Rat Brain Synaptosomal Membranes Research Articles

Identification of the catalytic subunit of brain adenylate cyclase: a calmodulin binding protein of 135 kDa.

The partial purification of the eukaryote adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] catalytic subunit has been achieved by a procedure based on the calmodulin (CaM) sensitivity of the enzyme. Small amounts of rat brain synaptosomal membranes depleted of CaM were solubilized with Lubrol and subjected to a three-step chromatographic procedure involving gel filtration, a CaM-Sepharose affinity step, and fast protein liquid chromatography. About 20% of the adenylate cyclase activity contained in the membranes was recovered in the final enriched fraction with a specific activity of 200 nmol X mg-1 X min-1. The alpha subunits of the adenylate cyclase stimulatory proteins NS were absent from this final fraction. The addition of CaM, of forskolin, or of preactivated NS-containing fractions to this preparation greatly increased the enzyme activity. A CaM-binding polypeptide of 135,000 Da copurified with the adenylate cyclase activity in each of the three steps. Polyacrylamide gel electrophoresis of the final fraction showed that this polypeptide represented 35% of the total protein. We propose that this polypeptide is likely to be the adenylate cyclase catalytic subunit. This enzyme would represent close to 0.5% of the synaptosomal membrane proteins. Its low turnover number would be due to the absence of the alpha subunits of the NS regulatory proteins and would correspond to the enzymic basal level.

Is Ca2+-calmodulin-dependent protein phosphorylation in rat brain modulated by carboxylmethylation?

Calmodulin stimulation of protein kinase activity in calmodulin-depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein-O-carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+-calmodulin-dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate-gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/micrograms protein or 0.15-1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8-azido-ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+-calmodulin protein kinase may modulate the activity of this enzyme in rat brain.

The binding of somatostatin to rat synaptosomal membranes: Effects of detergent solubilisation and of bacitracin

Specific high and low affinity binding sites for somatostatin have been demonstrated in rat brain synaptosomal membranes using 125I [Tyr″] somatostatin tracer. When incubations were carried out in the presence of low concentrations of bacitracin (0.01% w/v), a 66% increase in association constant of the high affinity binding site to K a = 1.71 × 10 10 M −1 and a 70% increase in the K a of the low affinity site to 0.034 × 10 10 M −1 were observed; a similar (50%) increase in the binding capacity of the low affinity site but no increase in the binding capacity of the high affinity site was seen. These results may indicate a direct effect of bacitracin on the membrane that is distinct from its effect as an inhibitor of tracer degradation. The somatostatin-binding capacity of the membranes was solubilised by treatment with Triton X-100 and the apparent molecular weight of the receptor-ligand-detergent micelle complex was estimated by gel filtration to be approximately 250,000.

Degradation of NAD by synaptosomes and its inhibition by nicotinamide mononucleotide: implications for the role of NAD as a synaptic modulator.

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.

Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate.

The muscarinic cholinergic receptor present in synaptosomal membranes of rat brain was covalently labelled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labelled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulphate, and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulphate analysed. Provided both proteolysis and inter-chain disulphide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, as a broad band of apparent Mr 66000-76000. Two dominantly labelled polypeptides, of apparent Mr 68000 and 73000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labelled receptor readily formed intermolecular disulphide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulphide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent Mr 119000-128000) occurred. This probably explains previous reports in the literature of larger-Mr species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulphide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5% sodium dodecyl sulphate with an equivalent Stokes' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent Mr in gel electrophoresis of 43000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes' radius, eluting just behind reduced bovine serum albumin (Stokes' radius 8.5 nm; apparent Mr in electrophoresis 67000). Thus the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behaviour in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycans of synaptosomal plasma membrane glycoproteins from adult rat forebrain

Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.

Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na + + K +)-ATPase (ouabain-sensitive), Mg 2+-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPase, for example 7.9 and 10 mM tetracaine for Mg 2+-ATPase and (Na + + K +)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F 1-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.

Gamma-hydroxybutyric acid binding sites in rat and human brain synaptosomal membranes

The binding of γ-hydroxy[2,3- 3H]butyric acid (GHB) was characterized in rat and human brain synaptosomal membranes. Binding was shown to be saturable, pH dependent, and linear with protein concentration. There was a distinct regional distribution of binding sites in both rat and human brain, with the hippocampus being the richest and the cerebellum the poorest, in density of [ 3H]GHB binding sites. Competition and saturation experiments revealed two different populations of binding sites, a high-affinity site with a K D1 of 580 nM and a B max1 of 1.8 pmoles/mg protein and a low-affinity site with a K D2 of 2.3 μM and a B max2 of 11.3 pmoles/mg protein. [ 3H]GHB binding was not inhibited by γ-aminobutyric acid (GABA), GABA receptor agonists, opiate antagonists or anticonvulsant drugs. These data suggest that GHB may play a role as a neurotransmitter or neuromodulator in brain independent of GABA.

Circulating digitalis-like substance is increased in doca-salt hypertension

Blood pressure and digitalis-like substance were measured in the plasma of control, salt-treated, and DOCA-salt treated rats. Blood pressure in DOCA-salt treated rats was significantly higher than that of either control or salt-treated animals. Digitalis-like activity was measured by two methods, radioimmunoassay for digoxin, and a receptor binding assay employing a rat brain synaptosomal membrane fraction. Digoxin-like immunoreactivity in plasma was not detected in either control or salt-treated rats, but was detected in DOCA-salt treated rats. Receptor binding activity in salt-treated rats was slightly but significantly higher than that of control rats. In DOCA-salt treated rats, receptor binding activity was significantly higher than that of salt-treated rats. Partial purification of the digitalis-like substance in plasma was performed by gel filtration using Sephadex G-25. Two peaks containing digoxin-like immunoreactivity were observed. Receptor binding activity, as well as Na +-K + ATPase inhibitory activity, was detected only in the second peak, in which approximately 70% of the digoxin-like immunoreactivity was eluted. These results indicate that a circulating digitalis-like substance is increased in DOCA-salt hypertension.

Protective effect of dimercaptosuccinic acid on methylmercury and mercuric chloride inhibition of rat brain muscarinic acetylcholine receptors

The reactivation of the rat brain muscarinic acetylcholine receptor (mACh-R) binding with dimercaptosuccinic acid (DMSA) after in vitro and in vivo inhibition by mercuric chloride (HgCl 2) and methylmercuric chloride (MeHg) was investigated. Receptor binding was estimated by the potent and specific antagonist l-[ 3H]quinuclidinyl benzilate ([ 3H]QNB). Rat brain synaptosomal membranes were exposed to HgCl 2 and MeHg. At 1 × 10 −4 M. HgCl 2 caused complete inhibition of the [ 3H]QNB binding. The inhibition of [ 3H]QNB binding by HgCl 2 was still higher than 50% at 1 × 10 −8 M. MeHg caused less inhibition of [ 3H]QNB binding than HgCl 2. The inhibited receptors showed a significant degree of reactivation when treated with DMSA. The recovery was almost complete after MeHg inhibition or with the lower HgCl 2 concentrations. Generally, the reactivation was dependent on the concentration of DMSA. When rats injected with either early or delayed doses of DMSA following administration with five consecutive daily doses (8 mg/kg body wt, Gavage method) of MeHg or HgCl 2, the inhibition of [ 3H]QNB binding was less than untreated ones. The early treatment with DMSA decreased the inhibition of [ 3H]QNB binding due to MeHg or HgCl 2 intoxication. However, DMSA was more effective in reducing HgCl 2 inhibition than MeHg either in vitro or in vivo treatment. The ability of DMSA to reactivate the mACh-R after inhibition with the mercurials emphasizes the involvement of essential sulfhydryl groups in [ 3H]QNB binding sites, and also shows that these sulfhydryl groups are the primary target for the MeHg and HgCl 2 inhibition of the rat brain muscarinic receptors.

Iminoglycine transport system in synaptosomes and its interaction with enkephalins.

Evidence is presented which suggests that proline, pipecolic acid, and glycine are accumulated by a common transport system in rat brain cortical synaptosomes and synaptosomal plasma membrane vesicles. This system is Na+ dependent and appears to be similar to the iminoglycine transport system present in renal tubules and in renal brush border membranes. The opioid pentapeptides Leu- and Met-enkephalin specifically inhibit the uptake of these three imino/amino acids, presumably by interaction with a nonopioid receptor, since the inhibition is not affected by the opiate antagonist naloxone and occurs with des-tyrosyl enkephalins as well as with the intact pentapeptides.

In vitro and in vivo displacement of [3H]-diazepam binding by purine derivatives in developing rat brain.

In vitro inhibiton of [3H]-diazepam binding to developing rat brain synaptosomal membranes has been studied. Adenosine, inosine, and hypoxanthine display approximately equal potencies as inhibitors of [3H]-diazepam binding to young rat brain membranes. In adult, inosine and hypoxanthine exhibit greater inhibitory potency. Data are also presented which show that the xanthine stimulants caffeine and theophylline competitively inhibit [3H]-diazepam binding with an equal potency throughout the development. In vivo study is also reported which shows that [3H]-diazepam binding to membranes from 5-day-old rats is displaced when female rats have been daily injected with caffeine or theophylline during gestation and lactation.

Interactions of some anaesthetic, convulsant, and anticonvulsant drugs at GABA-benzodiazepine receptor-ionophore complexes in rat brain synaptosomal membranes.

The effects of several anaesthetic, convulsant and anticonvulsant drugs were studied upon high affinity [3H]GABA and [3H]diazepam binding to rat brain synaptosomal membranes in chloride-containing incubation buffers at 25 degrees C, conditions under which pentobarbitone extensively enhanced binding of both ligands to GABA-benzodiazepine-receptor-ionophore complexes. Of the compounds studied, only (+)-etomidate enhanced both GABA and diazepam binding; the sedative-hypnotic glutethimide weakly enhanced GABA binding while inhibiting diazepam binding. Several drugs, including beta-butyl-beta-methyl-glutarimide, phenobarbitone, pentylenetetrazole, and ketamine reversed the enhancement of GABA binding by pentobarbitone (500 microM) while not altering basal GABA or diazepam binding. Enhancement of high affinity GABA binding does not appear to be a general property of sedative or anticonvulsant drugs.

Actions of pentobarbitone and derivatives with modified 5-butyl substituents on GABA and diazepam binding to rat brain synaptosomal membranes.

The effects of a variety of factors known to influence the enhancement of GABA binding by diazepam, were studied upon pentobarbitone stimulation of GABA binding to washed synaptosomal membranes prepared from whole rat brains. The differential kinetics of, and effects of temperature, chloride ions, a benzodiazepine receptor antagonist (Ro15-1788) and picrotoxinin upon pentobarbitone and diazepam enhancement of GABA binding, suggest that these drugs exert their actions upon GABA binding at different loci. The degree of enhancement of diazepam binding and of high affinity GABA binding in chloride-containing media at 25 degrees C by members of a series of twelve side chain methyl substituted and/or unsaturated derivatives of 5-butyl-5-ethyl-barbituric acid (pentobarbitone analogs) correlated significantly. For the sedative members of the series, enhancement of high affinity GABA binding correlated with their anaesthetic but not their anticonvulsant activities. It appears likely that the anaesthetic and anticonvulsant activities of barbiturates arise from different molecular actions.

Met-enkephalin binding to opiate receptors is not functionally coupled to biodegradation

Using synaptosomal rat brain membranes, the degradation of Met-enkephalin in both free and receptor-bound form was measured, together with the dissociation of Met-enkephalin from the receptors. The results show that the degradation rate of initially receptor-bound Met-enkephalin is significantly smaller than either the rate of dissociation from the receptors or that of the degradation of free Met-enkephalin. These data suggest that intact Met-enkephalin dissociates from the receptors and then it becomes a target for the same membrane-associated peptidases that split free Met-enkephalin. This view is supported by the good fitness of the measured degradation of initially receptor-bound Met-enkephalin to the predicted degradation curve calculated from the dissociation rate of receptor-bound Met-enkephalin and the degradation rate of free Met-enkephalin.

An electron-spin-resonance spin-label study of the interaction of purified Mojave toxin with synaptosomal membranes from rat brain.

The structural properties of isolated purified rat brain synaptosomal membranes, both in the presence and absence of purified active toxin of the Mojave snake Crotalus scutulatus scutulatus, were studied by spin-label electron spin resonance techniques. The spectra from eight different positional isomers of nitroxide-labelled stearic acids, a rigid steroid androstanol, and a spin-labelled phosphatidylcholine intercalated into the synaptosomal membranes, were obtained as a function of temperature from 4-40 degrees C. The flexibility gradient (from spin-label order parameters) and polarity profile (from isotropic splitting factors) across the synaptosomal membranes, was characteristic for lipid bilayers. The nitroxide spin-labelled steroid, androstanol, intercalated into the synaptosomal membrane, revealed the abrupt onset of rapid cooperative rotation about the long axis of the molecule at 12 degrees C showing that the lipid molecules are rotating rapidly around their long axes at physiological temperatures. The presence of the Mojave toxin affected the synaptosomal membrane in a complex manner, depending upon the temperature and the position of the nitroxide label on the alkyl chain of the stearic acid probe. Mojave toxin exerted little effect on the flexibility gradient of the synaptosomal membrane at 20 degrees C, a temperature at which the acyl chain labels detected a structural change in the membranes. At temperatures lower than 20 degrees C, the Mojave toxin produced a change in the flexibility gradient of the synaptosomal membrane which indicated an increased disordering in the upper region of the membrane and a concomitant increased ordering of the acyl chains in the deeper regions of the membrane. At temperatures higher than 20 degrees C, the order profile of the synaptosomal membrane was shifted by the presence of the Mojave toxin in a manner which indicated that the outer parts of the membrane were more rigid and the inner regions more fluid, than in controls. A cross-over point for the perturbation occurred at C8-9, which is about 12-14 A into the membrane. This is the approximate depth of the hydrophobic pocket shown in pancreatic phospholipase A2 [Drenth et al. (1976) Nature (Lond.) 264, 373-377], a protein likely to be homologous to the basic subunit of the toxin. At all temperatures, rotational lipid motion was inhibited by the toxin as indicated by the steroid probe. The electron spin-resonance spin-label results are interpreted in terms of the partial penetration of the basic subunit of the intact toxin into the membrane, disordering the ordered chains at low temperature and ordering the disordered chains at physiological temperatures. The purified individual toxin subunits did not perturb the membrane lipids at physiological temperatures implying that both subunits must be associated for activity of the toxin which is confirmed by toxicity studies.

Radioiodination of Botulinum Neurotoxin Type A with Retention of Biological Activity and Its Binding to Brain Synaptosomes

1 A homogeneous preparation of botulinum neurotoxin from Clostridium botulinum type A was labelled with [125I]iodine to high specific activity (e.g. 1750 Ci/mmol) with chloramine-T and shown to retain appreciable biological activity (e. g. 1.2 × 108 mouse LD50/mg). 2 Both of the toxin's subunits were iodinated as shown by sodium dodecyl sulphate gel electrophoresis; the behaviour of native and labelled toxin on Ouchterlony double immunodiffusion was identical. 3 Saturable binding of the 125I-labelled neurotoxin to rat brain synaptosomal membranes was demonstrated. A small fraction of these sites (60 fmol/mg protein) exhibited high affinity for the toxin (Kd= 0.6 nM) whilst the majority displayed a much weaker affinity. Kinetic analyses of the interaction of toxin with synaptosomes were found to be complex, consistent with the existence of heterogeneity. 4 Treatment of the membranes with proteases or heating prevented all the specific binding, suggesting the involvement of protein component(s). In addition, the ability of neuraminidase, and to a lesser extent gangliosides, to reduce the extent of binding to both the high-affinity and low-affinity sites, suggests the involvement of sialic residues in toxin binding. 5 The two individual subunits of native neurotoxin were separated and renatured; only the larger (Mr 97000) competed with the binding of labelled neurotoxin. Tetanus toxin was capable of decreasing the binding, albeit with low efficacy. 6 The binding component(s) appear selective as binding was unaltered by botulinum type B neurotoxin, other pure presynaptically-active neurotoxins (α-latrotoxin, β-bungarotoxin or dendrotoxin) and known antagonists of botulism. Taipoxin, crotoxin and bee venom phospholipase A2 did inhibit the binding; however, their relatively low potencies, which are decreased further in the absence of their phospholipase A2 activity, indicate a non-specific mechanism. 7 The function of the membraneous toxin binding component(s) is discussed in relation to the molecular basis of the neurotoxin's action.

The interactions of 1-alkyl-4,4-diphenylpiperidines with opiate receptors

1-Alkyl-4,4-diphenylpiperdines inhibit the binding of μ- and δ-opiate receptor agonists to crude synaptosomal membranes of rat brain. Opiate receptor affinity is dependent on the nature of the 1-alkyl substitution. In displacing [ 3H]etorphine, [ 3H]dihydromorphine and [ 3H][D-Ala 2,D-Leu 5]enkephalin, the 1-methyl-derivative was most effective ( IC 50 [ 3 H]etorphine = 9 μ M, IC 50 [ 3 H]dihydromorphine = 0.71 μ M, IC 50 [ 3 H][D-Ala 2, D-Leu 5] enkephalin = 2.1 μ M) . 1-t-Butyl-4,4-diphenylpiperidine and its 1-isopropyl analogue, used as antiparkinsonism drugs, exhibit distinctly lower inhibiting concentration values (50% inhibition, IC 50 values).

Brain receptor binding and lipophilic character of benzodiazepines

A series of measurements of the potency of 26 benzodiazepines (BDZs) to inhibit the binding of [ 3H]diazepam to rat brain synaptosomal membranes has been undertaken. All compounds studied are NO 2 or Cl 7-substituted, a class of BDZs known to have the highest biological activities and binding affinities (expressed as K i ). The results show that: (a) 2′-unsubstituted BDZs display a parabolic dependence of —log K i values on lipophilic character of the molecule (expressed by means of the chromatographic R m value); (b) 2′-halogen substituted BDZs show quite high binding affinities ( k i values in the range 2–5 nM) giving rise to another class of BDZs whose dependence on the lipophilic character remains to be studied; and (c) BDZs lacking the carbonyl oxygen at position 2 (see Table 1), and having an oxygen at position 4, show low or very low binding affinities ( K i values in the range 600–7000 nM). Moreover for the compounds under examination, statistically significant correlations have been obtained between -log K i values and psychopharmacological activity data.

ESR measurements on the effects of ethanol on the lipid and protein conformation in biological membranes

The effects of ethanol on the conformation of proteins or fluidity of lipids was studied in human erythrocyte ghosts and rat brain synaptosomal membranes. A maleimide nitroxide probe (MAL-6) was coupled to membrane protein, or 5-doxylstearic (5NS) was dissolved in the membrane lipid, and the electron spin resonance (ESR) spectra were recorded in the presence of increasing ethanol concentrations and at different temperatures from 4° to 37°. The lipid probe at all temperatures studied showed an increase in fluidity in the membrane lipid when ethanol was added in concentrations over 20 mM. The protein probe however required a high concentration of ethanol (200 mM) to produce an increase in the rigidity of the protein conformation at 4° in both erythrocyte and synaptosomal membranes. A decrease in protein rigidity was observed of the protein ethanol concentrations at 37° only in the erythrocytes. Thus an effect of ethanol on the conformation of membrane protein was observed using MAL-6 only at high ethanol concentrations and depended on the membrane used and the temperature of measurement.