Optical resolution of racemic 2-hydroxy octanoic acid by lipase-catalyzed hydrolysis in a biphasic membrane reactor

Racemic 2-hydroxy octanoic acid methyl ester was optically resolved by lipase-catalyzed hydrolysis in a biphasic membrane reactor using hydrophilic/hydrophobic capillary membranes. In a buffer/hexane biphasic membrane reactor using hydrophilic ultrafiltration membranes, (S)-2-hydroxy octanoic acid was recovered from the aqueous phase at 59–67% yield and 0.9–0.92 enantiomeric excess (ee), and the ester of (R)-isomer was recovered from the organic phase at 73–75% yield and 0.92–0.99 ee.     

Transport of p-aminohippuric acid,uric acid and glucose in highly purified rabbit renal brush border membranes

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na⁺ + K⁺)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (E_A = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

Arrhenius plot characteristics of adipocyte-plasma-membrane adenylate cyclase activity in lean and genetically obese (ob/ob) mice

Arrhenius plots of fluoride- and guanine-nucleotide-stimulated adenylate cyclase activity were linear in adipocyte plasma membranes from lean and obese (ob/ob) mice . Arrhenius plots of isoprenaline-stimulated adenylate cyclase activity in hepatic plasma membranes biphasic in both groups. The results were biphasic in membranes from Jean mice but linear in membranes from obese mice. In contrast, Arrhenius plots of glucagon-stimulated adenylate cyclase activity in hepatic plasma membranes were biphasic in both groups. The results suggest that the coupling between the -receptor and the regulatory unit of adenylate cyclase, which has been observed to be defective in adipocyte plasma membranes from obese mice, is influenced by a different lipid environment in membranes from obese animals.     

Immobilisation of pig liver esterase in hollow fibre membranes

Different methods were evaluated to immobilise Pig Liver Esterase (PLE) in hollow fibre membranes. Four covalent bonding techniques (using epoxy, imidazol, amino and carboxylic acid terminal groups) were tested to link the enzyme to microporous nylon membranes. Physical immobilisation was also studied, by entrapment of the enzyme inside the microporous structure of a polysulfone asymmetric ultrafiltration membrane. The entrapment method lead to a higher retention of enzymatic activity for a longer period of time. This technique was selected to be used in a biphasic membrane bioreactor where the microporous hydrophilic membrane, containing the enzyme, is used to separate an aqueous from an organic phase, in which the substrate is dissolved. Different enzyme loading procedures were studied in the biphasic reactor and the resulting axial and radial enzyme distribution in the hollow fibre module were related to the global enzymatic activity.     

Biphasic effect of sodium fluoride and guanyl nucleotides on binding to prostaglandin E2 receptors in rat epididymal adipocyte membranes

Both NaCl and NaF promoted PGE₂ binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the ‘salt effect’ of sodium on PGE₂ binding, the effects of Mg²⁺ and guanyl nucleotides on PGE₂ binding in the presence of NaCl or NaF were compared. Mg²⁺ decreased PGE₂ binding; high NaF concentration abolished this inhibition, while increased NaCl concentratipns did not affect the Mg²⁺ inhibition. In the presence of Mg²⁺ the effects of NaCl and NaF were additive. The enhancement of PGE₂ binding by fluoride, unlike sodium, was dependent on the presence of Mg²⁺. Induction of the membranes with GDPβS, Gpp(NH)p, GTP or GTPγS increased PGE, binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE₂ binding at low NaF concentrations and inhibition of PGE₂ binding at higjh NaF concentrations. No changes in the stimulatory action of NaCl on PGE₂ binding were observed in the simulatenous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE₂ binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE₂ binding. The implications of these findings are discussed.     

The role of fatty acids in anti-inflammatory effects of low-intensity extremely high-frequency electromagnetic radiation

The effects of low‐intensity extremely high‐frequency electromagnetic radiation (EHF EMR; 42.2 GHz, 0.1 mW/cm², exposure duration 20 min) on the fatty acid (FA) composition of thymic cells and blood plasma in normal mice and in mice with peritoneal inflammation were studied. It was found that the exposure of normal mice to EHF EMR increased the content of polyunsaturated FAs (PUFAs) (eicosapentaenoic and docosapentaenoic) in thymic cells. Using a model of zymosan‐induced peritoneal inflammation, it was shown that the exposure of mice to EHF EMR significantly increased the content of PUFAs (dihomo‐γ‐linolenic, arachidonic, eicosapentaenoic, docosapentaenoic, and docosahexaenoic) and reduced the content of monounsaturated FAs (MUFAs) (palmitoleic and oleic) in thymic cells. Changes in the FA composition in the blood plasma were less pronounced and manifested themselves as an increase in the level of saturated FAs during the inflammation. The data obtained support the notion that MUFAs are replaced by PUFAs that can enter into the thymic cells from the external media. Taking into account the fact that the metabolites of PUFAs are lipid messengers actively involved in inflammatory and immune reactions, we assume that the increase in the content of n‐3 and n‐6 PUFAs in phospholipids of cellular membranes facilitates the realization of anti‐inflammatory effects of EHF EMR. Bioelectromagnetics 32:388–395, 2011. © 2011 Wiley‐Liss, Inc.     

Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out. During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor. After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively. A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L). After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield). Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time. The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity.     

Redox reactions of tonoplast and plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis

Redox reactions were studied in more than 90% pure tonoplast and plasma membranes isolated by free-flow electrophoresis from soybean (Glycine max) hypocotyls. Both types of membrane contained a b-type cytochrome (_max = 561 nm) and a noncovalently bound flavin, two possible components of a transmembrane electron-transport chain. Isolated tonoplast and plasma membranes reduced ferricyanide, indophenol and various iron complexes with NADH or NADPH as electron donors. The redox activity was inhibited in tonoplast membranes by about 60% by 10 μM p-chloromercuribenzene sulfonate, 8% by 500 μM lanthanum nitrate and 10% by 100 μM nitrophenyl acetate. In contrast, the redox activity of isolated plasma membranes was inhibited by about 60% by 500 μM lanthanum nitrate or 100 μM nitrophenyl acetate, but only 25% by 10 μM p-chloromercuribenzene sulfonate. The results show that both tonoplast and plasma membranes of soybean contain active electron-transport systems, but that the two systems respond differently to inhibitors.     

Effect of lysophosphatidylcholine on the NADH-ferricyanide reductase activity associated with the plasma membranes of corn roots

Plasma membranes from corn roots (Zea mays L.) were isolated by aqueous two-phase partitioning. A fraction enriched in a vanadate-sensitive ATPase showed characteristics of a plasma membrane ATPase. The sidedness of these vesicles was 89% right-side-out, as evaluated by the ATPase latency. A NADH-ferricyanide reductase was associated with these plasma membrane vesicles. The rate of ferricyanide reduction was 1.3 μmol · min⁻¹·mg⁻¹ protein and was strongly enhanced by the addition of lysophosphatidylcholine (LPC). The effect of this detergent on membrane solubilization and reductase activity was particularly studied. This type of detergent treatment revealed two pH optima (7.0 and 5.0) for the reductase activity, which exhibited biphasic kinetics in the absence or presence of the detergent. These data suggest that two or more reductases could be involved. In addition, membrane vesicle solubilization and determination of ATPase and reductase latency were simultanously studied. From these experiments, it is postulated that the reductase, which exhibits an optimum pH at 7.0 and is slightly stimulated by LPC, could be located on the external side of the plasmalemma. In contrast, the reductase at pH 5.0 strongly stimulated by the detergent treatment, is probably located on the internal side of the membrane, such as the catalytic site of ATPase. Finally, a possible direct action of LPC on the enzymes, is discussed.     

Effect of dicyclohexylcarbodiimide on the proton conductance of thylakoid membranes in intact chloroplast

The effects of dicyclohexylcarbodiimide, a potent inhibitor of chloroplast ATPase, on the light-induced electric potential changes in intact chloroplasts of Peperomia metallica and of a hornwort Anthoceros sp. were investigated by means of glass microcapillary electrodes. The characteristics of potential changes induced by flashes or continuous light in chloroplasts of both species are similar except for the phase of potential rise in continuous light, which is clearly biphasic in Anthoceros chloroplasts. Dicyclohexylcarbodiimide at concentration 5 · 10⁻⁵ M completely abolishes the transient potential undershoot in the light-off reaction but has little effect on the peak value of the photoelectric response. The membrane conductance in the light and in the dark was tested by measuring the decay kinetics of flash-generated potential in dark-adapted and preilluminated chloroplasts. In the absence of dicyclohexylcarbodiimide, preillumination causes a significant acceleration of the potential decay. The light-induced changes in the decay kinetics of flash-induced responses were abolished in the presence of dicyclohexylcarbodiimide, whereas the rate of potential decay in dark-adapted chloroplasts was not altered by dicyclohexylcarbodiimide. The results are consistent with the notion that dicyclohexylcarbodiimide diminishes H⁺ conductance of energized thylakoid membranes by interacting with the H⁺ channel of ATPase. The occurrence of a lag (approx. 300 ms) on the plot of potential undershoot (diffusion potential) versus illumination time might suggest the increase in H⁺ permeability coefficient of thylakoid membrane during illumination.     

Two cholesterol pools in Acholeplasma laidlawii membranes

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.     

Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the in vitro conversion resembles the in vivo interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90°C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.     

Hydrogen peroxide induced by modulated electromagnetic radiation protects the cells from DNA damage

It is believed that non-ionizing electromagnetic radiation (EMR) and low-level hydrogen peroxide (H₂O₂) may change nonspecific resistance and modify DNA damage caused by ionizing radiation. To check this assumption, the combined effects of extremely high-frequency EMR (EHF EMR) and X-rays on induction of DNA damage in mouse whole blood leukocytes were studied. The cells were exposed to X-rays with or without preliminary treatment with EHF EMR or low-level H₂O₂. With the use of enhanced chemiluminescence, it was shown for the first time that pulse-modulated EHF EMR (42.2 GHz, incident power density of 0.1 mW/cm², exposure duration of 20 min, modulation frequency of 1 Hz) induced H₂O₂ at a concentration of 4.6 ± 0.3 nM L^?1 in physiological saline. With the use of an alkaline comet assay, it was found that the exposure of cells to the pulse-modulated EHF EMR, 25 min prior to treatment with X-rays at a dose of 4 Gy reduced the level of ionizing radiation-induced DNA damage. Continuous EHF EMR was inefficient. In turn, it was shown that low-level H₂O₂ (30–500 nM L^?1) protected the cells against X-irradiation. Thus, the mechanisms of radiation protective effect of EHF EMR are connected with the induction of the adaptive response by nanomolar concentrations of reactive oxygen species formed by pulse-modulated EHF EMR.     

Membrane fluidity of platelets and erythrocytes in patients with Alzheimer's disease and the effect of small amounts of aluminium on platelet and erythrocyte membranes

The membrane fluidity of platelet and erythrocyte membranes in 10 Alzheimer's disease patients and 9 age-matched controls was studied. The platelet membranes of patients with Alzheimer's disease were found to be significantly more fluid than those of controls (p<0.02). However, erythrocyte membranes of Alzheimer patients were less fluid (more viscous) than those of controls (p<0.05). On further investigation of platelet and erythrocyte membranes obtained from healthy volunteers, the fluidity was found to change with increasing aluminium concentrations. When aluminium ammonium sulphate (0.01–10 M) was added to membrane suspensions, the fluidity of platelet membranes was increased, whereas the fluidity of erythrocyte membranes was decreased (i.e. the microviscosity was increased).     

Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine

Summary The osmotic water permeabilityP _f of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations.P _f values for BBM and BLM preparations of small intestine were equal and amount to 60 m/sec. For renal preparations,P _f values amount to 600 m/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) m/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3±0.6 and in renal membranes, 1.0±0.3 kCal/mole, between 25 and 35°C. The mercurial sulfhydryl reagentpCMBS inhibited completely and reversibly the highP _f value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the highP _f values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to 1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.     

Isolation of luminal and antiluminal membranes from dog kidney cortex

Luminal (brush border) and antiluminal (basal-lateral) membranes were isolated from canine renal cortex. The enzyme marker for luminal membrane, alkaline phosphatase was enhanced 19-fold and the antiluminal enzyme marker, (Na⁺ + K⁺)-ATPase, was enhanced 22-fold in their respective membrane preparation, while the amount of cross contamination was minimal. Contamination of these preparations by enzyme markers for lysosomes, endoplasmic reticulum and mitochondria was also low. Routinely, more than 50 mg membrane protein was isolated for each membrane. Electron micrographs showed that the membranes were uniform in size, appearance, and vesicular in nature. An examination of the orientation of these membranes showed that 76.5% of the antiluminal membranes and 86% of the luminal membranes were right-side out.     

Pharmacological analysis of the antiinflammatory effects of low-intensity extremely-high-frequency electromagnetic radiation

The antiinflammatory effect of low-intensity extremely-high-frequency electromagnetic radiation (EHF EMR, 42.0 GHz, 0.1 mW/cm²) was studied in comparison to the effects of the antiinflammatory drug sodium diclofenac and the antihistamine clemastine in acute inflammatory reaction in mice of NMRI outbred stock. The local inflammatory reaction was induced by intraplantar injection of zymosan to the left hind paw. Intraperitoneal injections of 2, 3, 5, 10, and 20 mg/kg of sodium diclofenac or 0.02, 0.1, 0.2, 0.4, and 0.6 mg/kg of clemastine were made 30 min after the initiation of inflammation. An hour after the initiation of inflammation, animals were whole-body exposed to EHF EMR for 20 min. The inflammatory reaction was assessed 3–8 h after initiation by measuring the footpad edema and hyperthermia of the inflamed paw. Sodium diclofenac (5–20 mg/kg) reduced the exudative edema by ～26% compared to the control. Hyperthermia of the inflamed paw decreased by 60% with an increase in the diclofenac dose to 20 mg/kg. EHF EMR reduced both the footpad edema and hyperthermia by ～20%. This was comparable to the effect of a single therapeutic dose of diclofenac (3–5 mg/kg). The combination of diclofenac and exposure to EHF EMR produced a partial additive effect. Clemastine (0.02–0.4 mg/kg) did not affect the exudative edema, but at a dose of 0.6 mg/kg, edema was reduced by 14–22% five to eight hours after zymosan injection. Clemastine caused a dose-dependent increase in hyperthermia of inflamed paw at doses 0.02–0.2 mg/kg and did not affect the hyperthermia at doses 0.4 and 0.6 mg/kg. A combination of clemastine and EHF EMR exposure resulted in a dose-dependent abolishment of the antiinflammatory effect of EHF EMR. Our results suggest that both arachidonic acid metabolites and histamine are involved in the achievement of the antiinflammatory effects of low-intensity EHF EMR.     

The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus

The relationship between the delta 9-desaturase activity of the psychrophilic bacterium Micrococcus cryophilus grown at different temperatures and the physical state of its membrane lipids as measured by ESR spectroscopy has been studied. Arrhenius plots of desaturase activity were biphasic with a discontinuity at a temperature which depended upon the bacterial growth temperature. Changes in the desaturase activation energy, which increased as the growth temperature was lowered, are discussed in the context of membrane lipid fluidity adaptation to changing environmental temperature. The fluidity of membranes and isolated lipids was measured using nitroxide-labeled fatty acids. The spectra of 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl in membranes indicated that there were two lipid environments within the membrane whose relative proportions were dependent both on temperature of measurement and on bacterial growth temperature. In contrast, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl spectra showed a single lipid environment and plots of log order parameter (S3) vs 1/T were biphasic with inflexion temperatures which were closely related to the bacterial growth temperature. As with membranes, plots of log S3 vs 1/T for total lipids, phosphatidylglycerol and cardiolipin, but not phosphatidylethanolamine, were biphasic and showed inflexions which correlated well with bacterial growth temperature. These results are interpreted as being consistent with a location for the desaturase within the bulk lipid of the membrane rather than in association with specific lipid types.     

The Effects of Low-Intensity Millimeter-Wavelength Radiation and Electromagnetic Shielding on Pain Sensitivity in Rats

Here, we studied changes in pain sensitivity in rats subjected to low-intensity millimeter-wavelength electromagnetic radiation (EMR MM) of 7.1 mm and 0.1 mW/cm² in the occipital-collar region with daily exposure of 30 min over 21 days. As well, this radiation was combined with moderate electromagnetic shielding (EMS) which had the following parameters. The shielding coefficients of the constant component of the magnetic field along the vertical and horizontal constituents were 4.4- and 20-fold, respectively, with an exposure of 22 h/day over 21 days. The pain sensitivity was estimated with algometric tests, that is, the hot plate, flick-tail, and algesimeter-pincher tests; these allowed observation of the pain impulse at different regulatory levels. The algological effects of both individual and combined EMR MM and EMS were demonstrated. It was shown that EMR MM has an antinociceptive property when combined with EMS, as well as a modulation effect caused by shielding during hyperalgesia. At the same time, shielding reduces the antinociceptive effect of EMR MM.

     

Dual Action of Pentobarbitone on GABA Binding: Role of Binding Site Integrity

The effects of pentobarbitone on the binding of gamma-aminobutyric acid (GABA) to crude synaptosomal rat brain membranes were studied. In extensively washed P2 membranes, pentobarbitone had a biphasic action: at concentrations ranging between 12.5 and 500 microM, pentobarbitone enhanced GABA binding in a concentration-dependent manner; at concentrations greater than 500 microM, this enhancement was progressively reversed towards control levels of GABA binding. The effect of pentobarbitone seen at higher concentrations may reflect a GABA-mimetic action, since similar concentrations enhanced diazepam binding to washed P2 membranes, an effect antagonized by bicuculline methochloride and picrotoxinin. When washed P2 membranes were incubated in 0.5% Triton X-100 (30 min at 37 degrees C), the enhancement of GABA binding by low concentrations of pentobarbitone was abolished, while at higher concentrations GABA binding was progressively inhibited, suggesting that the GABA-mimetic action is retained. When washed P2 membranes were subjected to high-frequency homogenization, the biphasic dose-response relationship for pentobarbitone was markedly shifted to the right. The choice of membrane preparation appears to be a critical factor in examining drug-receptor interactions in vitro, at least for those involving GABA and the barbiturates.