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PubMed1981

Solubilization and reconstitution of the adenosine 5'-triphosphate dependent noradrenaline uptake system of bovine chromaffin granule membrane.

The ATP-dependent catecholamine uptake system of chromaffin granule membrane has been solubilized and reconstituted in phospholipidic vesicles. The activity of the vesicles implies that both the ATP-dependent H+-translocase and the noradrenaline carrier have been successfully reconstituted. The membrane was solubilized by sodium cholate in presence of asolectin and the asolectin to cholate ratio appeared to be critical. Omission of asolectin resulted in reconstitution of vesicles with an active H+-pump and an inactive transport system. The detergent was removed from the solubilized membranes by filtration on Sephadex G-50 and it has been verified that the residual detergent of the reconstituted preparation was below the concentration inhibitory to the ghost H+-pump. The pH-dependence, Km for ATP and Km for noradrenaline of the reconstituted vesicles were similar to those of the ghosts, but their specific activity and reserpine-resistance were somehow variable. Vesicle activity was limited by transporter reconstitution, thus suggesting that reconstitution of the complete system might be used as an assay for the transporter. The noradrenaline carrier is not physically linked to dopamine beta-hydroxylase and bears no wheat germ agglutinin binding sites.

Adenosine Triphosphate↗

PubMed1999

A novel, endogenous inhibitor of 7-ethoxyresorufin-O-deethylase activity isolated from liver cytosolic fractions of bream (Abramis brama L.).

The cytosolic supernatant of bream (Abramis brama L.) liver homogenates inhibits the 7-ethoxyresorufin-O-deethylase (EROD) activity of pike (Esox lucius) microsomal fractions. The inhibitor shows no activity against 7-ethoxycoumarin-O-deethylase and benzo(a)pyrene hydroxylase indicating a high isoenzyme specificity. The inhibiting component is a heat-sensitive substance (56 degrees C for 5') which is not self regenerating after subsequent cooling. It can be isolated from the cytosolic fraction using two combined steps of ion exchange chromatography. The purification factor is 500-fold with a recovery rate of 70%. SDS-PAGE of the purified fractions indicate that electrophoretic purity was not achieved. However, a prominent band at about 97 kDa was present in all fractions in a close intensity activity relationship. The molecular weight of the native form of the purified protein was determined to be 175 +/- 35 kDa using gel filtration on a Sephacryl S 300 HR column. So far the inhibitor can be characterized as a protein. It shows strong tendencies to aggregate due to lipophilic interactions. These interactions can be repressed by the addition of 1% sodium cholate. The inhibitor has an optimum activity at 25 degrees C and pH 8.0. The inhibitor does not correspond to any of the known cytosolic, endogenous inhibitors of EROD activities in fish, including proteases, cytosolic phosphatases, kinases and resorufin reductase (e.g. DT-diaphorase), although a non-dicoumarol (10 microM)-inhibited menadione oxidoreductase activity of up to 46.7 +/- 0.4 nmol/min per mg inhibitory protein was measured. Kinetic studies using Michaelis-Menten kinetics with purified inhibitor fractions prove a non-competitive mode of inhibition. As this kind of inhibitor is not described yet it is named CERODIP (cytosolic, EROD-inhibiting protein).

Animals↗

PubMed2000

Skin delivery of oestradiol from lipid vesicles: importance of liposome structure.

The aim of this study was to investigate the importance of liposome structure in oestradiol skin delivery as a tool for understanding the delivery mechanism from lipid vesicles. Liposomes of phosphatidylcholine (PC) (1), PC, sodium cholate; 86:14 w/w (II), PC, Span 80; 86.7:13.3 w/w (III) and PC, oleic acid: 84:16 w/w (IV) with 1 mg/ml radiolabelled drug were prepared. Saturated radiolabelled oestradiol solutions containing the components of I-IV were separately prepared in 90% w/w propylene glycol in water. In addition, saturated solutions containing cholate, Span, oleic acid and ethanol at the same concentrations used in vesicles were formulated. Oestradiol permeation through human epidermis was studied. Formulations I-IV increased oestradiol flux by 8.6, 17, 17 and 13-fold when used as vesicles compared with control and by 2.9. 4.0, 4.7 and 6.9-fold when used in solution with drug. Testing individual components in solution, relative fluxes were 2.9, 0.87, 1.1, 2.9 and 1.1 for PC, cholate. Span, oleic acid and 7% ethanol, respectively. Accordingly, it is important to prepare phospholipids as vesicles for efficient oestradiol skin delivery even after inclusion of oleic acid. Penetration enhancement is not the main mechanism for improved flux. Liposome components in solution have additive effect with a possible synergism in some cases.

Aged↗

PubMed2002

In-vitro release of diclofenac diethylammonium from lipid-based formulations.

This article presents the preparation and topical performance of some new lipid-based formulations of diclofenac, namely (a) a diclofenac aqueous gel containing mixed micelles (sodium cholate:egg lecithin molar ratio 0.55); (b) diclofenac lotion that contains soya lecithin, ethanol and buffer; and (c) diclofenac lipogel containing egg lecithin, isopropyl myristate, propylene glycol and ethanol. Gel formulations were prepared using Carbomer 934. Release of diclofenac from all formulations was monitored via dialysis through Spectra/por membrane into phosphate buffer (0.2 M pH=7.4) using a Franz cell. Drug release profile and diffusion coefficients were compared with brand formulation (Geigy's Vlotaren Emulgel). Statistical analysis of data show that the diffusion coefficient of the drug from these formulations rank according to the following order: Diclofenac lotion (D=5.308x10(-7) cm(2)/s) >lipogel (D=2.102 x 10(-7) cm(2)/s) >Voltaren Emulgel (1.518 x 10(-7) cm(2)/s) >aqueous gel mixed micelle (0.966 x 10(-7) cm(2)/s). These results show that diclofenac lotion and lipogel maybe more suitable formulations than the conventional topical dosage form.

Administration, Topical↗

PubMed1996

Sparfloxacin worldwide in vitro literature: isolate data available through 1994.

Sparfloxacin is a piperazinyl, cyclopropyl-fluoroquinolone with broad-spectrum antibacterial activity. Compared to other quinolones, sparfloxacin displays improved activity against a variety of pathogens including Staphylococcus, Streptococcus, Enterococcus, Chlamydia, Mycoplasma, Ureaplasma, and Mycobacteria species. Other susceptible organism group include Haemophilus, Legionella, Moraxella, Neisseria, Aeromonas, Acinetobacter, Bordetella, Brucella, Campylobacter, Gardnerella, and Helicobacter species. Most Enterobacteriaceae are also susceptible, whereas most isolates of Pseudomonas aeruginosa are not. Sparfloxacin is bactericidal. Activity is generally stable to variations of inoculum, pH, and cation concentration, and it is unchanged in the presence of 5% sodium cholate or 70% human serum. Susceptibility to the drug is diminished in urine. Cross-resistance, although incomplete, has been documented with other quinolones, but not with other antimicrobic classes.

Anti-Infective Agents↗

PubMed2001

Phospholipid abnormalities in postmortem schizophrenic brains detected by 31P nuclear magnetic resonance spectroscopy: a preliminary study.

It has been hypothesized that schizophrenia arises from cell membrane abnormalities due to changes in phospholipid (PL) composition and metabolism. We have used high resolution, in vitro 31P nuclear magnetic resonance (NMR) to characterize the PLs in left frontal cortex (gray matter) of postmortem brain from four schizophrenics and five controls. High resolution 31P NMR spectra were obtained in an organic-solvent system to resolve PL classes (headgroups) and in a sodium-cholate, aqueous dispersion system to resolve phosphatidylcholine (PC) molecular species. Multivariate analysis which included the major PC molecular species and phosphatidylinositol (PI) showed a significant difference between schizophrenics and controls. Analysis of specific interactions showed that the PI was significantly higher in the schizophrenic group than in the control group. There were no differences between the two groups for other individual PL classes, or for individual PL subclasses determined by the linkage type at the sn-1 position on glycerol. There was a trend for total PL content to be higher in schizophrenics than in controls. There was no evidence for elevated lysophosphatidylcholine or lysophosphatidylethanolamine in schizophrenia. The intensity of the PC peak representing molecular species with one saturated and one unsaturated (one or two double bonds) acyl chain was higher for the schizophrenic group than for the control group. Although these results are not in complete agreement with previous studies, they support the idea that PL abnormalities occur in the brain in schizophrenia and that fatty acid metabolism may be abnormal.

Adult↗

PubMed2002

Solubilization of cholesterol and polycyclic aromatic compounds into sodium bile salt micelles (part 2).

The aqueous solubility of cholesterol was determined over the temperature range from 288.2 to 318.2 K with intervals of 5 K by the enzymatic method. The solubility was (3.7+/-0.3)x10(-8) mol dm(-3) (average +/- S.D.) at 308.2 K. The maximum additive concentrations of cholesterol into the aqueous micellar solutions of sodium deoxycholate (NaDC), sodium ursodeoxycholate (NaUDC), and sodium cholate (NaC) were spectrophotometrically determined at different temperatures. The cholesterol solubility increased in the order of NaUDC<NaC<NaDC; for example, 0.10 for NaUDC, 0.61 for NaC, and 2.99 mmol dm(-3) for NaDC at the concentration of 60 mmol dm(-3) and at 308.2 K. The same solubilization experiments were made at 308.2 K using polycyclic aromatic compounds (benzene, naphthalene, anthracene, pyrene) as a reference. Their solubility increase for the bile salts was in the same order as above. Thermodynamic analysis was made for the solubilization, where a micelle was regarded as a chemical species. The average number of solubilizate per micelle was less than unity throughout the experiments. From the Gibbs energy change for solubilization at the different mean aggregation numbers, the function of bile salt micelles was discussed from the viewpoint of molecular structure of solubilizates. The DeltaG(0) value for cholesterol was most negative among the solubilizates studied, which reflected that solubilization of cholesterol into bile salt micelles brought about largest thermodynamic stabilization.

Anthracenes↗

PubMed2003

Purification and some properties of cholesterol oxidase stable in detergents from gamma-proteobacterium Y-134.

Cholesterol oxidase (CHO) with high stability in detergents was found from an isolated strain, Y-134, belonging to the gamma-subclass of Proteobacteria. CHO production reached its maximum by incubation at 30 degrees C for 12 d. It was purified from cell-free extract prepared by mixing the cells with 0.4% Triton X-100. The absorption spectrum of the purified enzyme exhibited maxima at 274 and 410 nm, and a shoulder at 330 nm. The molecular mass was 115 kDa with two identical subunits of 58 kDa. The enzyme oxidized cholest-5-en-3beta-ol (cholesterol) and 5alpha-cholestan-3beta-ol (dihydrocholesterol) at a high reaction rate, and the K(m) value for cholesterol was 65 microM. The stability of the enzyme was higher than other CHOs in nonionic detergents with high values of hydrophilelipophile balance (HLB) such as Triton X-450 and sodium cholate. NH2-terminal sequence analysis showed a high similarity to CHO from Burkholderia cepacia, but not to CHOs from Streptomyces or Brevibacterium.

Journal Article↗

PubMed1995

Chemical selectivity in micellar electrokinetic chromatography: characterization of solute-micelle interactions for classification of surfactants.

The influence of surfactant type on migration behavior and chemical selectivity in micellar electrokinetic chromatography (MEKC) is investigated through linear solvation energy relationships (LSER) and functional group selectivities. In LSER modeling, solutes' capacity factors are correlated with their structural descriptors such as size, dipolarity, and hydrogen-bonding abilities. Using the LSER methodology, useful information about the nature of solute interactions with different types of surfactant aggregates can be obtained since capacity factor in MEKC is directly related to solute distribution between the bulk aqueous solvent and micelles. High correlations were observed for different LSER models of migration behavior in MEKC for a group of 60 uncharged aromatic compounds of non-hydrogen bonding (NHB), hydrogen-bonding acceptor (HBA) bases, and hydrogen-bonding donor (HBD) acids. In two anionic, hydrocarbon micellar systems of sodium dodecyl sulfate (SDS) and sodium cholate (SC), retention is primarily influenced by the size of molecules and their hydrogen bond accepting basicity. Their dipolarity/polarizability and hydrogen bond donating acidity play minor roles. Capacity factors of solutes in SDS and SC systems increase with their size and decrease for stronger hydrogen bond acceptor bases. These results are similar to those observed for other systems where hydrophobic interactions play a major role, e.g., solute distribution in the 1-octanol-water solvent system or retention in reversed phase LC. In MEKC with an anionic fluorocarbon surfactant, lithium perfluorooctanesulfonate (LiPFOS), however, size and solute HBD acidity are the two predominant factors. The LSER results indicate that compounds find the SDS micellar environments slightly less cohesive (i.e., more apolar) than the SC micelles, while the LiPFOS micelles are the most cohesive among the three surfactant aggregates and 1-octanol provides the least cohesive environment. The fluorocarbon micelles of LiPFOS, on the other hand, are the strongest hydrogen bond donor acids, followed by SDS, SC, and 1-octanol, respectively. The SC micelles have the most hydrogen bond acceptor basic characteristics, followed by 1-octanol, SDS, and LiPFOS micelles. It can be concluded that selectivity differences between these surfactant types in MEKC is primarily due to hydrogen-bonding interactions rather than the dipolar interactions. Comparing the perfluorinated and the hydrocarbon surfactants, even solute size can play a role in selective migration patterns. In addition, information from polar and hydrophobic group selectivities confirm the LSER conclusions about the underlying interactions that control migration behavior and chemical selectivity in MEKC.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatography, Liquid↗

PubMed2003

Syntheses of immobilized G protein-coupled receptor chromatographic stationary phases: characterization of immobilized mu and kappa opioid receptors.

Opioid receptors are members of the superfamily of G protein-coupled receptors (GPCRs). Liquid chromatographic stationary phases containing either the human mu or kappa opioid receptor subtypes have been developed to study the binding between the opioid receptors and their ligands. The receptors were obtained from Chinese hamster ovary cells stably transfected with human mu or kappa receptor subtypes. The receptors were isolated through partial solubilization with sodium cholate detergent, and the partially purified receptor complex was immobilized in the phospholipid analogue monolayer of an immobilized artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in the on-line chromatographic determination of drug/ligand binding affinities to the immobilized opioid receptors. Preliminary on-line binding studies employing frontal chromatographic techniques were conducted with the known mu antagonist (naloxone) and a kappa agonist (U69593). The calculated dissociation constants (Kd) were 110 nM for naloxone and 84 nM for U69593. The results indicate that the immobilized receptors retained their ability to specifically bind ligands and were active for 1200 column volumes applied over two weeks. Zonal chromatographic experiments were also conducted, and competitive displacements of the marker ligands were observed. The results suggest that the immobilized opioid receptor stationary phases can be used to qualitatively assess the binding affinities of compounds to the immobilized receptors and represent the first example of the use of immobilized GPCRs in a chromatographic system.

Animals↗

PubMed2000

Dynamic micellar electrokinetic chromatography. Determination of the enantiomerization barriers of oxazepam, temazepam, and lorazepam.

The temperature-dependent enantiomerization barriers of oxazepam, temazepam, and lorazepam have been determined between 0 and 30 degrees C by dynamic micellar electrokinetic chromatography (DMEKC) in an aqueous 20 mM borate/phosphate buffer system at pH 8 with 60 mM sodium cholate as chiral surfactant. Interconversion profiles featuring plateau formation and peak broadening were observed and simulated by the new program ChromWin based on the theoretical plate as well as on the stochastic model using the experimental data plateau height, hplateau, peak width at half-height, wh, total retention times, tR, and electroosmotic breakthrough time, t0. Peak form analysis yielded rate constants k and kinetic activation parameters, deltaG double dagger, deltaH double dagger, and deltaS double dagger, of the enantiomerization of oxazepam, temazepam, and lorazepam. At 25 degrees C, the enantiomerization barrier, deltaG double dagger, was determined to be approximately 90 kJ mol-1 and the half-lives, tau, were determined to be approximately 21 min. The new approach allows the fast and precise determination of enantiomerization barriers in a biogenic environment and it mimics physiological conditions, as no organic modifiers or abiotic chiral stationary phases (CSP) are employed.

Algorithms↗

PubMed1992

Identification in bovine liver plasma membranes of a Gq-activatable phosphoinositide phospholipase C.

Phosphoinositide phospholipase C (PLC) activity extracted from bovine liver plasma membranes with sodium cholate was stimulated by GTP gamma S-activated G alpha q/G alpha 11, whereas the enzyme from liver cytosol was not. The membrane-associated PLC was subjected to chromatography on heparin-Sepharose, Q Sepharose, and S300HR, enabling the isolation of the G-protein stimulated activity and its resolution from PLC-gamma and PLC-delta. Following gel filtration, two proteins of 150 and 140 kDa were found to correspond to the activatable enzyme. These proteins were identified immunologically as members of the PLC-beta family and were completely resolved by chromatography on TSK Phenyl 5PW. The 150-kDa enzyme was markedly responsive to GTP gamma S-activated alpha-subunits of G alpha q/G alpha 11 or to purified Gq/G11 in the presence of GTP gamma S. The response of this PLC was of much greater magnitude than that of the 140-kDa enzyme. The partially purified 150-kDa enzyme showed specificity for PtdIns(4,5)P2 and PtdIns4P as compared to PtdIns and had an absolute dependence upon Ca2+. These characteristics were similar to those of the brain PLC-beta 1. The immunological and biochemical properties of the 150-kDa membrane-associated enzyme are consistent with its being the PLC-beta isozyme that is involved in receptor-G-protein-mediated generation of inositol 1,4,5-triphosphate in liver.

Animals↗

PubMed1994

Specific phospholipid requirements for the solubilization and reconstitution of D-1 dopamine receptors from striatal membranes.

We have reported the solubilization and reconstitution of functional D-1 dopamine receptors from rat striatal tissue, using sodium cholate as detergent [Sidhu, A. (1988) Biochemistry 27, 8768-8776]. Critical to our method of extraction was the absolute requirement for the persistent presence of a crude extract of phospholipids (PLs) from bovine brain, during both solubilization of membranes and reconstitution of the soluble extract into PL vesicles. In the absence of PLs, fewer than 10% of the receptors were recovered, while in the presence of PLs, 40% of the receptors were reconstituted into vesicles. To probe the composition of PLs required by D-1 dopamine receptors during these extraction procedures, specific PLs of a defined composition were used during either solubilization or reconstitution alone or during both solubilization and reconstitution. Phosphatidylcholine (PC), when used during the solubilization procedure alone or during both solubilization and reconstitution, resulted in recovery of 41-48% of the D-1 dopamine receptors but was 3.7-fold less effective when present during reconstitution alone (11%). Phosphatidylethanolamine (PE), when used during reconstitution alone, resulted in recovery of nearly 25% of the D-1 dopamine receptors. When PE was present during either solubilization or both solubilization and reconstitution, 6-11% of the receptors were recovered. If PE was used with PC in ratios of 1:1 or 2:1, respectively, 28-38% of the receptors were recovered. When PL vesicles of PE:PC were present in ratios of 1:2 during both solubilization and reconstitution, the maximum theoretical (74-87%) recovery of total receptor binding sites was achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Lipocortin 2 (annexin 2) is a major substrate for constitutive tyrosine kinase activity in chondrocytes.

Treatment of cultured bovine articular chondrocytes with 100 microM orthovanadate, in the absence of serum, results in the production of a single major tyrosine phosphorylated protein with an apparent molecular mass of 36 kDa (p36). Chondrocytes were found to contain proteins reactive with anti-lipocortin 1, 2, and 5 antibodies. p36 comigrated on SDS-polyacrylamide gels with lipocortin 2, but not with other members of the lipocortin family. The distribution of p36 between the particulate and soluble cell fractions was also similar to that of lipocortin 2. p36 that was purified on an anti-phosphotyrosine immunoaffinity column cross-reacted with anti-lipocortin 2 antibodies. Similarly, lipocortin 2 purified on an anti-lipocortin 2 immunoaffinity column reacted with anti-phosphotyrosine antibodies. Furthermore, cyanogen bromide cleavage fragments of purified lipocortin 2 and p36 were similar. These data demonstrate that the major constitutively tyrosine phosphorylated protein, in chondrocytes, is lipocortin 2. Tyrosine phosphorylated p36 required SDS buffers for extraction due to a loss of the tyrosine phosphate group under other solubilization conditions using Triton X-100 or sodium cholate. This study provides a system for the study of the effects of tyrosine phosphorylation on lipocortin 2 function. What role lipocortin 2 plays in chondrocyte biology remains to be determined.

Animals↗

PubMed1991

Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization.

A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B coupled with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-alpha-phosphatidylcholine with [1 beta-3H,4-14C]-androstenedione as substrate. The specific activity of purified aromatase was 65.0 (50.6-74.3) nmol.min-1.(mg of protein)-1 or a turnover rate of 5.0 (4.3-5.9) min-1. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The Km of immunoaffinity-purified aromatase was 12, 210, 41, and 2830 nM for androstenedione, 16 alpha-hydroxyandrostenedione, testosterone, and 16 alpha-hydroxytestosterone, respectively. The very high Km value for 16 alpha-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80 degrees C.

Animals↗

PubMed1982

Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes.

By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.

Acetylcholine↗

PubMed1982

Rhodopsin-phospholipid reconstitution by dialysis removal of octyl glucoside.

Recombinant membranes were prepared from phospholipid-free rhodopsin and egg phosphatidylcholine (PC) under a wide variety of conditions employing an octyl beta-D-glucoside (OG) dialysis procedure. Two bands were consistently observed after sucrose density centrifugation of these recombinants. The major band, which was protein rich, had a molar phospholipid:protein ratio that was in the range of 30:1 to 50:1, even when the molar phospholipid:protein ratio of the solubilized solution prior to OG removal was as high as 300:1. Similar results were obtained when dioleoyl-PC, 1-palmitoyl-2-oleoyl-PC, disk lipids, or diphytanoyl-PC was used instead of egg PC. These results can be explained in terms of a lower stability of the OG-phospholipid micelles relative to the OG-phospholipid-rhodopsin micelles. Of the phospholipids that were used in the OG dialysis procedure, only saturated dimyristoyl-PC produced a protein-rich recombinant band with a phospholipid:protein ratio close to that of the initial solubilized solution. In contrast to the results obtained by using OG, when solubilized disks supplemented with egg PC were reconstituted from sodium cholate or dodecyltrimethyl-ammonium bromide, the resulting recombinant membranes had initial and final phospholipid:protein ratios which were similar.

Animals↗

PubMed1983

Solubilization of the catecholamine carrier of chromaffin granule membranes in a form that binds substrates and inhibitors of uptake.

The component of bovine chromaffin granule membrane responsible for the binding of [2-3H]dihydrotetrabenazine has been solubilized by treatment with detergents. The binding activity of the soluble material was measured either by equilibrium dialysis or by precipitation of the protein by poly(ethylene glycol) and filtration of the precipitate. The best yields and activities were obtained with sodium cholate, which solubilized more than 70% of the binding sites; deoxycholate had deleterious effects on the soluble activity, and among nonionic detergents, octyl beta-glucoside was the most efficient. With cholate, [3H]dihydrotetrabenazine binding occurred on soluble sites as judged by centrifugation of the bound material and by filtration on Sepharose 6B columns. Binding could be measured in the presence of cholate, but high detergent concentrations had a reversible inhibitory effect. The [3H]dihydrotetrabenazine binding characteristics of the soluble material were similar to those of the granule membrane, with one class of binding sites (KD = 23 nM, Bmax = 90 pmol/mg of protein). The association and dissociation rate constants were 0.072 X 10(6) M-1 s-1 and 1.1 X 10(-3) s-1, respectively. The pharmacological properties of the binding sites were also similar to those of the membranes. Our results thus support the view that the catecholamine carrier has been solubilized in an active conformation that binds substrates and inhibitors of uptake.

Adrenal Medulla↗