Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

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.     

Preparation of chick brain synaptosomes and synaptosomal membranes

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome $\text{c}$: oxygen oxidoreductase (EC 1.9.3.1.), monoamine: oxygen oxidoreductase (deaminating) (EC 1.4.3.4), rotenoneinsensitive NADH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.3), NADPH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, $\text{(}\text{Na}{}^{\mathrm{+}}\text{?}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Highly purified glutamine transaminase from rat brain. Physical and kinetic properties.

Glutamine transaminase from rat brain was purified to a high degree. The isolated enzyme appeared to be homogeneous by electrophoresis on polyacrylamide gel. The molecular weight was found to be approximately 98 000; the enzyme is probably composed of two subunits. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents are strong indications for the presence of pyridoxal phosphate. The enzyme showed maximal activity at pH 9.0 to 9.2. Of the amino acids tested, none could replace glutamine in the transamination reaction. Glyoxylate and phenylpyruvate was found to be the best amino acceptors. The Km values for glutamine and glyoxylate were 0.6 and 1.5 mM, respectively.     

Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.     

Time-dependent cadmium-neurotoxicity in rat brain synaptosomal plasma membranes

• 1.1. The modulation of lipid dynamics and lipid protein interactions were studied in rat brain synaptosomal plasma membranes (SPM) up to 24 hr after exposure to cadmium (Cd).
• 2.2. The activity of acetylcholinesterase and adenylate cyclase showed a considerable decrease after 6 hr of Cd exposure, followed by a progressive increase up to 24 hr.
• 3.3. SPM chemiluminescence showed a maximum decrease at 12 hr, demonstrating a considerable increase in lipid peroxidation.
• 4.4. SPM of Cd-exposed animals showed a statistical significant increase in fluorescence anisotropy parameter [(r₀/r) — 1]⁻¹ at 18 and 24 hr compared to SPM of the control, indicating a decrease of membrane fluidity.

     

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μmoles·(mg protein)^?1·h^?1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.     

Highly purified mitochondria from rat brain prepared by phase partition

Mitochondria and synaptosomes from adult rat forebrain can easily be separated by counter-current distribution in an aqueous two phase system composed of Dextran T500 and poly(ethylene glycol) 4000. Both particles may also be separated by a batch procedure in which the same phase system is used. Electron micrographs and enzymatic activities show a high purity of the mitochondria obtained from the dextran-rich lower phase. Electron micrographs and enzymatic activities also show that intact synaptosomes can be obtained from the poly (ethylene glycol)-rich upper phase.The mitochondria purified by this method show good ADP/O ratios, respiratory control ratios, and state 3 rates. Synaptosomes showed a state 2-state 3 transition with no recuperation to state 4.     

Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity

Summary Preparations of intestinal epithelial cell basal lateral plasma membranes were analyzed with free flow electrophoresis and density pertubation with digitonin. The initial basal lateral membrane preparations were obtained by equilibrium density gradient centrifugation after two different schemes of homogenization and differential sedimentation (A.K. Mircheff, C.H. van Os, and E.M. Wright. 1978.Membr. Biochem. 1: 177, and A.K. Mircheff, S.D. Hanna, M.W. Walling, and E.M. Wright. 1979.Prep. Biochem. 9:33. In these preparations, Na,K-ATPase, a marker for the basal lateral mambrane, was purified 16- to 18-fold over the initial homogenate. The preparations were also enriched in NADPH-cytochromec reductase, alkaline phosphatase, acid phosphatase, and galactosylstransferase.Both free-flow electrophoresis, which separates on the basis of surface charge, and density perturbation with digitonin, which depends on a specific interaction of digitonin with cholesterol-rich membranes, resolved the preparation into three populations of particles. The major population, which represented basal lateral membranes purified 20- to 32-fold with respect to the initial homogenate, contained Na,K-ATPase, alkaline phosphatase, adenylate cyclase, and acid phosphatase. A second population was defined by its content of NADPH-cytochromec reductase, and the third was defined by its content of galactosyltransferase. Guanylate cyclase appeared to be partitioned between the Na,K-ATPase-rich and NADPH-cytochromec reductase-rich populations. Galactosyltransferase is also present in fractions which contain the Na,K-ATPase-rich membranes, but the present data cannot exclude the possibility of spillovers by the adjacent, galactosyltransferase-rich population. This work emphasize the importance of multiple, physical criteria for purity in the isolation of subcellular components.     

Biochemical and immunological characterization of A1 adenosine receptors purified from human brain membranes.

The A1 adenosine receptor was purified approximately 13,000-fold to apparent homogeneity from human cerebral cortex membranes using a novel affinity-chromatography system developed for the purification of rat brain and rat testis A1 adenosine receptors [Nakata, H. (1989) J. Biol. Chem. 264, 16,545-16,551; Nakata, H. (1990) J. Biol. Chem. 265, 671-677]. The purified human brain receptor showed the ligand-binding specificity expected of the A1 adenosine receptor. The Bmax and Kd for the purified receptor with a specific A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, were approximately 16 nmol/mg protein and 2 nM, respectively. SDS/PAGE of the purified receptor preparation showed one broad protein band of molecular mass of approximately 35 kDa, which is very similar to that of purified A1 adenosine receptor from rat brain membranes. Endoglycosidase F treatment of the purified receptor reduced the molecular mass to approximately 30 kDa, suggesting that the human brain A1 adenosine receptor is a glycoprotein. Comparison of the purified human and rat brain A1 adenosine receptors by peptide mapping after the proteolytic digestion showed minor differences between these receptors. Immunological comparisons of the human brain A1 adenosine receptor with rat brain A1 adenosine receptor using polyclonal antibodies against the purified rat brain A1 adenosine receptor showed that the antibodies react preferentially with the rat brain receptor and weakly with human brain receptor.     

Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

Sensitivity of high-conductance potassium channels in synaptosomal membranes from the rat brain to intracellular pH

High-conductance potassium channels have been studied in inside-out patches excised from proteoliposomes reconstituted from giant liposomes and rat brain synaptosomes. Acid pH in the medium reduced single channel current amplitude and increased the mean open probability and the frequency of channel opening. This was accompanied by a shortening of the open time constant at positive potential and by shortening of the longer closed time constant. The decrease of channel amplitude, the increase of the open probability and the decrease in the longer closed time constant can be explained by neutralization of negative charges of the membrane and by a decrease in the surface membrane potential which mimics membrane depolarization. The shortening of the mean open time is apparently due to a channel blockade by protons. Correspondence to: H. Zemková     

Influence of dietary fat on the lipid composition of rat brain synaptosomal and microsomal membranes.

The modulation of rat brain microsomal and synaptosomal membrane lipid by diet fat was examined. Brain synaptosomal and microsomal membrane composition was compared for rats fed on diets containing either soya-bean oil (SBO), SBO plus choline, SBO lecithin, sunflower oil (SFO), chow or low-erucic acid rape-seed oil (LER) for 24 days. Cholesterol and phosphatidylcholine levels in both membranes were altered by diet. Diet fat also affected the microsomal content of sphingomyelin. Change in membrane phosphatidylcholine level was related to the relative balance of omega-6, omega-3 and monounsaturated fatty acids within the diets fed. The highest phosphatidylcholine levels appeared in membranes of animals fed on SBO lecithin and the lowest in those fed on LER. Microsomal membrane cholesterol and sphingomyelin content increased by feeding on SBO lecithin. In both synaptosomal and microsomal membranes a highly significant correlation was observed between membrane phosphatidylcholine and cholesterol content. The fatty acyl composition of phospholipids from both membranes also altered with diet and age. Alteration in fatty acid composition was observed in response to dietary levels of omega-6, omega-3 and monounsaturated fatty acids, but the unsaturation index of each phospholipid remained constant for all diet treatments. These changes in lipid composition suggest that dietary fat may be a significant modulator in vivo of the physicobiochemical properties of brain synaptosomal and microsomal membranes.     

Spin label studies on synaptosomal membranes of rat brain cortex during aging

Membrane lipids of brain synaptosomes of 2, 12 and 24 months old rats were labeled by stearic acid spin probes with a doxyl group in the C-5 and C-16 position, respectively. We demonstrated that the hydrophobic region of synaptosomal membranes became more rigid during the life span studied, and the region located nearer the surface of membranes displays a significantly decreased fluidity only in the oldest group. Membrane proteins labeled with a nitroxide derivative of maleimide suggest that conformational changes take place during aging.