Postsynaptic membranes in the electric tissue of Narcine: III. Isolation and characterization

Postsynaptic membranes in homogenates of the electric tissue of Narcine were identified by labelling nicotinic acetylcholine receptors in the membranes with radioactive alpha-bungarotoxin. Various media and centrifugation conditions were examined in an attempt to obtain highly purified postsynaptic membranes. The main criterion for purification was approach towards the specific activity of the pure receptor protein, 9–10 nmol toxin-sites/mg protein. Isolation of tissue microsomes with Tris buffer, EDTA and the protease inhibitor phenylmethylsulfonylfluoride (PMSF), conditions which preserve the receptor molecules optimally, yielded about 50 % of the tissue toxin-sites, 5 % of the protein, 4 % of the ATPase and less than 2 % of the acetylcholinesterase (AChE). Further separation of vesiculated membranes in continuous density gradients of sucrose showed that the major contaminants of postsynaptic membrane vesicles were damaged mitochondria and tubular vesicles of dorsal electroplaque membranes rich in ATPase. Mitochondria were effectively removed from homogenates by ‘differential’ centrifugation, and ATPase-rich vesicles could be largely removed by causing their agglutination with calcium ions, or by controlled proteolysis in the absence of PMSF. Partially purified postsynaptic membranes were obtained having about 7 nmol toxin-sites/mg membrane protein. Further purification appears possible by affinity techniques.     

THE ISOLATION OF CHOLINERGIC SYNAPTIC VESICLES FROM BOVINE SUPERIOR CERVICAL GANGLION AND ESTIMATION OF THEIR ACETYLCHOLINE CONTENT

Abstract— A method is described for the isolation of relatively pure cholinergic synaptic vesicles by hypo-osmotic shock and density gradient centrifugation of a synaptosome fraction prepared from bovine superior cervical ganglia. Vesicles from this source were found to sediment to a density equivalent to 0·3–0·41 M-sucrose. The vesicle subfraction from the gradient had an acetylcholine (ACh) content of 4.4 nmol/mg of protein and were subject to leakage of ACh. By a 'tagging' technique, the vesicles were counted under the electron microscope and their numbers related to their ACh concentration. After correction for leakage, an ACh content of 1630 molecules/vesicle was estimated.     

EFFECT OF ELECTRICAL STIMULATION ON THE YIELD AND COMPOSITION OF SYNAPTIC VESICLES FROM THE CHOLINERGIC SYNAPSES OF THE ELECTRIC ORGAN OF TORPEDO: A COMBINED BIOCHEMICAL, ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL STUDY

Abstract— The effect of stimulating the electric organ of Torpedo marmorata , anaesthetized with 0.01% Tricaine methane sulphonate, by means of electrical stimulation (5/s) administered via an electrode placed on the electric lobe has been studied electrophysiologically, biochemically and morphologically. The response of the organ declined to about 50 per cent of its initial value after about 500 stimuli, by a further 10 per cent after another 500 stimuli and then to about 12 per cent of the initial value after a further 1000 stimuli. Thereafter the response fell off progressively. However, even when the response was less than 1 per cent of its initial value, the organ had considerable powers of recuperation during a 30-s rest period, to 30–50 per cent of its initial value.
The fall in response was accompanied by a reduction in vesicle size and number, an increase in the area of the presynaptic membrane and a fall in the protein, total nucleotide, ATP and acetylcholine content of the vesicle fraction isolated from the stimulated tissue. However, whereas vesicle numbers and the protein and total nucleotide content of the vesicle fraction fell by only about 50 per cent, vesicular ATP and acetylcholine levels were reduced to about 10 per cent. An analysis of the covariance of vesicular ATP and acetylcholine showed an initial loss of an acetylcholine-rich (relative to ATP) population of vesicles. The early loss of vesicular protein and nucleotide and vesicle numbers as well as the morphological changes seen would be consistent with a loss of vesicles due to fusion with the external membrane. The preferential loss of acetylcholine and ATP from the vesicle fraction indicates that the vesicles surviving the stimulation procedure have been utilized in a number of cycles causing the progressive fall in vesicle volume, and acetylcholine and ATP content.     

PRELIMINARY ESTIMATES OF THE DOPAMINE β-HYDROXYLASE CONTENT AND ACTIVITY IN PURIFIED NORADRENERGIC VESICLES1

The dopamine β-hydroxylase (DβH) content and activity of large dense-core noradrenergic vesicles purified from bovine splenic nerve were determined using two assay procedures : enzymic activity expressed in Units per mg protein and homospecific activity based on radioimmunoassay expressed in Units per mg DβH antigen. Approximately two-thirds of the total enzyme activity is latent in these vesicles, even after various treatments designed to compromise vesicle integrity. DβH can be completely unmasked by brief treatment with 0.01-0.05% Triton X-100 and activity increases from 0.20 to 0.64 Units per mg vesicle protein. Calculations based on both assay methods suggested that an average of 7% (range 3-15%) of the total vesicle protein was DβH and that the average vesicle contained about 4 molecules of enzyme (range 2-9 molecules). The estimated homospecific activities indicated an average of 25 and 50% (range 18-72%) of the vesicle enzyme was inactive in the various samples using the two antibodies. The vesicle can synthesize up to 30 molecules of noradrenaline/s per molecule of DβH at near optimal substrate concentration, and 60-270 molecules of norepinephrine/s per vesicle. The assumptions used in the various calculations were critically analyzed and, based on the methods employed, it is tentatively considered to be unlikely that there could be more than 5-12 molecules of DβH per vesicle. The possibility that circulating DβH originates primarily, if not exclusively, from the large dense-core vesicle type is considered and the functional implications of the data support the concept of vesicle reuse during several cycles of exocytosis involving a quantal size equal to a fraction of the vesicle transmitter content.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: Isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus

A convenient, large-scale method for the isolation of membrane vesicles permeable to specific inorganic ions has been developed. The general principle of this method involves the exchange of Na⁺ within the vesicles for external Cs⁺. Vesicles in which this exchange rapidly occurs can be separated on the basis of their density from vesicles in which the exchange occurs slowly (G. P. Hess and J. P. Andrews (1977) Proc. Nat. Acad. Sci. USA74, 482–486). This approach has been adapted to develop a method suitable for the large-scale isolation of vesicles that contain functional acetylcholine receptors from the Electrophorus electricus electroplax. The new procedure involves a discontinuous sucrose gradient for an initial purification of the vescles. This allows the use of a low-speed centrifuge, which has a capacity up to 30 times greater than the Beckman ultracentrifuge previously used. A self-forming CsCl-Percoll gradient and low-speed centrifugation are then used for the isolation of the functional acetylcholine receptor-containing vesicles. The isolation step leads close to the theoretically possible fourfold purification of the vesicles that contain functional receptors. The yield, up to 12 mg membrane protein/centrifugal run, is about 100-fold higher than the yield from the sucrose-CsCl density gradient previously (Hess and Andrews, see above) used. The gradients are self-forming and an equilibrium is reached after centrifugation for only 30 min. In 12 experiments with membrane preparations from 12 different ceis, the functional vesicles had an internal volume of 2.0 ± 0.3 μl/mg vesicle protein and a receptor concentration of 1.2 ± 0.02 μm (1.2 μmol/liter of internal volume). Electron micrographs of these vesicles show an average vesicle radius of 1600 ± 300 Å. From these results, an average of 12 receptor molecules/membrane vesicle is calculated.     

Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles

The effect of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium ionophore A23187 on Ca2+ release from bovine adrenal medullary secretory vesicles and microsomes was examined. Ins(1,4,5)P3 released 3.5 nmol of Ca2+/mg protein from secretory vesicles and 1.5 nmol of Ca2+/mg protein from microsomes as measured by a Ca2(+)-selective electrode. However, A23187 promoted Ca2+ uptake into vesicles while releasing Ca2+ from microsomes. Ins(1,4,5)P3-induced Ca2+ release from secretory vesicles was rapid, but the released Ca2+ was absorbed within 3 min during which the Ins(1,4,5)P3-releasable pools were refilled. The in situ calcium content of secretory vesicle measured by atomic absorption spectrometry was 112 +/- 6.3 nmol/mg protein indicating the potential importance of secretory vesicles as an intracellular Ca2+ store. The high Ca2(+)-buffering capacity of secretory vesicles is presumed to be due to the high Ca2(+)-binding capacity of chromogranin A, the major intravesicular protein, which has calsequestrin-like properties.     

Isolation of Cholinergic Synaptic Vesicles from the Myenteric Plexus of Guinea-Pig Small Intestine

The acetylcholine-rich myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine has been subjected to subcellular fractionation using modifications of both classical methods and that originally devised for bulk isolation of cholinergic synaptic vesicles from the electromotor nerve terminals of Torpedo marmorata by means of density gradient centrifugation in a zonal rotor. The latter method gave a vesicle fraction with the highest acetylcholine content so far recorded for a mammalian particulate fraction, 30.9 × S.E.M. 1.8 (5) nmol of acetylcholine × mg of protein^?1. Electron-microscopical examination showed that it consisted of a homogeneous preparation of vesicles of mean spherical diameter 61 ×sd 4 (108) nm, with little or no contamination with other lipoprotein membrane structures, mixed how-ever with considerable amounts of actomyosin fibrils, presumably derived from the longitudinal muscle. Slab-gel electrophoresis in sodium dodecyl sulphate showed that, in addition to prominent peaks attributable to actin and myosin, there was a relatively simple pattern of (presumably) vesicle protein among which all the proteins thought to be characteristic of Torpedo synaptic vesicles were present. Dowe G. H. C. et al. Isolation of cholinergic synaptic vesicles from the myenteric plexus of guinea-pig small intestine. J. Neurochem. 35, 993–1003 (1980).     

Norepinephrine:adenosinetriphosphate ratios in purified adrenergic vesicles.

Norepinephrine (NE):adenosinetriphosphate (ATP) ratios were studied in a highly purified fraction of large dense core vesicles isolated from the bovine splenic nerve. Vesicles prepared from nerves chilled approximately 10 and 30 min post mortem were compared. The NE:ATP molar ratio decreased from 6.3 to 4.8, p less than 0.005; NE decreased from 61 to 42 nmol, while ATP decreased only from 9.6 to 8.8 nmol/mg protein. Animals weighing 180-360 kg were compared with heavier ones weighing 400-700 kg. NE increased from 42 to 68 nmol and ATP increased from 5.9 to 13.2 nmol/mg protein, while the NE:ATP molar ratio decreased from 7.2 to 5.2, p less than 0.005. Changes during vesicle maturation were studied by comparing vesicles identically prepared from equal weights of a proximal nerve segment close to the coeliac ganglion and a distal, intrasplenic segment. NE increased from 45 to 70 nmol while ATP remained unchanged at 10.0 nmol/mg protein and the NE:ATP molar ratio increased from 4.5 to 7.0, p less than 0.005. It was interpreted that vesicle ATP content, like dopamine beta-hydroxylase, was established early in the cell body and remained unchanged during axoplasmic transport. ATP was in a complex which was relatively stable to post mortem hydrolysis at least between 10 and 30 min prior to chilling the nerves. The addition of newly synthesized NE into a readily releasable pool during axoplasmic transport occurs without ATP and can account for the increased ratio above 4:1 in the distal segment vesicles.     

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.     

Lipid peroxidation and active calcium transport in inside-out vesicles of red blood cells from preeclamptic women

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca++ uptake by inside-out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49+/-3% in the preeclamptic women as compared to the gestational controls ( 8.06 +/- 0.11 nmol Ca++/mg protein min, gestational controls; 4.08 +/- 0.1 nmol Ca++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05 +/- 0.96 nmol Pi/mg protein min, gestational controls; 8.85 +/- 0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.     

Norepinephrine: Adenosinetriphosphate ratios in purified adrenergic vesicles

Norepinephrine (NE):adenosinetriphosphate (ATP) ratios were studied in a highly purified fraction of large dense core vesicles isolated from the bovine splenic nerve. Vesicles prepared from nerves chilled ～10 and 30 min post mortem were compared. The NE:ATP molar ratio decreased from 6.3 to 4.8, p < 0.005; NE decreased from 61 to 42 nmol, while ATP decreased only from 9.6 to 8.8 nmol/mg protein. Animals weighing 180-360 kg were compared with heavier ones weighing 400-700 kg. NE increased from 42 to 68 nmol and ATP increased from 5.9 to 13.2 nmol/mg protein, while the NE:ATP molar ratio decreased from 7.2 to 5.2, p < 0.005. Changes during vesicle maturation were studied by comparing vesicles identically prepared from equal weights of a proximal nerve segment close to the coeliac ganglion and a distal, intrasplenic segment. NE increased from 45 to 70 nmol while ATP remained unchanged at 10.0 nmol/mg protein and the NE:ATP molar ratio increased from 4.5 to 7.0, p < 0.005. It was interpreted that vesicle ATP content, like dopamine β-hydroxylase, was established early in the cell body and remained unchanged during axoplasmic transport. ATP was in a complex which was relatively stable to post mortem hydrolysis at least between 10 and 30 min prior to chilling the nerves. The addition of newly synthesized NE into a readily releasable pool during axoplasmic transport occurs without ATP and can account for the increased ratio above 4:1 in the distal segment vesicles.     

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

Development of sarcoplasmic reticulum in cultured chicken muscle.

The development of sarcoplasmic reticulum membranes was studied in vivo and in tissue culture in chicken pectoralis muscle cells. The concentration of the calcium- and magnesium-activated ATPase measured by selective labeling of the enzyme with [32P]ATP in whole muscle homogenates was found to increase in developing chicken pectoralis muscle in vivo from 0.01 nmol/mg of protein in 12-day embryos to 0.3 to 0.4 nmol/mg of protein in 1-month-old chicks, where it constitutes about 3% of the total protein content of muscle. In cultured muscle cells the concentration of calcium-sensitive phosphoprotein increased from 0.015 nmol/mg of protein at 2 days to 0.04 to 0.05 nmol/mg of protein after 5 days of culture. This amount represents about 0.5% of the protein content of the muscle cells. The accumulation of Ca2+ transport ATPase began during fusion and continued with a linear rate during 8 days of culture. The density of 75 A intramembranous particles seen by freeze-etch electron microscopy on fracture faces of sarcoplasmic reticulum membranes is about 4,000/mum2 in adult chick pectoralis muscle but only 400/mum2 in cultured muscle cells in rough proportion to the concentration of Ca2+-sensitive phosphoprotein. The Ca2+, Na+, and K+ concentration of the medium and addition of ouabain, caffeine, or the calcium ionophores A23187 and X537A sharply influence the concentration of calcium transport ATPase in cultured muscle cells, parallel with their effect upon cell fusion and growth. These observations are consistent with the proposition that the gene expression leading to the accumulation of Ca2+ transport ATPase during development in culture may be regulated by intracellular ion concentrations.     

An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

ON THE DEVELOPMENT OF SYMPATHETIC NERVE TRUNK VESICLES DURING AXONAL TRANSPORT: DENSITY GRADIENT ANALYSIS OF DOPAMINE β-HYDROXYLASE IN BOVINE SPLENIC NERVE

—Dopamine β-hydroxylase was used as a marker enzyme for sympathetic nerve vesicles which were studied by density gradient technique in bovine splenic nerves. The enzyme analyses were complicated by the occurrence of inhibitors which had to be carefully neutralized with copper. The inhibitor was mainly found in the soluble fraction and no evidence for the occurrence of endogenous inhibitors in the nerve vesicles was obtained. A great variation in density of the dopamine β-hydroxylase containing particles was observed. This was probably mainly due to the variation in vesicle maturation since dopamine β-hydroxylase was distributed more towards the lighter gradient fractions in the proximal nerve segment preparations compared with intrasplenic nerve segment preparations. Noradrenaline/protein and noradrenaline/dopaminc β-hydroxylase ratios were found to be increased about 1·7-fold in the vesicle fraction isolated from the proximal nerve segments to those from the intrasplenic segments. A further increase of the noradrenaline/dopamine β-hydroxylase ratio was observed in a fraction with the same density isolated from the spleen. On the basis of these findings the noradrenaline/protein ratio was calculated to be about 500-600 nmol/mg in the nerve terminal vesicles.     

A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles

A monoclonal antibody, tor70, recognizes an antigenic determinant on the inside surface of synaptic vesicles, purified from the electric organ of Narcine brasiliensis. The antigenic determinant appears to be unique to vesicles since it co-purifies with vesicle content and is blocked by an antiserum specific for synaptic vesicle antigens. Immunoblotting of vesicle proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antigen has a low heterogeneous electrophoretic mobility and corresponds to a major protein component of pure synaptic vesicles. Synaptic vesicles contain a proteoglycan-like material since proteolytic digestion yields a ruthenium red-binding material that migrates during electrophoresis with a mammalian heparin standard. The only major vesicle component with which the proteoglycan-like material co-elutes during chromatography on Sepharose 6B is the material recognized by tor70. The antigen adsorbs specifically to beads coated with the lectin wheat germ agglutinin. Isolation of the tor70 antigen by velocity sedimentation in sodium dodecyl sulfate-sucrose gradients shows it to contain glucosamine (0.75 nmol/microgram of protein) and uronic acid but no galactosamine. Earlier work has shown that specific antiserum to pure synaptic vesicles could be used to identify nerve terminals, quantitate vesicle components, purify membranes, and monitor exocytosis. We now know that one of the components recognized by the antiserum is a molecule with properties of a proteoglycan, attached to the inside surface of vesicle membranes.     

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).     

Characterization of Na+−Ca2+ exchange activity in plasma membrane vesicles from postmortem human brain

Procedures were developed for measurement of Na⁺/Ca²⁺ exchange in resealed plasma membrane vesicles from postmortem human brain. The vesicle preparation method permits use of stored frozen tissue with minimal processing required prior to freezing. Vesicles prepared in this manner transport Ca²⁺ in the presence of a Na⁺ gradient. The kinetic characteristics of the Na⁺/Ca²⁺ exchange process were determined in membrane vesicles isolated from hippocampus and cortex. The K_act for Ca²⁺ was estimated to be 32 M for hippocampal and 17 M for cortical tissue. The maximal rate of Ca²⁺ uptake (V_max) was 3.5 nmol/mg protein/15 sec and 3.3 nmol/mg protein/15 sec for hippocampal and cortical tissue, respectively. Exchange activity was dependent on the Na⁺ gradient, and was optimal in the high pH range. Therefore, membranes in which Na⁺-dependent o Ca²⁺ transport activity is preserved can be isolated from postmortem human brain and could be used to determine the influence of pathological conditions on this transport system.     

Matrix free Ca2+ in isolated chromaffin vesicles

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS)