Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.     

Subcellular distribution of marker enzymes and of neurotoxic esterase in adult hen brain.

Neurotoxic esterase (NTE) is now regarded as the site of the primary biochemical lesion in the delayed neuronal degeneration produced by certain organophosphorus esters. Since hens are the species of choice in studies of this neuropathy the subcellular distribution of NTE and marker enzymes in adult hen brain was carried out. Up to 70%, of NTE was recovered in a microsomal fraction (P₃) which was also enriched in 5′-nucleotidase (5′-ribonucleotide phosphohydrolase EC 3.1.3.5), a plasma membrane marker. The protein content of this fraction (31% of the parent homogenate) is double that of equivalent mammalian brain fractions. The LDH distribution suggests that the P₃ fraction contained many small synaptosomes. Subfractionation of microsomes by rate and equilibrium centrifugation on sucrose density gradients segregated the RNA but failed to separate the NTE. 5′-nucleotidase and glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase EC 3.1.3.9) from each other. NTE was considerably concentrated (2–5 times) in subfractions of the P₂ fraction, which are believed to be enriched in synaptosomal membranes. A similar localization of NTE and AChE was found in subfractions of P2 from neonatal chick brain. Axon fragments contained a significant amount of NTE which was not associated with the myelin. Nuclear and mitochondrial fractions were low in NTE. Microsomes could be partitioned in biphasic aqueous polymer systems, but with little enrichment of NTE. The possible association of NTE with synaptosomal membranes suggests that early events in organophosphorus neuropathy may occur at the axonal (? synaptic) surface.     

Subcellular sites of enzymes catalyzing the phosphorylation-dephosphorylation of dolichol in the central nervous system

The subcellular locations of several enzymes involved in dolichyl monophosphate (Dol-P) metabolism in brain have been investigated. Dolichol kinase is highly enriched in a heavy microsomal fraction from calf brain, while 71% of the Dol-P phosphatase activity was recovered with the light microsomes. Lower amounts of the phosphatase activity were also found in the heavy microsomal, mitochondrial-lysosomal, and synaptic plasma membrane fractions. Since the light microsomal fraction also contained substantial acetylcholinesterase activity, an axon plasma membrane marker, an axolemma-enriched fraction, was prepared from rat brain by a second procedure. A comparison with microsomal and mitochondrial-lysosomal fractions revealed that the axolemma-enriched fraction contained the highest specific activity of Dol-P phosphatase, indicating that the enzyme was present in the axon plasma membrane. The tunicamycin-sensitive UDP-N-acetylglucosamine:Dol-P N- acetylglucosaminylphosphotransferase , glucosyl- phosphoryldolichol (Glc-P-Dol) synthase, Glc-P-Dol:oligosaccharide glucosyltransferase, and the oligosaccharyltransferase were all found predominantly in the heavy microsomes. These results indicate that the enzymes responsible for the initiation and termination of biosynthesis, as well as the transfer of dolichol-linked oligosaccharides, reside in the rough endoplasmic reticulum (ER) of central nervous tissue. Evidence that at least some Dol-P molecules formed by dolichol kinase are accessible to multiple glycosyltransferases in the rough ER of brain is also presented.     

Isolation of the plasma membrane of a trypanosomatid flagellate: General characterization and lipid composition

A technique is described for the isolation of a fraction that contains the plasma membrane of the trypanosomatid flagellate Leptomonas collosoma. This fraction has been investigated by electron microscopy and has been shown to be mostly membranes associated with microtubules, a known plasma membrane marker in this organism. The fraction is enriched in Mg²⁺-dependent ATPase but has a decreased specific activity of succinate dehydrogenase. Lipid has been extracted from whole cells and the isolated plasma membrane fraction. A fraction of the total lipid that is eluted from a silicic acid column by acetone is found to be concentrated in the plasma membrane. Also enriched in the plasma membrane fraction is a 5,7-diene sterol identified as ergosterol. The major phospholipids of the whole cell and the plasma membrane are phosphatidylethanolamine and phosphatidylcholine. Approximately 60% of the fatty acids of the cell and plasma membrane have a carbon chain length of eighteen, and half of this is in the form of the mono-unsaturated fatty acid.     

Interaction of calmodulin with synaptic plasma membranes isolated from sheep brain

Calmodulin binding to a membrane fraction enriched in synaptic plasma membranes of sheep brain cells was investigated with [¹²⁵I]calmodulin. Calmodulin binding to these membranes is Ca²⁺-dependent with a half maximal saturation at the pCa value of about 5.5. The binding is reduced by replacing Ca²⁺ with Mg²⁺, but it is significantly enhanced when both cations are present in the medium. Cation-dependent binding is specific and saturable with an apparent K_D of about 47–50 nM and a maximal capacity of about 4 pmol mg⁻¹ protein. The results indicate that synaptic plasma membranes isolated from sheep brain cells interact with calmodulin in a Ca²⁺-dependent, Mg²⁺-facilitated manner.     

Correlation of glutamate binding activity with glutamate-binding protein immunoreactivity in the brain of control and alcohol-treated rats

Specific antibodies raised against a glutamate binding protein purified from bovine brain were used to trace the immunoreactivity of this protein in rat brain subcellular fractions. In the subcellular fractions obtained from whole brain homogenates, the synaptic membranes had the highest immunochemical reactivity towards the anti-glutamate-binding protein antibodies. The combination of measurements of glutamate binding activity and glutamate-binding protein immunoreactivity indicated that in brain synaptic membranes from control animals the highest activity in these two measures was associated with a synaptic plasma membrane subfraction that was enriched with synaptic junctions. In animals treated with ethanol for 14 days, there was a significant increase in the density of synaptic membrane glutamate binding sites. This increase in glutamate binding capacity was correlated with a greater than two-fold increase in the glutamate binding activity and binding protein immunoreactivity of the light synaptic membrane subfraction, a subfraction which does not contain many recognizable synaptic junctions. Acute administration of ethanol to rats produced a moderate but non-significant decrease in glutamate binding capacity of synaptic membranes. The increase in the number of glutamate binding protein subunits in brain plasma membranes may be an adaptive response of central nervous system neurons to the acute effects of ethanol on glutamate synaptic transmission.     

Synaptic proteome changes in mouse brain regions upon auditory discrimination learning

Changes in synaptic efficacy underlying learning and memory processes are assumed to be associated with alterations of the protein composition of synapses. Here, we performed a quantitative proteomic screen to monitor changes in the synaptic proteome of four brain areas (auditory cortex, frontal cortex, hippocampus striatum) during auditory learning. Mice were trained in a shuttle box GO/NO-GO paradigm to discriminate between rising and falling frequency modulated tones to avoid mild electric foot shock. Control-treated mice received corresponding numbers of either the tones or the foot shocks. Six hours and 24 h later, the composition of a fraction enriched in synaptic cytomatrix-associated proteins was compared to that obtained from na?ve mice by quantitative mass spectrometry. In the synaptic protein fraction obtained from trained mice, the average percentage (±SEM) of downregulated proteins (59.9 ± 0.5%) exceeded that of upregulated proteins (23.5 ± 0.8%) in the brain regions studied. This effect was significantly smaller in foot shock (42.7 ± 0.6% down, 40.7 ± 1.0% up) and tone controls (43.9 ± 1.0% down, 39.7 ± 0.9% up). These data suggest that learning processes initially induce removal and/or degradation of proteins from presynaptic and postsynaptic cytoskeletal matrices before these structures can acquire a new, postlearning organisation. In silico analysis points to a general role of insulin-like signalling in this process.     

Isolation and partial characterization of chick brain synaptic plasma membranes

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones. D. H. and Matus. A. I. (1974) Biochim. Biophys. Acta 356, 276–287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organcelles. Fraction 2 was recovered from the 28.5–34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na⁺+K⁺)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5′-nucleotidase (EC 3.1.3.5) were, respectively, 4.5. 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Isolation and partial characterization of chick brain synaptic plasma membranes.

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones, D. H. and Matus, A. I. (1974) Biochim. Biophys. Acta 356, 276-287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organelles. Fraction 2 was recovered from the 28.5-34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na+ +K+)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5'-nucleotidase (EC 3.1.3.5) were, respectively, 4.5, 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Isolation of rat brain subcellular fraction enriched in putative neurotransmitter receptors and synaptic junctions

A simple reliable method was developed for the rapid isolation of a synaptic plasma membrane-enriched fraction from rat brain. The procedure involves the direct lysis of a crude mitochondrial fraction followed by a combined flotation-sedimentation density gradient centrifugation in a fixed-angle centrifuge rotor. All fractions have been characterized with respect to relative enrichment of (Na⁺–K⁺) ATPase activity as well as putative cholinergic neurotransmitter receptors determined by [¹²⁵I]-bungarotoxin and [³H]quinuclidinyl benzilate binding. The 2-to 4-fold relative enrichment of putative receptor binding sites correlated well with the 4-fold enrichment of morphologically identifiable synaptic junctions in the synaptic plasma membrane enriched fraction.     

Localization in the synaptic junction of the cyclic amp stimulated intrinsic protein kinase activity of synaptosomal plasma membranes

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggest that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.     

Localization in the synaptic junction of the cyclic AMP stimulated intrinsic protein kinase activity of synaptosomal plasma membranes.

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggests that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.     

Influence of pentobarbital on synaptic plasma membrane protein synthesis

The effects of acute and chronic pentobarbital treatment on the incorporation of ³H-lysine into rat brain synaptic plasma membranes (SPM) were examined by means of SDS polyacrylamide gel electrophoresis. The data suggest the major effects of both drug treatments are found in a unique population of SPM derived from a light population of nerve ending particles (NEP). These light NEP preferentially accumulate labelled GABA, suggesting this fraction is enriched in GABA containing nerve terminals.     

Characterization of the cyclic 3',5'-nucleotide phosphodiesterase actitivty associated with synaptosomal plasma membranes and synaptic junctions.

Some characteristics of the cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) activity associated with the synaptosomal plasma membrane (synaptic membrane) and the synaptic junction fractions of rat brain are reported. Kinetic analysis revealed that only one type of phosphodiesterase activity, with a Km of 2.10 19(-4) M for cyclic AMP, is associated with both fractions. The specific activities of the phosphodiesterase in synaptic membranes and synaptic junctions have been estimated at 23.4 nmol/min per mg protein and 22.5 nmol/min per mg protein, respectively. The synaptic junction-associated activity undergoes a 30% stimulation by Ca2+ while no Ca2+ sensitivity of the synaptic membrane-associated activity could be detected. Cytochemical studies performed on the synaptic membrane fraction demonstrated a predominant localization of phosphodiesterase activity over postsynaptic densities, while dense deposits were sometimes observed over the synaptic cleft region.     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

Subcellular Fractionation on Percoll Gradient of Mossy Fiber Synaptosomes: Morphological and Biochemical Characterization in Control and Degranulated Rat Hippocampus

Abstract: A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaption of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well-preserved morphology (large size 3 μm, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1–8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.     

Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-1 (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dlg and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.