Subcellular distribution of adenosine system enzymes in the hypothalamus and hippocampus of the rat brain

The subcellular distribution of 5'-nucleotidase and adenosine deaminase in rat brain hypothalamus and hippocampus was studied. In the hippocampus the 5'-nucleotidase activity was shown to be much higher than in the hypothalamus, while the adenosine deaminase activity, contrariwise, is nearly two times as high as that in the hypothalamus. During the analysis of subcellular distribution 5'-nucleotidase and adenosine deaminase were detected in all fractions under study, i. e., in nuclear, soluble, myelin fractions as well as in synaptic membranes, synaptosomes and "pure" mitochondria. The highest 5'-nucleotidase activity was found in the myelinic and synaptic fractions both in the hypothalamus and in the hippocampus. The highest adenosine deaminase activity was detected in the soluble fraction of the above structures. The enzyme activity in synaptic membranes and synaptosomes was nearly two times as low.     

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.     

Subcellular changes in the cortex of the motor analyzer and hippocampus of dogs with locally manifested neuroses

In experiments on dogs with local neurosis-continuous flexion of the foreleg-changes were revealed in the beta-rhythm amplitude and the frequency of mean unit activity in the motor cortex, and the appearance and increased amplitude of the theta-rhythm in the hippocampus. Specific activity of Na+-K+-activated, and Mg2+-dependent ATPase decreases in subcortical fractions of the experimental animals' cerebral cortex by 55.0% in the synaptic membranes and 2 to 2.5 times in light and heavy synaptosomes, respectively. In similar fractions of the dorsal hippocampus, the activity of the enzyme decreases by 30.0% in the synaptic membranes and increases by 16.6% in the light synaptosomes and by 6.6% in the heavy ones.     

Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain

We have made a monoclonal antibody which specifically recognizes smg p25A among many ras p21/ras p21-like GTP-binding proteins thus far purified from bovine brain membranes. By use of this antibody, we have investigated the localization and subcellular distribution of smg p25A in rat brain by light and electron microscopic immunocytochemistry and by immunoblotting. By light microscopic immunocytochemistry, specific immunoreactivity is widely distributed, most abundant in neuropil, weak in neuronal somata, and absent from white matter. By electron microscopic immunocytochemistry, intense labeling is demonstrated on most of the synapses and concentrated in the presynaptic area where synaptic vesicles are observed. Presynaptic plasma membranes are weakly labeled but mitochondria, postsynaptic plasma membranes, and postsynaptic densities are unlabeled. In subcellular fractionation analysis of cerebrum, about one-fifth of smg p25A is found in the soluble cytosol fraction and the rest is found in the particulate fraction. About half of the particulate-bound smg p25A is recovered in the P2 fraction containing synaptosomes, mitochondria, and myelin, among which a major portion of smg p25A is recovered in the synaptosomal fraction. In the synaptosomal fraction, smg p25A is concentrated about 8-fold in the fraction containing synaptic vesicles and about 3-fold in the fraction containing synaptic plasma membranes compared with the original homogenate. smg p25A is present at a low level in the fraction containing synaptosomal soluble substances but almost absent from the fractions containing intrasynaptosomal mitochondria or post-synaptic densities. These results suggest that smg p25A plays important roles in the regulation of synaptic functions such as exo-endocytotic recycling of synaptic vesicles during neurotransmitter release.     

THE SUBCELLULAR DISTRIBUTION OF CARBONIC ANHYDRASE IN HOMOGENATES OF PERFUSED RAT BRAIN

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.
Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.
Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten-fold increase in the measurable carbonic anhydrase activity of myelin fragments.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

大鼠大脑皮层中钙调神经磷酸酶活力的时空变化

以ＰＮＰＰ为底物测定了超离心制备的大鼠出生后早期和成年大脑皮层亚细胞各组分中钙调神经磷酸酶的活力。实验结果表明：（ｌ）钙调神经磷酸酶活力广泛地存在于胞液和突触部分,并且各亚细胞组分有明显差异。成年大鼠大脑皮层中ＣａＮ活力相对最高水平是在突触体,突触质,胞液,重的和轻的突触膜部分。（２）大鼠大脑皮层突触体中ＣａＮ活力在出生后第２周和第３周出现高峰的平台期,这与突触发生的高峰期是一致的。在胞液和重的突触膜中ＣａＮ活力最高水平是在出生后的第７ｄ,而在突触质和轻的突触膜中是在第２０ｄ。总之,这些发现证实,在脑发育期间,ＣａＮ活力是依照区域和时间性控制的,提示ＣａＮ可能参与了突触功能作用。     

Autophosphorylation of chick brain synaptic polypeptides. Phosphorylation of proteins during incubation of intact synaptosomes with 32PO4

Chick brain synaptosomes incorporated phosphate into proteins when incubated in physiological buffer containing energy sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that three synaptosomal polypeptides were significantly phosphorylated after 15 sec incubation while at least fifteen polypeptides were active kinase substrates after 15 min incubation. Labeled synaptosomes were hypotonically lysed and separated by centrifugation into soluble, membrane, and mitochondrial fractions. Every fraction exhibited significant phosphate incorporation. Electrophoresis revealed that each fraction had several unique phosphorylated polypeptides and a distinctive phosphorylation pattern. The same polypeptides appear to be labeled whether MgATP was added to synaptic plasma membranes or synaptic plasma membranes were isolated after synaptosomal autophosphorylation.     

GANGLIOSIDE COMPOSITION AND CONTENT OF RAT-BRAIN SUBCELLULAR FRACTIONS

Abstract— The composition and content of gangliosides from rat-brain microsomal, synaptosomal, mitochondrial and myelin fractions were studied. Outer membranes of synaptosomes were also isolated, separated into subfractions and investigated. Of all the fractions studied the outer membranes of synaptosomes are richest in gangliosides, in one of their sub-fractions the concentration of gangliosides per mg of protein is five times higher than in the homogenate. Microsomes are rich in gangliosides as well, but to a lesser degree, whereas the mitochondrial fraction contains considerably smaller amounts of gangliosides per mg of protein than does the homogenate. The ganglioside pattern of outer membranes of synaptosomes and of their subfractions is somewhat different from that of the homogenate; the outer membranes contain approximately one-third less monosialogangliosides. On the contrary a very high content of monosialogangliosides is characteristic of the ganglioside pattern of the myelin fraction. In this fraction monosialoganglioside G_MI (nomenclature of Svennerholm, 1963) constitutes 60–63 per cent of ganglioside sialic acid, or 75–80 molar per cent of gangliosides, the content of di- and trisialogangliosides being much lower than in other fractions. Fatty acid and long chain base composition of gangliosides from synaptosomal and microsomal fractions and homogenate is very similar, almost identical. In gangliosides from myelin fractions the relaitve content of palmitic and monoenoic acids is higher and that of arachinic acid and C₂₀-sphingosine—lower than in other fractions studied. The difference in ganglioside composition of synaptosomes and their outer membranes and on the other hand of myelin appears to reflect the difference in ganglioside composition of neuronal and oligodendroglial plasma membranes.     

Identification and Topography of Substrates for Protein Carboxyl Methyltransferase in Synaptic Membrane and Myelin-Enriched Fractions of Bovine and Rat Brain

The major components of crude brain synaptosomes (synaptic membranes, mitochondria, and myelin) have been separated and analyzed by polyacrylamide gel electrophoresis for the presence of proteins that serve as substrates for protein carboxyl methyltransferase. Of the three fractions, synaptic membranes contain the largest number of individual methyl acceptors (at least seven), while mitochondria contain no well-defined methyl acceptors. Undisrupted myelin contains a single major methyl acceptor with a very low apparent molecular weight. The patterns of protein methylation in synaptic membranes prepared from cerebral cortex, hippocampus, striatum, thalamus, and tectum showed marked differences; however, these differences could largely be explained by differential degrees of myelin contamination in synaptic membranes from the different regions. The effect of trypsin pretreatment on the carboxyl methylation of intact and lysed synaptosomes was studied to estimate the sidedness of the major methylation sites on synaptic membranes. One of the methyl acceptors (Mr 48K) appears to be facing the intracellular surface of the synaptosome, but most sites appear to be outward facing.     

Mature and immature synaptosomal membranes have a different lipid composition

Subfractionation of the optic tectum in chick embryos results in the isolation of two fractions enriched in synaptosomes (fraction A and fraction B). In chicks after hatching, this fractionation results in the isolation of a single synaptosomal fraction (fraction B) and of a fraction enriched in myelin membranes devoid of synaptosomes (fraction A). The lipid composition of synaptosomal fractions (A and B) and corresponding synaptosomal plasma membranes has been analyzed and compared to the lipid composition of similar fractions isolated from 2–3 day-old chicks. The phospholipid composition of fraction A in embryos was mainly represented by phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PE content was significantly lower than that of PC, which accounted for by approximately 50%. Sphingomyelin (SP) and phosphatidylinositol (PI) accounted for by only 6% of the total membrane phopsholipids. Fraction A isolated from the young chicks showed many significant changes. PC accounted for by approximately 40% and PE made up 35%. The amount of phosphatidylserine (PS) and SP increased. These data parallel our previous morphological observations, which showed that fraction A contains immature synaptosomes in embryos but myelin membranes and no synaptosomes in the young chicks. Fraction B has been shown to contain synaptosomes at all stages considered. It possessed in embryos a lipid composition similar to fraction A, except that PC content was higher in young embryos. The analyses on membrane fractions confirmed these results. On the contrary, this fraction showed many significant changes after hatching. The content of PC was significantly reduced, PE/PC ratio was significantly increased as well as ethanolamine plasmalogen (PLE) content. The percentage of PS, PI and SP were increased. The composition of fatty acids of the total fraction of phospholipids was also examined. The results parallel the observations on phospholipid classes.     

Comparison of cholinergic systems in Ep neocortical regions of cats with high and low cognitive ability

Cats were behaviorally tested for the ability to solve the abstraction and generalization tasks. Fractions of light (C) and heavy (D) synaptosomes of the associative temporal (Ep) areas were prepared, and subfractions of synaptic membranes and synaptoplasm were isolated. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity and the content of protein and protein sulphydric (SH-) groups were measured in synaptic subfractions. All the studied characteristics were lower in subfractions C of cats with higher cognitive abilities. In subfractions D, the ChAT activity was correlated neither with ChAT activity in the respective C fraction, nor with cognitive abilities of cats. It is suggested that cholinergic terminals originating from neurons of the basal magnocellular nuclei are concentrated in the C fractions, and those from the cortical cholinergic neurons are concentrated in the D fractions. Physiological significance of the "deficiency" of cholinergic inputs of the Ep areas from the basal magnocellular nuclei in animals with higher cognitive abilities is discussed.     

Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging

The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed.     

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.     

Alterations in brain lipid composition of mice made physically dependent to ethanol

The ability of chronic ethanol treatment to alter CNS membrane lipids was tested. Adult male C57/BL6 mice were given a liquid diet containing ethanol for eight days. This regimen produced strong physical dependence as judged by withdrawal seizures, tremors and concomitant hypothermia. Analyses were performed on cholesterol, total phospholipid content and total phospholipid acyl composition of myelin, crude (P2), light and heavy synaptosomes as well as synaptosomal plasma membranes. Chronic ethanol treatment had no effect on total phospholipid levels nor phospholipid acyl composition in any of the above subcellular fractions. In ethanol dependent mice, significant increases in cholesterol content and cholesterol/ phospholipid ratios were observed only in synaptosomal plasma membranes.     

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in cat peripheral nerve

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in desheathed, saline perfused cat sciatic nerve is reported. CNPase specific activity was enriched in the total particulate (P₂) fraction and was low in the soluble (S₂) fraction. “Light-myelin” floating above the 0.60 M sucrose phase had the highest CNPase activity, 2.5-fold over the crude homogenate (CH). By contrast, enzyme activity in “heavy myelin” floating above the 0.85 M sucrose interface was equal to that of the CH and accounted for only 12% of total activity. CNPase activity in the membranes floating above the 0.25 and 0.60 and 0.85 M sucrose phases comprised nearly 70% of the total CNPase activity. The “light myelin” fraction floating above the 0.60 M sucrose accounted for approx. 51% of the total CNPase activity. SDS-PAGE of membranes individually harvested from above the 0.25 and 0.60 and 0.85 M sucrose phases revealed the presence of myelin-specific proteins (P₀, P₁; and P₂). Electron microscope examination demonstrated the presence of myelin in each membrane fraction. The results of this study show that the majority of CNPase activity is associated with “light myelin” in cat peripheral nerve.     

Variation of proteins, enzyme markers and gangliosides in myelin subfractions

A discontinuous sucrose gradient was used to separate adult rat brain myelin into light, medium and heavy subfractions. Basic proteins decreased sharply, proteolipid potein changed very little, and high molecular weight proteins increased from the light to the heavy fraction. The concentration of monosialoganglioside G_M1 was the highest in the middle fraction. The amount of carbohydrate in the major myelin-associated glycoprotein per mg total myelin protein increased 3.5-fold from the light to the heavy fraction. 2′,3′-Cyclic nucleotide 3′-phosphohydrolase, which is related to myelin or the oligodendroglial membrane, and acetylcholinesterase, which is in neural membranes such as the axolemma, both increased between the light and the heavy fraction, although their relative distributions among the three fractions were different. The glycoprotein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase had similar distributions suggesting that they were concentrated in similar locations, possibly in the loose myelin and oligodendroglial plasma membrane. Electron microscopic examination of the subfractions was consistent with this interpretation.     

硒、谷胱甘肽过氧化物酶和不饱和脂肪酸在鼠脑亚细胞中的分布

采用梯度离心和放射性同位素等方法从鼠脑中分离得到髓磷脂、突触囊、轻突触体、重突触体、线粒体６个亚细胞组分。分别测定了各亚细胞中硒－７５、谷胱甘肽过氧化物酶和不饱和脂肪酸的含量,结果表明这些成分在鼠脑亚细胞中的分布呈现明显的相关性,同时首次在突触囊、线粒体和微粒体中检测到三种不同的谷胱甘肽过氧化物酶的活性峰,其中之一可能是红细胞谷胱甘肽过氧化物酶（ＥＣ１．１１．１．９）．还就机体的自我保护机制和硒在脑组织中的重要作用进行了讨论。     

The content and composition of gangliosides in brain subcellular fractions of the frog Rana temporaria

Ganglioside distribution in various frog brain subcellular fractions (myelin, microsomes, mitochondria, synaptosomes, plasma membranes of nerve endings and synaptic vesicles) was investigated. The synaptosomes and plasma membranes of nerve endings were found to be the main places of ganglioside localization, ganglioside concentration being 2.42 and 1.79 times higher than that in homogenates. Gangliosides were shown to be present in synaptic vesicles. The characteristic features of gangliosides from frog brain and its subcellular fractions are the predominance of polysialogangliosides with 3-5 sialic acid residues (up to 57.4%), low content of monosialogangliosides (not more than 7%) and the presence of disialogangliosides with short carbohydrate chain. The increase of ganglioside content per one nerve cell during phylogenetic development of vertebrates is discussed.     

Ratio and composition of the plasmalogen and diacylated forms of phospholipids in subcellular fractions of the avian brain

Studies have been made on the specific content of plasmalogen and diacylated forms of phosphatidylethanolamine and phosphatidylcholine in subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) from the brain of pigeons, as well as in the myelin fraction from the brain of the crow Corvus cornix and the hawk Accipiter gentelis. Fatty acid composition and fatty aldehyde composition of these two main phospholipids of the brain were studied in the subcellular fractions obtained. It was shown that plasmalogen forms of phospholipids are localized in birds mainly in the myelin fraction which exhibits the highest plasmalogen concentration as compared to the same fraction of all the vertebrates investigated. With respect to fatty acid and fatty aldehyde composition, as well as to the degree of their unsaturation, myelin plasmalogens from birds are similar to those from other cold-blooded and warm-blooded animals. This fact indicates that high relative content of plasmalogens together with their high unsaturation account for normal functional activity of myelin membranes in all vertebrates.