the CYP2B subfamily P450 in rats.

We have indicated that strychnine and brucine induce concurrently an unknown P450 (2B-UP) which cross-reacts with anti-CYP2B1 antibody (Fujisaki et al., J. Pharmacol. Exp. Ther., 268, 1024-1031, 1994). We purified 2B-UP from brucine-treated rats and characterized it in this study. The purification was achieved by solubilization with sodium cholate followed by successive chromatographic steps with omega-aminooctyl-Sepharose 4B, DEAE-Sephacel and hydroxyapatite. The minimum molecular weight of purified 2B-UP was calculated to be 48000 by sodium dodecyl sulfate-gel electrophoresis. This preparation showed no Soret peak in the ultraviolet absorption spectrum indicating absence of heme. The amino terminal sequence of 2B-UP up to the 10th residue was consistent with the deduced amino acid sequence of CYP2B3 cDNA, but did not agree with the sequences of CYP2B1/2. The result strongly suggests that 2B-UP is CYP2B3. Thus, we indicated here that Strychnos alkaloid, brucine, is a potent inducer of the CYP2B3 or the closely related P450.

Amino Acid Sequence↗

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↗

PubMed1981

Rat liver ferrochelatase. Purification, properties, and stimulation by fatty acids.

Ferrochelatase from rat liver mitochondria was purified 628-fold with a 25% yield to apparent homogeneity. The purification procedure involved solubilization of the enzyme with sodium cholate, followed by ammonium sulfate fractionation and blue Sepharose CL-6B column chromatography. The molecular weight of the enzyme was estimated to be 42,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Sepharose 6B gel filtration gave a molecular weight of 240,000. The purified enzyme was analyzed for amino acid composition and shown to have abundant amounts of lysine (11%) and hydrophobic amino acid residues (48%). The enzyme was quite stable in a buffer containing 20% glycerol and 1 mM dithiothreitol. When the enzyme was assayed in the presence of palmitic acid, the specific activity for mesoheme synthesis was approximately 12,000 nmol formed/30 min/mg of protein at 37 degrees C; for protoheme synthesis, a value of 3,500 nmol formed/30 min/mg of protein was obtained. The optimum pH for the reaction was 7.8, and the Km values for the substrates were as follows: protoporphyrin IX, 28.5 microM; mesoporphyrin IX, 26.7 microM; iron with protoporphyrin IX, 33.1 microM; and iron with mesoporphyrin IX, 37.4 microM. Enzyme activity was inhibited by metals such as Co, Zn, Pb, Cu, or Mn and was highly sensitive to sulfhydryl inhibitors. The purified enzyme contained fatty acids, and its activity was markedly stimulated by their addition. Phospholipids slightly stimulated enzyme activity. Short chain carbonic acids and neutral lipids produced no effects.

Amino Acids↗

PubMed1995

Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization.

We have reported previously that human keratinocytes synthesize and secrete acetylcholine and that muscarinic cholinergic drugs have effects on keratinocyte proliferation, adhesion, and migration. This study defines the location of muscarinic acetylcholine receptors in human epidermis and describes some pharmacologic and molecular properties of these receptors. Confocal microscopy employing the anti-muscarinic receptor monoclonal antibody M35 visualized the receptors in the intercellular areas of normal human epidermis. Using immunoelectron microscopy, the receptors appeared to be attached to the keratinocyte plasma membranes. Functional, high-density (Bmax = 8.3 nmol/2 x 10(6) cells) and high-affinity (Kd = 21.5 nM) muscarinic receptors were demonstrated by saturable binding of the reversible radioligand [3H]quinuclidinyl benzilate to the surfaces of freshly isolated epidermal cells at 0 degrees C. Receptor proteins were separated by gel electrophoresis. An apparent isoelectric point of pH 4.3 was determined in immunoblots of sodium-cholate-solubilized receptors separated on isoelectric-focusing gels. Three protein bands, two at approximately 60 kDa and one at 95 kDa, were visualized in immunoblots of membrane-bound or solubilized receptors separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The covalent, irreversible ligand [3H]propylbenzilylcholine mustard confirmed these results. Thus, human keratinocytes express a heterogeneous population of muscarinic cholinergic receptors. Because human keratinocytes also express nicotinic cholinergic receptors, endogenously secreted acetylcholine may control different biologic processes in these cells by activating different types of their cholinergic receptors.

Antibodies, Monoclonal↗

PubMed1987

Purification and characterization of human placental aromatase cytochrome P-450.

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.

Androgens↗

PubMed1981

Early induction of rat colonic epithelial ornithine and S-adenosyl-L-methionine decarboxylase activities by N-methyl-N'-nitro-N-nitrosoguanidine or bile salts.

The responses of male noninbred rat colonic epithelial ornithine decarboxylase (EC 4.1.1.17) (ODC) and S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) (SAMD) activities following topical administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or bile salts were studied. A single intrarectal installation of 13 mumol of MNNG resulted in a significant (p < 0.001) 20-fold peak ODC activity after 4 hr, with a prompt return to control levels by 12 hr. Stimulation of SAMD activity was less pronounced but significant (p < 0.01), with a broad 2-fold peak over controls. No significant responses of colonic epithelial enzyme activities were detected following a single intrarectal instillation of N-methyl-N'-nitroguanidine, a noncarcinogenic and nonmutagenic metabolite of MNNG, at a dose equimolar to that of MNNG. Bile salts significantly (p < 0.001) induced ODC with almost the same kinetic pattern as that observed after MNNG administration in the following order: sodium deoxycholate > sodium chenodeoxycholate > sodium cholate. Activations of SAMD were similar for these 3 bile salts. Glycine- or taurine-conjugated deoxycholate showed ODC and SAMD enzyme activations similar to that of nonconjugated deoxycholate. No significant enzyme response was seen after sodium dehydrocholate treatment. Stimulation of activities of both enzymes was directly dependent on bile salt dose. Induced ODC and SAMD activities were principally localized in colonic epithelium. Deoxycholate-stimulated enzyme activities were significantly inhibited by cycloheximide. Enzyme stimulations by active compounds were accompanied by morphological changes such as mucosal cell degeneration, mucus depletion, submucosal congestion, and punctate hemorrhage, followed by submucosal leukocytic cellular infiltration. These data support the concept that initiating and promoting events may be involved in colon carcinogenesis.

Adenosylmethionine Decarboxylase↗

PubMed1988

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids.

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.

3-Hydroxysteroid Dehydrogenases↗

PubMed2002

Cholate feeding counteracts calcium-induced depression of apparent fat digestibility in rats.

This study tested the hypothesis that cholate feeding would counteract the earlier described calcium-induced inhibition of fat digestion. Rats were fed semipurified diets; either low (0.25%, w/w) or high (1.0%) in calcium, the latter diets being without or with 0.5% added sodium cholate. Apparent fat digestibility was 95.6% of intake in the rats fed the low-calcium diet. Calcium feeding significantly lowered apparent fat digestibility to 82.6%, but in the presence of cholate it was 91.2%. It is concluded that the inhibitory effect of calcium on fat digestion is mediated by diminishing the availability of bile acids.

Animals↗

PubMed1975

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.

Amylases↗

PubMed1995

A lysophosphoinositide-specific phospholipase C distinct from other phospholipase C families in rat brain.

Distinct phospholipase C activities capable of hydrolyzing lysophosphatidylinositol (lysoPI-PLC) or phosphatidylinositol (PI-PLC) have been demonstrated in rat brain membranes. Treatment of brain membranes with 1 M NaCl or 1% sodium cholate solubilized a majority of PI-PLC activity from the membranes, whereas a significant level of lysoPI-PLC activity still remained membrane-associated. Most of the lysoPI-PLC activity was recovered in a 0.5% sodium deoxycholate-0.25 M NaCl extract which contained only low levels of PI-PLC activity. Using the separated fractions, differences between lysoPI-PLC and the known PI-PLC isoforms were examined. A specific peptide inhibitor of PI-PLC, which was previously shown to interact with active site regions common to known PI-PLC activity. Immunoblot analysis of both the lysoPI-PLC-rich and PI-PLC-rich fractions revealed that an antiserum against PI-PLC delta 1 cross-reacted with other PI-PLC isoforms, but not significantly with lysoPI-PLC. Furthermore, lysoPI-PLC was more resistant to sulfhydryl reagents than was PI-PLC. Our results indicate that lysoPI-PLC is an enzyme distinct from PI-PLC and that lysoPI-PLC possesses a different active site than known PI-PLC isoforms.

Amino Acid Sequence↗

PubMed2005

Percolation phenomenon in mixed reverse micelles: the effect of additives.

The conductivity of AOT/IPM/water reverse micellar systems as a function of temperature, has been found to be non-percolating at three different concentrations (100, 175 and 250 mM), while the addition of nonionic surfactants [polyoxyethylene(10) cetyl ether (Brij-56) and polyoxyethylene(20) cetyl ether (Brij-58)] to these systems exhibits temperature-induced percolation in conductance in non-percolating AOT/isopropyl myristate (IPM)/water system at constant compositions (i.e., at fixed total surfactant concentration, omega and X(nonionic)). The influence of total surfactant concentration (micellar concentration) on the temperature-induced percolation behaviors of these systems has been investigated. The effect of Brij-58 is more pronounced than that of Brij-56 in inducing percolation. The threshold percolation temperature, Tp has been determined for these systems in presence of additives of different molecular structures, physical parameters and/or interfacial properties. The additives have shown both assisting and resisting effects on the percolation threshold. The additives, bile salt (sodium cholate), urea, formamide, cholesteryl acetate, cholesteryl benzoate, toluene, a triblock copolymer [(EO)13(PO)30(EO)13, Pluronic, PL64], polybutadiene, sucrose esters (sucrose dodecanoates, L-1695 and sucrose monostearate S-1670), formamide distinctively fall in the former category, whereas sodium chloride, cholesteryl palmitate, crown ether, ethylene glycol constitute the latter for both systems. Sucrose dodecanoates (L-595) had almost marginal effect on the process. The observed behavior of these additives on the percolation phenomenon has been explained in terms of critical packing parameter and/or other factors, which influence the texture of the interface and solution properties of the mixed reverse micellar systems. The activation energy, Ep for the percolation process has been evaluated. Ep values for the AOT/Brij-56 systems have been found to be lower than those of AOT/Brij-58 systems. The concentration of additives influence the parameters Tp and Ep for both systems. A preliminary report for the first time on the percolation phenomenon in mixed reverse micelles in presence of additives has been suggested on the basis of these parameters (Tp and Ep).

Journal Article↗

PubMed1996

Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process involving transcytosis. In the present study, we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells. The endothelial cell membranes were isolated from cultured cells by differential centrifugation and solubilized with sodium cholate and urea. The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.

Animals↗

PubMed1982

Enzymic differentiation of neurologic and nonneurologic forms of Gaucher's disease.

This study explores the biochemical basis that may distinguish neurologic and nonneurologic forms of Gaucher's disease. Crude membrane preparations from spleens of controls and patients representing the three clinical categories of Gaucher's disease were delipidated by extraction with sodium cholate and n-butanol. Total beta-glucosidase activity was estimated using 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) as substrate, and glucocerebrosidase activity was determined using (3H)-glucocerebroside. beta-Glucosidase and glucocerebrosidase activities were reconstituted by inclusion of sodium taurocholate or phosphatidylserine in the assay medium. When assays contained phosphatidylserine, residual beta-glucosidase activity in delipidated spleen preparations from type 1, nonneurologic cases were five times greater than cases of neurologic Gaucher's disease (82.3 vs. 11.3 units per mg protein). However, beta-glucosidase assays using sodium taurocholate did not discriminate Gaucher's disease subtypes. Similar results were obtained when spleen preparations were analyzed for glucocerebrosidase using glucocerebroside as the substrate. Brain beta-glucosidase from patients representing the three classes of Gaucher's disease showed a similar pattern of sensitivity toward phosphatidylserine. The specific activity of beta-glucosidase in an extract of brain from the one case of type 1 Gaucher's disease analyzed was five times greater than the mean residual specific activity of brain beta-glucosidase measured in five cases of type 2 and type 3 Gaucher's disease. These findings suggest that, in patients with type 1 Gaucher's disease, glucocerebrosidase may show greater activity in the presence of acidic phospholipids than glucocerebrosidase does in patients with neurologic forms of the disease. The ability of the brain enzyme from a type 1 case to be profoundly stimulated by an acidic phospholipid may explain why such individuals are spared central nervous system involvement.

Adult↗

PubMed2001

Discontinuous electrophoretic stacking system for cholate-based electrokinetic chromatographic separation of 8-hydroxy-2'-deoxyguanosine from unmodified deoxynucleosides.

The stacking and baseline-resolved separation of the oxidative damage marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), from unmodified deoxynucleosides in under 4 min is reported. Separations of 8-OHdG from 2'-deoxyadenosine, 2'-deoxycytosine, 2'-deoxyguanosine, and thymidine are accomplished using micellar electrokinetic capillary chromatography with sodium cholate. Importantly, the use of sulfate, intentionally added to the sample matrix, results in effective stacking of 8-OHdG and other analytes. This work extends electrokinetic stacking injection of neutral analytes to include deoxynucleosides. The procedure works well with either electrokinetic or hydrodynamic injection. The separation buffer and sample matrix composition were optimized to effect stacking conditions with an uncoated 50 microm fused-silica capillary. The lower limit of detection for the analytes is in the nanomolar range, and is more than an order of magnitude lower than without stacking. With 30 s (5.7 cm) electrokinetic injections, stacking and baseline separation of 8-hydroxy-2'-deoxyguanosine from the unmodified nucleosides is accomplished, even in the presence of a 400-fold excess of unmodified deoxynucleosides.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1989

Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae.

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.

Cholic Acid↗

PubMed1986

Purification and characterization of a single form of cytochrome P-450 from the spiny crab Maja crispata.

Microsomes from Maja crispata hepatopancreas contain all the components of the functional mixed function oxidase system: cytochrome P-450 (0.47 nmol/mg), the activity of NADPH cytochrome c reductase (12.25 nmol/mg/min) and benzo[a]pyrene monooxygenase activity (6.58 pmol/mg/min). Solubilization of hepatopancreas microsomes with sodium cholate, and affinity chromatography on omega-amino-n-octyl Sepharose 4B, gave a single cytochrome P-450 peak eluting with 0.2% Emulgen 913. DEAE cellulose chromatography of this cytochrome peak gave rise to a single haemoprotein peak, with apparent monomer Mr = 53,500, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Animals↗

PubMed1987

Molecular cloning of bile acid 7-dehydroxylase from Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal bacterium which contains an inducible bile acid 7-dehydroxylase. Two-dimensional polyacrylamide gel electrophoresis showed that at least four new polypeptides were synthesized after exposure of growing cells to sodium cholate. One of these, of molecular weight 27,000 (PP-27), was implicated in 7-dehydroxylase catalysis. PP-27 was purified to greater than 95% homogeneity by DEAE-cellulose chromatography, high-pressure liquid chromatographic gel filtration, high-pressure liquid chromatography-DEAE chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 33 amino acid residues of the N terminus of PP-27 were determined with a gas-phase sequencer, and a corresponding mixed oligonucleotide (17-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.2-kilobase fragment which hybridized to the 32P-labeled 17-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA, ligated into the bacterial plasmid pUC8, and used to transform Escherichia coli HB101. Transformants containing the putative 7-dehydroxylase gene were detected with the 32P-labeled 17-mer by colony hybridization techniques. The insert was 2.2 kilobases in length and contained the first 290 bases of the PP-27 gene. Preliminary nucleic acid sequence data correlate with the amino acid sequence. The entire gene was cloned on a 1,150-base-pair TaqI fragment. Western blot analysis of E. coli strains containing these plasmids indicated that PP-27 is expressed in E. coli but is not regulated by bile acids under the conditions used.

Amino Acid Sequence↗

PubMed1978

Reconstitution of neutral amino acid transport system from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.

Alanine↗