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.     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

The distribution of A1 adenosine receptor and 5′-nucleotidase in pig brain cortex subcellular fractions

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [³H]R-PIA binding, the activity of adenosine metabolizing enzymes (5-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A₁ adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A₁ adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding paramters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.     

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.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a ?nerve ending fraction” consisting of at least 85% well-preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a ?synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na⁺-K⁺ ATPase, 5′-nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the ?nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone-insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5- and 0.7-fold that in the ?nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P₂ fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the ?synaptosomal membrane fraction”, referred to the ?nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds -fetuin. The K_m values for CMP-NeuNAc were different using lactosylceramide and ds -fetuin as acceptor substrates (0.57 and 0.135 mm , respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds -fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds -fetuin (and glycoprotein acceptors). Ganglioside GM₃ was recognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.     

Distribution of selected phospholipid modifying enzymes in rat brain microsomal subfractions prepared by density gradient zonal rotor centrifugation

A rat brain P₃ fraction enriched in ER derived microsomes was centrifuged through a 20–40% linear sucrose gradient in a Beckman Ti-14 Zonal rotor and 11 fractions were obtained. The distribution of marker enzyme activities and protein were determined in these 11 subfractions. NADPH-Cytochrome C reductase, choline phosphotransferase were employed for endoplasmic reticulum, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were employed for plasma membrane, 2, 3-cyclic nucleotide phosphohydrolase was employed for myelin. The bulk of the protein was recovered in the 24–34% sucrose fractions, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were in the 22–38% sucrose fractions while NADPH-cytochrome C reductase and CNPase were enriched in the 20–22% sucrose fractions. The ethanolamine and the serine base exchange activities had a bimodal distribution, with highest specific activities in sucrose fractions 32–34% and 20–24%. Choline base exchange activity was nearly undetectable in all the fractions. The specific activities of CDP-choline phosphotransferase, and phospholipid-N-methyltransferase were highest in the 20–22% sucrose fraction. Phospholipid-N-methyltransferase activity was significantly stimulated in the presence of exogenous phospholipid acceptors as phosphatidylethanolamine or phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine, however, the greatest response was with phosphatidylmonomethylethanolamine. The rat brain P₃ fraction yielded a population of a membrane at the light end of the sucrose gradient which has a buoyant density similar to myelin but seemed to be enriched with NADPN cytochrome C reductase and phospholipid modifying enzymes. This is in contrast to liver microsomes submitted to a similar fractionation.     

ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M-sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W₁ and W₂) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac-tions of W₁ and W₂ were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was up to 2-fold higher than that of myelin in the heavier subfractions of W₁ and W₂. The major myelin-associated glycoprotein was also increased in these subfractions as determined by periodic acid-Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W₁ and W₂ subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome-depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.     

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.     

Absence of 5′-nucleotidase in porcine polymorphonuclear leucocyte membranes

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess $\text{p-}\text{nitrophenyl}$ phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5′-nucleotidase inhibitor α,β-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5′-nucleotidase. An antibody against mouse liver 5′-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5′-nucleotidase and the necessity for distinguishing between true 5′-nucleotidase and non-specific phosphatase activity is discussed.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

SUBCELLULAR FRACTIONS OF NORMAL HUMAN SUBSTANTIA NIGRA AND CAUDATE NUCLEUS; A STUDY OF THEIR MORPHOLOGY AND SOME ENZYMES INCLUDING GLUTAMATE DECARBOXYLASE AND CHOLINE ACETYLTRANSFERASE

Abstract— Subcellular fractions have been prepared from normal human caudate nucleus and substantia nigra by a standard fractionation technique and the fractions assayed for the following enzymes, which were studied because of their relevance to neurotransmission and pathological change: glutamate decarboxylase (EC 4.1.1.15), choline acetyltransferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), acid phosphatase (EC 3.1.3.2) and succinate dehydrogenase (EC 1.3.99.1). The distribution of these enzymes was assessed in relation to the morphology of the fractions as observed by electron microscopy. As with preparations from animal cerebral cortex, acetylcholinesterase and acid phosphatase were found mainly in fractions known to contain plasma membranes, synaptosomal membranes and microsomes. The levels of choline acetyltransferase in fractions from the substantia nigra were too low to measure but, in the caudate nucleus, the enzyme was concentrated in the crude mitochondrial fraction (P₂), especially in the P₂B and P₂C subfractions. A high proportion of the glutamate decarboxylase activity was present in the P₂ fractions of the substantia nigra and caudate nucleus and, although the synaptosomal (P₂B) fraction contained the enzyme, significant amounts were found in the mitochondrial (P₂C) fraction. This may have been due to some contamination of the mitochondria with small synaptosomes. Succinate dehydrogenase showed a conventional bimodal distribution between synaptosomes and mitochondria with a concentration in the latter.     

Actomyosin-like ATPase extracted from plain synaptic vesicle fractions

Ca²⁺-sensitive Mg²⁺-dependent ATP phosphohydrolase (EC 3.6.1.3, ATPase) was extracted from the plain synaptic vesicle fractions that were virtually devoid of contamination. The protein pattern of the ATPase preparation on SDS polyacrylamide gel electrophoresis closely resembled that of actomyosin from skeletal muscle. The finding suggests that the main components of the ATPase are actin- and myosin-like proteins of the brain (stenin and neurin). Microsome and synaptosomal plasmalemma fractions were extracted under the same conditions to examine the possibility that the ATPase extracted derived from contaminating particulates. An entirely different ATPase was extracted from microsomes, and no protein from plasma membranes. Although Ca²⁺-sensitive Mg²⁺-dependent ATPase was extracted from coated vesicle fraction, the electrophoretic pattern was dissimilar to that of the ATPase from plain synaptic vesicle fractions. It may be inferred that the whole complex of neurostenin is located in plain synaptic vesicles from the brain.     

Subfractionation of rat liver plasma membrane: Uneven distribution of plasma membrane-bound enzymes on the liver cell surface

Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na⁺+K⁺)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na⁺+K⁺)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Smooth muscle cell sarcolemma. Purification and properties of plasma membranes from the rat uterus

A membrane fraction with sarcolemmal properties was purified from the smooth muscle layers (myometrium) of rat uterus by successive differential and equilibrium centrifugation in sucrose. The putative sarcolemmal fraction was identified by iodination with [^125I]iodosulfanilic acid, had an equilibrium density of 1.15, and was enriched in enzyme activities usually associated with the plasma membrane including 5′-nucleotidase (EC 3.1.3.5) and (Na⁺ + K⁺) ATPase (EC 3.6.1.3). These membranes were free of mitochondrial or nuclear membrane contamination, suggesting the relative enrichment of sarcolemmal membranes in the fraction. Proteins of the membranes were heterogeneous with respect to molecular weight, but only a few were labelled when intact muscle was radioiodinated. Uniform resistance of sarcolemmal proteins to trypsin digestion and salt extraction suggested many are tightly bound or intrinsic membrane proteins and was a further indication of the homogeneity of membranes in this fraction.     

Subcellular distribution of prostaglandin and gonadotrop in receptors in bovine corpora lutea.

The subcellular distribution of prostaglandin (PG) E₁, F₂α and gonadotropin receptors in bovine corpora lutea was critically examined by preparing various subcellular fractions, assaying for various marker enzymes to assess the purity and examining ³H-PGE₁, ³H-PGF₂α and ¹²⁵I-human lutropin (hLH) specific binding. The marker enzyme data suggested that subcellular fractions were relatively pure with little or no cross contamination. The binding of ³H-PGs and ¹²⁵I-hLH was markedly enriched in plasma membranes with respect to homogenate. The other subcellular fractions also exhibited binding despite very little or no detectable 5′-nucleotidase activity. If 5′-nucleotidase was assumed to lack sensitivity and reliability to detect minor contamination with plasma membranes and ³H-PGs or ¹²⁵I-hLH binding were used as sensitive plasma membrane markers, it was still difficult to explain binding in other fractions based on plasma membrane contamination. Therefore, these results lead to the inevitable conclusion that plasma membranes were primary (or one of the primary) but not exclusive sites for PGE₁, PGF₂α and gonadotropin receptors.     

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.     

Studies on the turnover of mouse brain synaptosomal proteins

(l) The half-lives of the proteins of various fractions of whole mouse brain increase with increasing insolubility; the supernatant and hypotonic-extractable proteins had half-lives of about 13 days, whereas the membrane proteins solubilized with Triton X-100 and SLS had half-lives of about 18 days. The proteins of the subfractions of synaptosomes had half-lives ranging from 15 to 19 days; those in the cytoplasm had a half-life of 18·3 days, in the membranes, about 17 days and in the synaptic vesicles, 15·6 days. (2) Although the half-life of the synaptic vesicles was not significantly different from that of other synaptosomal subfractions, the vesicles exhibited a different protein pattern on acrylamide gels, an observation which implies that the proteins of the vesicles are qualitatively different from those of other synaptic membranes. (3) The uptake of labelled lysine into the cytoplasm of the synaptosomes of youg mice in vivo was very rapid. (4) The data derived from the relative specific radioactivities of synaptosomal fractions compared with their whole brain analogs support the contention that a sizeable fraction of the synaptosomal cytoplasmic protein was transported to the synapse by axoplasmic flow. The relative specific radioactivities of synaptosomal membrane and synaptic vesicle proteins rose much more quickly than the comparable activities for the cytoplasmic material, and the alternate possibility of synthesis in situ is discussed.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Localization of adenylate cyclase in Dictyostelium discoideum. I. Preparation and biochemical characterizations of cell fractions and isolated plasma membrane vesicles.

A difference in the organization of adenylate cyclase and 3′5′-cyclic phosphodiesterase in isolated plasma membranes was observed. Observation of this difference was made possible by the development of a new technique for the lysis of Dictyostelium discoideum using the polyene antibiotic amphotericin B. A particulate fraction prepared from the cell lysate contains adenylate cyclase, 3′5′-cyclic phosphodiesterase and 5′-nucleotidase. The yield of adenylate cyclase is 40% higher than in paniculate fractions prepared from cells lysed by sonication or with Triton X-100. Purification of the particulate fraction on discontinuous sucrose gradient completely separates membranes from mitochondria and other cellular material as shown by electron microscopic analysis of different fractions. Biochemical characterization of the purified membrane fraction shows it contains adenylate cyclase, 3′5′-cyclic phosphodiesterase and 5′-nucleotidase activities while electron microscopic analysis shows a vesicular morphology. Additional studies on the purified membranes used Triton X-100, trypsin and phospholipase C to probe the relationship between membrane structural elements and enzymatic activities. The results of these studies show distinct differences in the organization of each enzyme molecule within the membrane.