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PubMed2002

Expressing and purifying membrane transport proteins in high yield.

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.

Animals↗

PubMed1991

Purification of a [3H]dihydrotetrabenazine-binding protein from bovine adrenal medulla.

A high affinity binding site for [3H]dihydrotetrabenazine is thought to be present on the monoamine transport protein from chromaffin granules. We describe a procedure for purification of this binding activity from frozen bovine adrenal tissue, and we partially characterize the purified preparation. Binding activity solubilized with sodium cholate and soybean lecithin was fractionated on wheat germ lectin-Sepharose, phenyl-Sepharose, Mono Q, and hydroxylapatite. Denaturing electrophoresis of the purified binding activity, followed by silver staining, revealed a single broad band centered at an apparent molecular weight of 85,000. This preparation bound [3H]dihydrotetrabenazine with an apparent dissociation constant of 2.7 nM and had a site density of 10 nmol/mg. Treatment of the purified protein with neuraminidase reduced the apparent molecular weight by 9000, indicating the presence of terminal sialic acids on the oligosaccharide portion of this molecule.

Adrenal Medulla↗

PubMed2002

Micelle formation of sodium deoxycholate and sodium ursodeoxycholate (part 1).

Micellization of sodium deoxycholate (NaDC) and sodium ursodeoxycholate (NaUDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle, where sodium cholate (NaC) was used as a reference. The fluorescence probe technique of pyrene was employed to determine accurately the CMC values for the bile salts, which indicated that a certain concentration range of CMC and a stepwise aggregation for micellization were reasonable. The temperature dependences of micellization for NaDC and NaUDC were studied at 288.2, 298.2, 308.2, and 318.2 K by aqueous solubility change with solution pH. Aggregations of the bile salt anions were analyzed using the stepwise association model and found to grow in size with increasing concentration, which confirmed that the mass action model worked quite well. The average aggregation number was found to be 2.5 (NaUDC) and 10.5 (NaDC) at the concentration of 20 mM and at 308.2 K. The aggregation number determined by static light scattering also agreed well with those by the solubility method in the order of size: NaUDC<NaC<NaDC at 308.2 K. The results indicated that the location of the OH group at C-7 and its orientation were the most important factors from the viewpoint of chemical structure for the growth of micelles. The activity measurement for sodium ions was made by a sodium ion selective electrode in order to confirm the low counterion binding to micelles and the validity of the present association model of bile salts, but the model did not hold good for NaDC at higher concentrations.

Deoxycholic Acid↗

PubMed1993

Characterization of the Na+/H(+)-antiporter of bovine renal cortex and its immunoaffinity purification in a reconstitutively active form.

In order to analyze the localization and pharmacological characteristics of the Na+/H(+)-antiporter of bovine kidney, renal cortex membranes were fractionated by 35-48% continuous sucrose density gradient centrifugation. Both the Na+/H(+)-antiporting activity and the 110-kDa peptide cross-reactive with a polyclonal antibody against a peptide representing the C-terminal 22 amino acid residues of human Na+/H(+)-antiporter (NHE1) were found in the same fractions as alkaline phosphatase, a marker enzyme of the brush-border membrane. Most of the Na+/H(+)-antiporter distributed among the fractions was found to be of the low amiloride-sensitive type. Brush-border membranes were solubilized with sodium cholate and the solubilized proteins were applied to an immunoaffinity matrix (Protein A Sepharose CL-4B coupled with the above antibody). The 110 kDa protein was adsorbed to the affinity matrix and then recovered in the fraction eluted from the column at pH 11.5. After reconstitution into proteoliposome, the fraction containing the 110 kDa protein was highly active in Na+/H(+)-antiport. A 125-fold increase in the specific activity of the Na+/H+ antiport reaction was attained with the present immunoaffinity purification procedure. This represents the highest degree of purification of the native Na+/H(+)-antiporter so far attained.

Amiloride↗

PubMed1970

Resistance of Escherichia coli to penicillins. 8. Physiology of a class II ampicillin-resistant mutant.

Class II ampicillin-resistant mutants of Escherichia coli are defined as having a twofold increase in penicillinase-mediated ampicillin resistance when determined by colony formation tests on plates. In this paper, one class II mutant has been compared to its parent strain. In liquid medium, the mutant was less resistant than the parent strain both in the absence and in the presence of R1 and R-factor mediating penicillinase activity. The penicillinase activity was found to be almost completely bound to the cells in the parent strain, whereas it was excreted to a great extent in the class II mutant strain. In liquid medium, resistance was well correlated to the cell-bound penicillinase activity, whereas the excreted penicillinases were also of great importance for survival on ampicillin plates. The mutant also had a changed resistance to a great number of other antibacterial drugs. The mutant was found to be more sensitive than the parent strain to osmotic shock, especially when treated with ethylenediaminetetraacetic acid or washed with sodium ions. However, the osmotic stability was restored by the presence of 1 mm Mg(2+) ions. The class II mutant was more sensitive than the parent strain to sodium cholate, and it adsorbed the phages T4 and T3-1 at a slower rate than did the parent strain. The two strains adsorbed T6 at the same rate. The class II phenotype could be gradually reversed by increasing concentrations of divalent cations. The pleiotropic changes in the phenotype are apparently unrelated to the specific targets for the antibacterial agents tested. They are secondary consequences of a cell envelope mutation. The findings indicate that the class II mutation mediates a structural change in the lipopolysaccharide of the cell envelope.

Ampicillin↗

PubMed1981

Covalent binding of polychlorinated biphenyls to proteins by reconstituted monooxygenase system containing cytochrome P-450.

Incubation in the presence of NADPH and molecular oxygen of 14C-labeled polychlorinated biphenyls (PCBs) and two tetrachlorobiphenyl (TCB) isomers with a reconstituted system containing NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital(PB)-pretreated rabbits, led to covalent binding of radioactive metabolites of PCBs and TCBs to the protein components of the system. A rabbit liver cytosol fraction added to the system provided more binding sites for the activated metabolites and thus increased the extent of binding markedly. The binding reaction depended absolutely on the reductase, cytochrome P-450 and NADPH, and required dilauroyl phosphatidylcholine and sodium cholate for maximal activity. A further stimulation of the binding was attained by including cytochrome b5 in the reconstituted system. Four forms of cytochrome P-450, purified from liver microsomes of PB- and 3-methylcholanthrene(MC)-treated rabbits and rats, were used to reconstitute the PCB- and TCB-metabolizing systems, and it was found that PB-inducible forms of the cytochrome from both animals were more active than those inducible by MC in catalyzing the PCB- and TCrome P-450, purified from liver microsomes of PB- and 3-methylcholanthrene(MC)-treated rabbits and rats, were used to reconstitute the PCB- and TCB-metabolizing systems, and it was found that PB-inducible forms of the cytochrome from both animals were more active than those inducible by MC in catalyzing the PCB- and TCrome P-450, purified from liver microsomes of PB- and 3-methylcholanthrene(MC)-treated rabbits and rats, were used to reconstitute the PCB- and TCB-metabolizing systems, and it was found that PB-inducible forms of the cytochrome from both animals were more active than those inducible by MC in catalyzing the PCB- and TCB-binding reaction. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis indicated that, in the system containing the reductase, cytochrome P-450 and cytochrome b5, PCB metabolites bound to the reductase and cytochrome P-450, but not to cytochrome b5. In the presence of the liver cytosol fraction, the binding took place to many cytosolic proteins in addition to the reductase and cytochrome P-450.

Animals↗

PubMed1984

Molecular properties of the apamin-binding component of the Ca2+-dependent K+ channel. Radiation-inactivation, affinity labelling and solubilization.

Radiation-inactivation was used to assess the functional size of the apamin-binding component of the Ca2+-dependent K+ channel. The amount of specific binding of 125I-apamin to receptors in synaptic membranes of rat cortex decayed exponentially with increasing doses of ionizing radiation and target size analysis was consistent with a relative molecular mass of 250 000 +/- 20 000 for the 125I-apamin receptor. Analysis on sodium dodecyl sulfate gels following covalent cross-linking of 125I-apamin to its receptor in a synaptosomal membrane preparation from rat cortex revealed a single labelled polypeptide chain of Mr = 33 000 +/- 2000 in the presence of protease inhibitors. Our results suggest that the Ca2+-dependent K+ channel from rat cortex is an oligomeric structure of Mr = 250 000 +/- 20 000 containing an apamin-binding subunit of Mr = 33 000 +/- 2000. The apamin-binding component of the Ca2+-dependent K+ channel from rat synaptosomes was solubilized using detergents such as sodium cholate or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate. Phospholipids did not increase the stability of the apamin-binding component during the solubilization. Binding of apamin to its solubilized receptor is reversible and saturable. The dissociation constant of the apamin-receptor complex is 40-150 pM, the rates constants of association and dissociation being 3.2 X 10(6) M-1s-1 and 1.4 X 10(-4)s-1 respectively. These binding characteristics are similar to those found for the membrane-bound apamin receptor.

Affinity Labels↗

PubMed2007

Semihydrodynamic injection for high salt stacking and sweeping on microchip electrophoresis and its application for the analysis of estrogen and estrogen binding.

In this work, a semihydrodynamic (SHD) injection method was introduced and coupled with high salt stacking and electrokinetic chromatography for the analysis of estrogen and estrogen binding using a simple cross microchannel. The SHD method allows all samples to be hydrodynamically injected and focused into the separation channel at a relatively high flow rate and without splitting and diffusion, leading to reproducible bias-free injections of larger sample volumes (up to 50 nL) within 3 s. Moreover, the injection method is initiated without voltage switching, leading to a reduced mixing effect. Such advantages are well suited for performing stacking and sweeping on a microchip. We investigated the stacking effect under continuous and discontinuous co-ion conditions as well as under sweeping conditions. Micellar sweeping effect alone was relatively weak (7-8 times), partly due to a lower sodium cholate concentration (30 mM) used for the running buffer. By combining the sweeping effect with high salt stacking, however, up to a 200-300-fold enhancement factor could be achieved, and the high-salt and low-surfactant contents for the running buffer were favorable for binding study under nonequilibrium conditions. To the best of our knowledge, this is the first demonstration of the hydrodynamic injection used for high salt sample stacking on a microchip, also for further combining micellar electrochromatography and affinity separation for the analysis of hydrophobic ligand binding using microchip electrophoresis.

Binding Sites↗

PubMed1990

Octyl glucoside extracts GTP-binding regulatory proteins from rat brain "synaptoneurosomes" as large, polydisperse structures devoid of beta gamma complexes and sensitive to disaggregation by guanine nucleotides.

GTP-binding regulatory proteins are generally purified from cholate-extracted membranes in the form of heterotrimers (G proteins) consisting of a GTP-binding subunit (alpha protein) complexed with a tightly interacted heterodimer termed beta gamma. In this study we extracted the proteins from rat brain "synaptoneurosomes" using the neutral detergent 1-octyl beta-D-glucopyranoside (octyl glucoside). Using specific antibodies for detection by immunoblotting and sucrose gradients for analyzing hydrodynamic properties, we found that each species of alpha protein (alpha subunits of stimulatory, inhibitory, and brain GTP-binding proteins) exhibited a broad range (4 S to greater than 12 S) of polydisperse structures with peak values (5 S to 7 S) considerably greater than that of heterotrimeric G proteins. The beta subunit proteins, for example, appeared as a homogeneous peak at 4.4 S within which only a fraction of the total alpha proteins can be associated. Incubation of octyl glucose extracts at 30 degrees C rapidly sedimented the alpha proteins but not the beta proteins. Incubation at 30 degrees C with guanosine 5'[gamma-thio]triphosphate (10-100 microM) prevented rapid sedimentation. Hydrodynamic analysis revealed that all alpha proteins were converted to approximately 4 S structures by the actions of guanosine 5'-[gamma-thio]triphosphate without change in the hydrodynamic properties of the beta proteins. Extraction of the membranes with sodium cholate instead of octyl glucoside resulted in complete loss of the large, polydisperse structures of the alpha proteins; the S values were approximately 4 S, in the range for beta proteins. These findings suggest that the transducing GTP-binding proteins in synaptoneurosomes exist as polydisperse, possibly multimer, structures of various size that are stable in octyl glucoside but destroyed by cholate. The polydisperse structures are not associated with beta gamma complexes and are sensitive to the disaggregating effects of guanosine 5'-[gamma-thio]triphosphate.

Animals↗

PubMed1984

Induction of catecholamine-responsive adenylate cyclase in HeLa cells by sodium butyrate. Evidence for a more efficient stimulatory regulatory component.

HeLa cells, when exposed to 5 mM sodium butyrate, increased their responsiveness to isoproterenol and their number of beta-receptors. As untreated HeLa cells have a substantial number of receptors but respond poorly to isoproterenol, the effect of butyrate could be due to quantitative or qualitative changes in beta-receptors or other components of the adenylate cyclase system. Receptors were analyzed by membrane/membrane and membrane/cell fusion techniques. HeLa donor membranes, treated to inactivate regulatory and catalytic components of adenylate cyclase, were fused with Fc cells, which lack beta-receptors. Isoproterenol-stimulated adenylate cyclase activity in the fusates was proportional to the number of receptors present. There appeared to be only quantitative but not qualitative differences in beta-receptors from control and butyrate-treated HeLa. Prostaglandin E1 receptors from neuroblastoma cell membranes were similarly coupled to HeLa adenylate cyclase. The hybrid prostaglandin E1-stimulated activity was lower when acceptor membranes were from control HeLa than when they were from butyrate-treated HeLa cells. These results suggested that butyrate was altering the ability of the regulatory component to interact with receptors. HeLa membranes were extracted with sodium cholate and the extracts used to reconstitute effector-stimulated adenylate cyclase activity in S49 cyc- membranes, which lack a functional regulatory component. Whereas extracts from control and butyrate-treated HeLa were equally effective in restoring NaF-stimulated activity in cyc- membranes, extracts from control HeLa were less efficient in reconstituting isoproterenol- and prostaglandin E1-stimulated activities. We conclude that the poor response of control HeLa to beta-agonists is due to a limited activity of the regulatory component but not the receptor. Butyrate induces quantitative changes in the receptor and qualitative changes in the regulatory component that facilitate its ability to couple to receptors but do not alter its ability to interact with the catalytic component of adenylate cyclase.

Adenosine Diphosphate Ribose↗

PubMed1996

Further characterization of rat brain flavin-containing monooxygenase. Metabolism of imipramine to its N-oxide.

Flavin-containing monooxygenase (FMO) activity was compared in rat liver and brain microsomes by estimating the actual amount of imipramine N-oxide relative to the corresponding activity, measured using substrate-stimulated rates of NADPH oxidation. The activities measured as NADPH oxidation rates were significantly higher than those estimated from the N-oxide formed. The brain FMO activity was detectable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The antibody to rabbit pulmonary FMO selectively inhibited imipramine N-oxidation. The antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect on imipramine N-oxidation, indicating the noninvolvement of cytochrome P-450 in the above metabolic pathway. A flavin-containing monooxygenase was partially purified from the rat brain microsomes using sequential chromatography on n-octylamino-Sepharose 4B, DEAE-Sephacel and 2',5'-ADP agarose. The purified FMO was resolved by SDS-PAGE into two bands (approximately 57 and 61 KDa, respectively) both of which cross-reacted with antibody to rabbit pulmonary FMO. The purified enzyme metabolized imipramine and the model substrate methimazole to their respective N-oxide and S-oxides.

Animals↗

PubMed1990

Immobilized cytochrome P-450LM2. Dissociation and reassociation of oligomers.

Subunit interactions in the purified hexameric cytochrome P-450LM2 have been studied using covalent binding of one of the 6 promoters to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (greater than 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.

Animals↗

PubMed1980

Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced cation channel regulation.

We report the purification of acetylcholine receptors with active agonist-regulated cation channels from Torpedo californica electric organ tissue by five methods. In one method, previously used by others, contaminating proteins were removed from partially purified membranes by alkaline extraction, preserving membrane integrity throughout the procedure. In the other four methods, acetylcholine receptors were purified after solubalization with sodium cholate. The continual presence of soybean lipid in mixed micelles with cholate was required to prevent irreversible inactivation of the cation channel. Solubilized receptors were purified by affinity chromatography using either Naga naja siamensis toxin III or concanavalin A coupled to agarose. Sucrose gradient centrifugation was also used to purify solubilized receptors. The best method combined affinity chromatography on toxin-agarose and concanavalin A agarose. Receptors purified by all five methods were incorporated into soybean lipid vesicles by the cholate dialysis technique. The agonist-regulated cation channels of the receptors were equally active after reconstitution, independent of the method used for purification. All reconstituted vesicle preparations were similar in preferential orientation of acetylcholine receptor toward the external surface, dose-response to carbamylcholine, carbamylcholine-induced desensitization, and carbamycholine-induced influx of 22Na+ per mol of receptor. Carbamylcholine-induced 22Na+ influx/receptor was greater after reconstitution than in native vesicles. This was because, in native vesicles, carbamylcholine-induced 22Na+ influx was limited by equilibration of the internal volume of the vesicles with the external 22Na+ concentration, whereas in reconstituted vesicles 22Na+ influx was limited by desensitization of the receptor molecule. We demonstrate that only one of the two toxin binding sites on the receptor monomer, the one which can be affinity alkylated with 4-(N-maleimido)benzyltrimethylammonium, controls the carbamylcholine-induced opening of the cation channel.

Acetylcholine↗

PubMed1982

Biochemical characterization of the tetrodotoxin binding protein from Electrophorus electricus.

Biochemical properties of a detergent-solubilized tetrodotoxin binding component from Electrophorus electricus have been examined and compared with those found for the membrane-bound protein. The toxin binding component was solubilized with high efficiency by a variety of nonionic detergents and with lower efficiency by sodium cholate and deoxycholate. Detergent-solubilized preparations bound tetrodotoxin and saxitoxin tightly and specifically, and this binding was observed to be rapidly and irreversibly blocked by carboxylate-modifying reagents. Inactivation by carbodiimide and glycine ester or by a trimethyloxonium salt could be prevented by tetrodotoxin occupancy of the binding site. Tetrodotoxin binding activity in both solubilized preparations and in membranes was found to be highly resistant to proteases. In contrast, the activity was extremely sensitive to the action of phospholipase A2. The biochemical properties of the tetrodotoxin binding component solubilized in mixed lipid-detergent micelles are similar to those found in native membranes, with respect to the characteristics of equilibrium toxin binding and to the sensitivity of toxin binding activity to chemical modification and degradative enzymes. There were some differences with respect to the kinetics of tetrodotoxin binding. In addition, the tetrodotoxin binding component from eel is shown to behave as a glycoprotein, being selectively absorbed to resins coupled to concanavalin A, wheat germ agglutinin, Lens culinaris lectin, and ricin with the appropriate glycoside.

Animals↗

PubMed1982

The incorporation of solubilized choline-transport activity into liposomes.

The choline-transport system has been solubilized from synaptic plasma membrane by using either sodium cholate or Triton X-100, and re-incorporated into unilamellar liposomes by using the technique of cholate dialysis. The criteria of choline-transport activity were saturability by excess choline, inhibition by hemicholinium-3, and trans-activation (i.e. stimulation of the uptake of [3H]choline into liposomes by preloading them with non-radioactive choline). Liposomes prepared from detergent extracts of synaptic plasma membrane and added lipid showed uptake of [3H]choline fulfilling these three criteria. Data on choline-transport activity of liposomes at various choline concentrations could be interpreted as implying that the transport system has two apparent Km values (2-5 microM and 50-100 microM), or alternatively that the system is composed of two or more negatively co-operating subunits (or units). It was shown by t.l.c. that the transported radioactivity was choline and that it was not significantly acetylated. Replacing Na+ by K+ on the outside of these liposomes partially inhibited uptake, and the formation of a potential gradient (inside negative) with valinomycin increased the total but not the saturable components of uptake when liposomes were prepared in a K+ medium, and transferred to an Na+ medium.

Adenosine Triphosphate↗

PubMed1998

Migration behavior and separation of tetracycline antibiotics by micellar electrokinetic chromatography.

The migration behavior and separation of six tetracyclines (TCs) were investigated by micellar electrokinetic chromatography (MEKC) in the pH range 5.0-9.0 using ammonium acetate buffer with the addition of sodium dodecyl sulfate (SDS). Mixed SDS-Brij 35, sodium cholate (SC) and tetradecyltrimethylammonium bromide (TTAB) were also used as surfactants. The influences of surfactant concentration and buffer pH on the separation of TCs were examined and the separations of TCs were optimized. Complete separation of six TCs was achieved within 8 min with 15 mM ammonium acetate buffer containing 20 mM SDS, with or without the addition of Brij 35 (0.135%, w/v), at pH 6.5 using a fused-silica capillary (42 cm x 75 microns I.D.) at 15 kV. In general, good linear correlations of the logarithm of migration factor (log k') versus the logarithm of octanol-water partition coefficient (log P(ow)) in these micellar systems, except for the TTAB-MEKC system, were obtained. The results indicate that the migration of TCs in MEKC is mainly based on hydrophobic interactions. However, hydrogen bonding interactions also play a significant role in influencing the chemical selectivity of TCs. In addition, the micelle-water partition coefficients (Pmw) of TCs, which are pH-dependent in the SDS-MEKC micellar system, are reported.

Anti-Bacterial Agents↗

PubMed1992

Molecular properties of 5-hydroxytryptamine3 receptor-type binding sites purified from NG108-15 cells.

5-Hydroxytryptamine3 (5-HT3) receptor-type binding sites were solubilised from NG108-15 mouse neuroblastoma x rat glioma hybrid cells using five different detergents [n-octyl-beta-D-glucoside, Triton X-100, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), sodium cholate, and deoxycholate] and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5-HT3 receptor-type binding sites (5-HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high-affinity antagonist GR119566X to an Affi-Gel 15 resin. The affinity of purified 5-HT3R for [3H]-GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (11S, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n-dodecyl-beta-D-maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8-9 nm diameter with a 2-nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.

Animals↗

PubMed2006

Fluorescence imaging of sample zone narrowing and dispersion in a glass microchip: the effects of organic solvent (acetonitrile)-salt mixtures in the sample matrix and surfactant micelles in the running buffer.

A mismatch in the EOF velocities between the sample zone and running buffer region is known to generate pressure-driven, parabolic flow profile of the sample plug in electrokinetic separation systems. In the present study, video fluorescence microscopy was employed to capture real-time dynamics of the sample plug (containing fluorescein as the probe molecule) in a discontinuous conductivity system within a glass microchip, in which the sample matrix consisted of a mixture of ACN and salt (NaCl), and the running buffer contained sodium cholate (SC) micelles as the pseudo-stationary phase (i.e., performing "ACN stacking" in the mode of MEKC). Upon application of the separation voltage, the video images revealed that zone narrowing and broadening of the probe molecules occurred as the sample plug headed toward the cathode during the initial time period, probably resulting in part from the stacking/sweeping, and destacking of the SC micelles at the boundaries between the sample zone and running buffer. Interestingly, a second sample zone narrowing event can be observed as the sample plug moved further toward the cathode, which could be attributed to the sweeping of the slower moving probe molecules by the faster moving SC micelles that originated from the anode. This phenomenon was studied as a function of pH, sample plug length, as well as the concentration of organic solvent and salt in the sample matrix. The data suggested that the presence of large amounts of an organic solvent (such as ACN or methanol) and salts in the sample matrix not only induces sample dispersion due to the formation of a pressure-driven (hydrodynamic) flow, but may also lead to the formation of a double sample zone narrowing phenomenon by altering the local EOF dynamics within the separation system.

Acetonitriles↗