Phospholipids and acyl groups in subcellular fractions from human cerebral cortex

Subcellular fractionation of human brain cortex obtained at autopsy yielded microsomal and synaptosome-rich fractions from the gray matter and microsomal and purified myelin fractions from the white matter. The phospholipids of myelin were high in plasmalogens, and the molar ratio of alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 4. The acyl groups of the myelin phosphoglycerides were enriched in monoenes (mainly 18:1 and 20:1) and a tetraene, 22:4(n - 6). The phospholipids in the synaptosome-rich fraction were high in diacyl sn-glycero-3-phosphorylcholine, and the molar ratio of the alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 0.88. The acyl groups of synaptosomal ethanolamine phosphoglycerides were rich in 22:6(n - 3) but contained a very low amount of 20:1. The lipid composition of microsomes from the gray matter was different from that of microsomes from the white matter but was nearly identical with that of the synaptosome-rich fraction. Except for a slightly lower proportion of alkenyl acyl sn-glycero-3-phosphorylethanolamine and sphingomyelin, the lipid composition of microsomes from the white matter was also similar to that of the myelin. There were also species-related differences between the brain lipid composition of human and subhuman primates and that of the rodents. Furthermore, the brain lipid composition in normal human subjects is rather constant and does not seem to be affected much by individual variations.     

Aminoacyl-tRNA-synthetase activity of subcellular fractions of cerebral cortex]

The total activity of aminoacyl-tRNA-synthetases of myelin, synaptic membranes, heavy and light synaptosomes, mitochondria and soluble fractions of rat cerebral cortex was studied. It was found that the highest activity of the enzymes is localized in the fractions of synaptic membranes and heavy and light synaptosomes and is practically absent in the myelin fraction. The specific activity of the total aminoacyl-tRNA-synthetase fraction in the soluble fraction is 2 times as low as compared to the synaptic membranes and light and heavy synaptosomes.     

Pre-incubation of subcellular fractions from rat cerebral cortex inactivates protein phosphorylation.

Pre-incubation of various subcellular fractions from rat brain caused a significant decrease in the phosphorylation of individual polypeptides. The rate and extent of this loss of labelling was not uniform and polypeptides whose phosphorylation was independent of cyclic AMP were primarily affected, whereas substrate availability remained unaltered. It is recommended that pre-incubation effects must be carefully monitored if valid conclusions are to be made about the physiological relevance of changes in protein phosphorylation observed in vitro.     

Distribution of endogenously phosphorylated proteins in subcellular fractions of rat cerebral cortex

The cerebral cortex from adult rats was separated into several subcellular fractions by using established methods of differential and sucrose density gradient centrifugation. Aliquots from each fraction were incubated with -³²P-ATP, in the presence and absence of adenosine 3,5-monophosphate (cyclic AMP), and its protein constituents were separated by means of SDS-slab gel electrophoresis. Fractions containing nuclei, synaptosomes, myelin, microsomes, and soluble proteins each showed a characteristic pattern of protein staining and of endogenously phosphorylated proteins detected by autoradiography of the gels. Cyclic AMP-stimulated phosphorylation of proteins with MW 78K and 84K can serve as markers for membranes of synaptic origin, while cyclic AMP-independent phosphorylation of low-molecular-weight proteins (15K–20K) is characteristic of myelin. The finding of different phosphoproteins in various subcellular fractions may be related to the diversity of cellular functions known to be regulated by phosphorylative activity.     

Activity and subcellular distribution of phospholipase A1 from neuronal cell-enriched fractions of the rabbit cerebral cortex

Glycerophosphatides, specifically labeled either in the 1 or in the 2 position, were used to measure the activity of neuronal phospholipase A₁ and to investigate the subcellular distribution of the enzyme. The microsomes were found to possess the highest phospholipase activity, with a threefold increase as compared to the cell homogenate. A considerable enzymatic activity could still be observed in the plasma membranes isolated from the neuronal-enriched cell fraction. Microsomal phospholipase possessed the highest activity with phosphatidylcholine, whereas phosphatidylserine was cleaved at a much lower rate. The rate of release of labeled fatty acids from the substrates by the microsomal phospholipase decreased with increasing degree of unsaturation of the fatty acids at the 1 position. The presence of plasmalogens and of alkylacyl analogues in the incubation mixture caused an appreciable inhibition of the hydrolysis of the diacyl glycerophosphatides.     

The lipid composition of ovine placental tissue homogenate and its subcellular fractions

Homogenates of the placental tissue of near term sheep were separated by differential centrifugation into mitochondrial, microsomal and cytosolic fractions. The relative proportions of the major neutral lipids and phospholipids, together with their fatty acid compositions, were determined in the homogenates and in each subcellular fraction. The cytosolic fraction contained the highest proportion of cholesteryl esters (CEs) and these possessed a fatty acid composition markedly different from the total CEs extracted from the homogenate. Both the mitochondrial and microsomal fractions contained significant proportions of solvent front phospholipid (SFP) and whereas the mitochondrial SFP displayed the relatively unsaturated fatty acid composition characteristic of diphosphatidylglycerol (cardiolipin), the fatty acids of the microsomal SFP were distinctly more saturated. These results are compared with those obtained from other mammalian tissues, both ruminant and non-ruminant, and discussed in terms of the function of the components of the subcellular fractions.     

Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of guinea-pig cerebral cortex in vitro

The addition of 10(-5) M to 10(-3) M neomycin to incubations of subcellular fractions of guineapig cerebral cortex increased the labelling of phosphatidylinositol phosphate and decreased the labelling of phosphatidylinositol diphosphate by [gamma-32P]ATP. The effect was observed in all subcellular fractions tested and depended on the cationic form of the antibiotic. Similar effects on lipid labelling were exerted by related aminoglycosidic antibiotics, by neamine, spermine and poly-L-lysine. Other neomycin fragments, antibiotics, local anesthetics or small polyamines were ineffective. Neomycin also inhibited the enzymatic hydrolysis of 32P-polyphosphoinositides. The addition of the drug to aqueous dispersions of these lipids increased the turbidity and lowered the pH of the suspensions. It is suggested that the effects of neomycin on polyphosphoinositide metabolism result from the formation of an ionic complex between the lipids and the antibiotic.     

Phosphatidylinositol synthetase activities in neuronal nuclei and microsomal fractions isolated from immature rabbit cerebral cortex

The synthesis of phosphatidylinositol was studied using a nuclear fraction N1, a microsomal fraction P3, rough (R) and smooth (S) microsomal fractions and a microsomal fraction P derived from isolated nerve cell bodies. Each fraction was prepared using cerebral cortices of 15-day-old rabbits. In assays using CDP-diacylglycerol (prepared from egg phosphatidylcholine) and myo[3H]inositol at pH 7.4, fraction N1 had the highest maximal specific rates of phosphatidylinositol synthetase (EC 2.7.8.11) (expressed per mumol phospholipid in the fraction). However the three microsomal fractions achieved maximal specific activities at liponucleotide concentrations close to 50 microM, while fraction N1 required 200 microM concentrations. In certain cases (25-120 microM CDP-diacylglycerol, and at higher pH values) fraction R had specific activities which equalled or surpassed those of N1. However, with respect to inositol, fraction N1 had a distinctly lower Km than was shown for fractions R or P3. Each of the microsomal fractions and N1 required Mg2+ for the reaction, but for N1, maximal rates could be sustained at 0.1 mM, while for the microsomal fractions the optimal Mg2+ concentration was 1 mM. For each fraction Mn2+ could not replace Mg2+ in the reaction and Mn2+ was inhibitory. The optimal pH for the reaction was between 8.0 and 9.0. Phosphatidylinositol synthetase could also be shown using fraction N1 enriched in endogenous CDP-diacylglycerol. The relatively high specific activities of fraction N1, and the differences found between N1 and the microsomal fractions, for optimal CDP-diacylglycerol and Mg2+ concentrations and for Km values for inositol, support the existence of a neuronal nuclear phosphatidylinositol synthetase.     

The association of calmodulin with subcellular fractions isolated from rat liver

Calmodulin associated with rat liver mitochondria has been found to belong to a contaminant membranous fraction which contains different subcellular membranes. The concentration of calmodulin in this fraction is relatively high, about 1.6 micrograms/mg protein, and can not be decreased with EGTA. The calmodulin-rich membranous fraction seems to contain cytoskeletal proteins which could be responsible for the binding of calmodulin.     

Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells

1. A procedure has been developed for the separation of intact metabolically active neuronal and glial cells in bulk from rat cerebral cortex. Separation depended on dispersion of the tissue in a Ficoll medium followed by centrifugation on a discontinuous Ficoll gradient. Up to 1.5x10(7) neuronal cells could be collected from 12 brains within 3hr. The morphological appearance of these cells seemed good, and the fraction was 8.5-fold purified in terms of dry weight. Average dry weight per neuron was 2300mumug. Maximum glial contamination of the neuronal fraction was 11% as determined by carbonic anhydrase measurements. The glial fraction was free from neurons but contained various subcellular contaminants. 2. Concentrations of nucleic acids, phospholipid, protein and phosphoprotein were determined in the separated fractions. The neuronal fraction was richer than the glial in all except phospholipid. Succinate dehydrogenase was equally distributed between neurons and glia but the neuronal fraction was 1.8-fold enriched in cytochrome oxidase. 3. Measurement of respiration by the cells showed an endogenous uptake of 117mmumoles of oxygen/mg./hr. in neurons, and 173mmumoles of oxygen/mg./hr. in glia. Addition of substrate at 10mm stimulated uptake to similar values in both fractions. With glucose it was 390, with pyruvate 355, and with glutamate 215mmumoles of oxygen/mg./hr. This represented a larger stimulation of neuronal than of glial respiration compared with the basal level. 4. Respiration in cell suspensions was 70-80% of that of slices, whereas fractionated tissue homogenates had respiratory rates of only one-third those of the cell suspensions. Lactate dehydrogenase content of cell suspensions was maintained during gradient centrifugation and washing. 5. The possible uses of isolated cell preparations are discussed.