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PubMed1996

Phospholipid composition of erythrocyte membranes and plasma of mammalian blood including Australian marsupials; quantitative 31P NMR analysis using detergent.

The phospholipid classes of erythrocyte membranes and plasma from several domestic animals and marsupials were quantified by 31P NMR using detergents. Washed erythrocyte samples were thoroughly haemolysed by tip-sonication and dissolved in sodium cholate; plasma samples were dissolved in Triton X-100. The species studied were: common wombat (Vombatus ursinus), black-striped wallaby (Macropus dorsalis), bandicoot (Isoodon macrocarpus), Eastern grey kangaroo (Macropus giganteus), Tammar wallaby (Macropus eugenii), sheep (Ovis aries), goat (Capra hircus), cattle (Bos taurus), horse (Equus caballus), dog (Canus familiaris) and rabbit (Orytolagus caniculus). There were considerable species variations in the relative abundance of erythrocyte and plasma phospholipid classes. The variations may be attributed to the habitats and diets of the animals as well as to their phylogenetic differences.

Animals↗

PubMed1992

A detergent-solubilized nicotinic acetylcholine receptor of Caenorhabditis elegans.

We have used a spin column assay to study the detergent-solubilized levamisole receptor, a nicotinic acetylcholine receptor of the nematode Caenorhabditis elegans. The receptor can be successfully solubilized in detergent solutions of Triton X-100, Lubrol PX, or sodium cholate. Centrifugal gel filtration assay using the tritiated ligand [3H]meta-aminolevamisole ([3H]MAL) provides a greater signal and a better signal-to-noise ratio for soluble levamisole receptor binding than either polyethylene glycol precipitation or DEAE filter assay with the same ligand. As for membrane-bound receptor, the detergent-solubilized levamisole receptor consists of more than one affinity state. Detergent solubilization appears to increase the affinity of all states for [3H]MAL (Kd for the highest affinity solubilized [3H]MAL binding state, 41 +/- 5 pM). Data is presented on the equilibrium binding and the association and dissociation reaction rates of the receptor. The similar relative efficacy with which various compounds inhibit specific [3H]MAL binding and deficiencies in solubilizable high affinity specific [3H]MAL binding in two receptor mutants show that the solubilized receptor is the same nicotinic acetylcholine receptor that is detected by assaying membrane-bound specific [3H]MAL binding. The detergent-solubilized levamisole receptor is stable at 0 degree to 4 degrees C, making receptor purification feasible.

Animals↗

PubMed1992

Bile salt independent retinyl ester hydrolases in the bovine eye.

Homogenates of bovine neuroretina and retinal pigment epithelium (RPE) were incubated with 11-cis and all-trans retinyl palmitate to study retinyl ester hydrolysis. The highest activity was found in RPE when 11-cis retinyl palmitate served as substrate (Km = 7.8 microM and Vmax = 44.8 pmol/min/mg). This retinyl ester hydrolase (REH) had an optimum activity at acidic pH (pH 5), which is in contrast to the neutral hydrolase (pH 8) found in the neuroretina. Similar to REH in the liver, REH activities in the bovine eye are not stimulated by bile salts because sodium cholate, taurocholate and deoxycholate did not enhance retinyl ester hydrolysis. Most REH activities in retinal homogenate were soluble, whereas in the RPE, these activities were membrane-bound. Divalent cations such as zinc and cadmium completely inhibited REH activities in the neuroretina. Our results show that bovine ocular tissues contain several retinyl ester hydrolases with distinct biochemical properties.

Animals↗

PubMed1982

Isolation and partial purification of phthalate ester hydrolyzing enzyme(s) from the brine shrimp, Artemia.

Isolation and 30-fold purification of a di-n-butyl phthalate (DNBP) hydrolyzing enzyme from synchronously hatched larvae of the brine shrimp, Artemia, are reported. The activity of the enzyme increases during larval development simultaneously with the acute toxic action of DNBP on the larvae. The enzyme is not stimulated by 10 mM sodium cholate, is stable for days to weeks in solution at 4 degrees C, indefinitely stable at -70 degrees C and is distinct from the previously reported trypsin- and chymotrypsin-like proteases [1].

Animals↗

PubMed1995

Separation of urinary estrogens by micellar electrokinetic chromatography.

Urinary estrogen levels are important for monitoring the normal pregnancy process as well as for the diagnosis of reproductive diseases. 17 beta-Estradiol and estrone are maintained at very low concentrations in urine and, therefore, are difficult to determine using standard chromatography with UV detection. In the present study, we describe a potential method for the determination of urinary estrogens (estrone, estradiol and estriol) using a solid-phase extraction and rapid capillary electrophoretic (CE) separations. Micellar electrokinetic chromatographic (MEKC) analysis was optimized by evaluating the number of surfactants in a 5 mM borate-5 mM phosphate separation buffer, of which sodium cholate (75-90 mM) was found to be optimal. Changing the hydrophobic character of the separation buffer with organic additives had a significant effect on the resolution of the three estrogens and an internal standard (d-equilenin). The addition of an organic additive (20% acetonitrile) was found to be necessary for the resolution of all components of the mixture. Substitution with 20% methanol provided a similar separation with better resolution but at the cost of increased analysis time. Analysis of two extracted urine samples from 18-weeks and 21-weeks pregnant women showed that, with the present technology, CE can provide adequate resolution and superior speed, but the sensitivity limits attainable with the existing technology may limit its utility to the measurement of estriol and estrone.

Chromatography, Liquid↗

PubMed1989

An ion-selective microelectrode for bile salts.

The development of a prototype ion-selective microelectrode for bile salts is described. The microelectrodes demonstrated Nernstian response (59 +/- 10 mV dec-1) with sodium deoxycholate in Tris (0.2 M, pH 9.0, 15-30 degrees C), HEPES (0.13 M, pH 7.5), and bicarbonate buffers (0.1 M, pH 7.5) (25 +/- 0.1 degrees C) were stable for several days, and the responses were highly reproducible. The microelectrodes were selective for bile salts over the physiologically important inorganic anions, bicarbonate and chloride. The response to sodium cholate (42 mV dec-1) was consistently lower than the ideal response (59 mV dec-1). This ion-selective microelectrode may show promise as a useful tool for the determination of intracellular bile salt activity.

Bile Acids and Salts↗

PubMed1995

Potentiometric determination of formation constants of copper(II)/bile acid/peptide in aqueous solution.

A potentiometric method was used to study the equilibria in aqueous solution for the systems copper(II)/bile acid salt/peptide [bile acid salt-sodium cholate or glycocholate; and peptide-glutamic acid (glutamate), glutamine or glutamylglutamic acid], specifically to assess cooperative binding between small peptides and bile acids in the presence of copper(II). The results obtained suggest large cooperativity in formation of ternary complexes with amino acids when they are coordinated to copper(II) as bidentate ligands.

Amino Acid Sequence↗

PubMedDate not supplied

In vitro activity of sparfloxacin (CI-978, AT-4140, and PD 131501). A quinolone with high activity against gram-positive bacteria.

Sparfloxacin (CI-978, AT-4140 and PD 131501) is a new antimicrobial agent of the piperazinyl quinolone class. Relative to other quinolones, it is a potent antistaphylococcal and antistreptococcal drug in vitro: The microbroth 90% minimum inhibitory concentration (MIC90) (in microgram/ml) was 0.25 vs 26 methicillin-resistant and -sensitive coagulase-positive and -negative staphylococci and 20 Streptococcus pneumoniae; 0.5 vs 20 strains each of S. pyogenes, S. agalactiae, and Enterococcus faecalis. The data indicate sparfloxacin to be generally superior to ciprofloxacin, ofloxacin, oxacillin, cefazolin, doxycycline, amikacin, and vancomycin against these Gram-positive bacterial groups. Additional MIC90s were determined for Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, and Neisseria gonorrhoeae (less than or equal to 0.03); Enterobacteriaceae (0.5); and Listeria monocytogenes (1). Activity was generally unchanged with light, 50% human serum, aerobic-anaerobic atmosphere, 5% sodium cholate, cation supplementation, and 100-fold increased or decreased inoculum; as with other quinolones, potency was measurably diminished with decreasing pH (pH less than or equal to 6.0) and in 100% urine. Naturally occurring resistant mutants occurred at frequencies of 10(-8) or lower.

Anti-Infective Agents↗

PubMedDate not supplied

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes.

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.

Animals↗

PubMed1996

Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain.

Flavin-containing monooxygenase (FMO), previously reported both from hepatic and extrahepatic tissues, including brain, catalyze the oxidation of certain xenobiotics and drugs that contain a nucleophilic heteroatom. Psychoactive drugs, including the antidepressant imipramine, are substrates for the brain FMO. Since FMO-mediated metabolism of these drugs might contribute to local pharmacodynamic modulation within the human brain, the metabolism of imipramine by human brain FMO was studied in further detail. In the present study, the FMO activity was determined in human brain microsomes by estimating the actual amount of imipramine N-oxide formed. It was then compared with the corresponding activity measured using substrate (imipramine)-stimulated rates of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation, which was significantly higher than the activity estimated as the amount of N-oxide assayed using high-pressure liquid chromatography (HPLC). The brain FMO activity was measurable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The activity was inhibited by an antibody to rabbit pulmonary FMO, but an antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect indicating that cytochrome P-450 was not involved in the above metabolic pathway. The optimum pH for N-oxidation of imipramine was found to be 8.5; thermolability experiments indicated that the FMO activity was completely lost only after the incubation of brain microsomes at 45 degrees C for 20 minutes. An FMO purified to apparent homogeneity from a human brain had a molecular weight of 71,000 Da. The purified enzyme cross-reacted with the antibody to rabbit pulmonary FMO and efficiently catalyzed the metabolism of imipramine to its N-oxide. The human brain clearly contains an active FMO system, and it is conceivable that such enzymes are significantly involved in the local metabolism and modulation of pharmacological and/or toxic effects of certain xenobiotics, including psychoactive drugs.

Antidepressive Agents, Tricyclic↗

PubMed1990

Solubilization of a 5-HT3 binding site from rabbit small bowel muscularis membranes.

A 5-HT3 binding site, with high affinity for (S-)[3H]zacopride, was solubilized from rabbit small bowel muscularis membranes utilizing 0.5% sodium cholate and 400 mM (NH4)2SO4. Approximately 72% of the (S-)[3H]zacopride binding activity was recovered in a form that retained the high affinity (Kd = 0.7 nM) and specificity for this radioligand that is characteristic of the membrane-bound receptor. ICS 205-930 and other 5-HT3 compounds were effective inhibitors and exhibited the same rank order of potency in the solubilized and membrane-bound preparations. The receptor-detergent complex did not sediment after centrifugation for 1 h at 150,000 x g and eluted between thyroglobulin (MW = 669,000) and apoferritin (MW = 443,000) when fractionated by high-performance liquid chromatography gel filtration. This is the first report of the solubilization of a 5-HT3 binding site.

Animals↗

PubMed1992

Reconstitution of alpha 1-adrenoceptors having high affinity for prazosin.

Using a variety of detergents we find that soluble alpha 1-adrenoceptor recovery from aortic and hepatic membranes is markedly enhanced if the receptor is first prelabelled with prazosin. Moreover, prelabelling prevents the reduction in prazosin affinity induced by solubilizing concentrations of digitonin, possibly by stabilizing the receptor's conformation. 20-25% of alpha 1-adrenoceptors solubilized in sodium cholate were reconstituted into brain lipids. Specific [3H]prazosin binding to the reconstituted receptor was saturable and of high affinity (KD = 0.019 +/- 0.008 nM). We conclude that prelabelling is essential to preserve the receptor in detergents. However, once the detergent is removed brain lipids alone are able to maintain the receptor in a form with high affinity for prazosin.

Animals↗

PubMed1993

Very long chain acyl-CoA dehydrogenase deficiency: identification of a new inborn error of mitochondrial fatty acid oxidation in fibroblasts.

A patient highly suspected of long chain fatty acid oxidation defect was investigated for membrane-bound palmitoyl-CoA dehydrogenation in a membrane extract from skin fibroblasts, using 1% sodium cholate as detergent. The profoundly decreased activity observed is consistent with a deficiency of the newly identified mitochondrial 'very long chain acyl-CoA dehydrogenase'.

Acyl-CoA Dehydrogenase, Long-Chain↗

PubMed2006

Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli.

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.

17-alpha-Hydroxyprogesterone↗

PubMed2004

Cholesterol amperometric biosensor based on cytochrome P450scc.

A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA microM(-1) and the detection limit is 300 microM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 microM and 6.9 nA microM(-1) with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5-2 min).

Biosensing Techniques↗

PubMed2005

Partial purification and characterization of pro-phospholipase A2 activating proteases from gill membranes of the red sea bream, Chrysophrys major.

We previously reported that gill group IB secretory phospholipase A(2) (sPLA(2)) exists as an inactive pro-sPLA(2) with the dipeptide Ala-Arg, at the N-terminus of mature sPLA(2) in mucous cells. Pro-sPLA(2) should be activated after being secreted to the surface of gill epithelia by trypsin-like protease. To clarify the above hypothesis, we investigated the existence of pro-sPLA(2) activating protease (PAP) in the gills of the red sea bream, using gill pro-sPLA(2) as a substrate. PAP was solubilized from the membrane fraction of the gills with 2% sodium cholate and partially purified by benzamidine-Sepharose chromatography and reversed-phase HPLC. Partially purified proteases, PAP1 and PAP2 showed a high molecular mass of about 200 kDa by gelatin zymography. PAP1 and PAP2 had optimal pH from 7 to 9 and were inhibited by trypsin inhibitors. These properties of PAP1 and PAP2 suggest that both enzymes belong to the membrane-associated trypsin-like serine protease family, such as enteropeptidase and corin. This is the first report verifying the existence of the activating protease of group IB pro-sPLA(2) isoforms in a non-digestive tissue.

Animals↗

PubMed2005

Selective determination of gamma-aminobutyric acid, glutamate and alanine by mixed micellar electrokinetic chromatography and fluorescence detection.

A mixed micellar electrokinetic chromatography method with fluorescence detection was developed to simultaneously monitor gamma-aminobutyric acid (GABA), glutamate (Glu) and alanine (Ala) in biological samples. Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of three NDA-labeled isomers (GABA, alpha-ABA, beta-ABA) was studied in detail with different micelles solutions such as sodium dodecyl sulfate (SDS), beta-cyclodextrin (beta-CD) and sodium cholate (SC). Simultaneous resolution of GABA, Glu and Ala from 21 amino acids was achieved within 5 min using 20 mM phosphate buffer at pH 8.7 containing 24 mM SC and 26 mM SDS. The detection limits were 4.0 x 10(-8), 1.1 x 10(-8) and 1.3 x 10(-8) M, for GABA, Glu and Ala, respectively, with S/N = 2. The method was applied to monitor the changes of amount of GABA, Glu and Ala in tobacco leaf in response to cold and dark stress.

Alanine↗

PubMed2004

Transcutaneous IL-2 uptake mediated by Transfersomes depends on concentration and fractionated application.

INTRODUCTION: Transfersomes (TF) are new, ultradeformable carriers with characteristics that enable them to penetrate the skin spontaneously. TFs are able to transport noninvasively both low- and high-molecular-weight molecules into the body. MATERIALS AND METHODS: TFs contain phosphatidylcholine and sodium cholate. Recombinant human interleukin-2 (Proleukin, Chiron) was added to the TFs and incubated for 24 h at 4 degrees C. The immunotransfersomes (ITF) were isolated from free interleukin-2 (IL-2) by filtration (Centrisart, Sartorius). Twenty-five thousand, 50,000 and 150,000 IU pure IL-2 and ITFs, which had been incubated with the same concentrations of IL-2, were applied subcutaneously (s.c.) (n = 8) and epicutaneously (e.c.) (n = 8) to mice. The IL-2 serum concentrations in the mice were then measured by ELISA after 2, 4, 6, 8, 10 and 24 h. Fractionation of the transdermal IL-2 application was also examined as a means of improving uptake. RESULTS: In concentrations of 25,000 and 50,000 IU IL-2, the subcutaneous application of ITFs resulted in a longer lasting IL-2 serum concentration than did the subcutaneous application of pure IL-2. While at 25,000 IU, the epicutaneous application of ITFs resulted in serum concentrations comparable to those resulting from s.c. application, at 50,000 and 150,000 IU, only 50% and 22.6% of the maximum serum concentration resulting from the s.c. application of pure IL-2 was obtained. Fractionating the transdermal IL-2 application improved uptake. CONCLUSION: We were able to show that biologically active IL-2 can be bonded to TFs up to 75%. It is possible to transport IL-2 through the skin using TFs. Both the concentration-dependent saturation of the TFs with IL-2 and fractionation of the application resulted in differing degrees of transcutaneous IL-2 uptake.

Administration, Cutaneous↗