SUBCELLULAR FRACTIONATION OF GANGLIOSIDE SIALIDASE FROM HUMAN BRAIN

—A subcellular fractionation was performed on forebrain cortex from three human brains and the fractions obtained were assayed for ganglioside sialidase and four p-nitrophenyl glycohydrolases. Differences in the sedimentation patterns of the enzymes were observed. From 53 to 77 per cent of the recovered sialidase activity was found in the synaptosomal fraction, while the p-nitrophenyl glycosidases were mainly recovered in the lysosome-enriched fraction. Three possible interpretations of the sialidase sedimentation pattern are suggested: (1) The ganglioside sialidase is bound to the limiting membrane structure of the nerve ending. (2) The ganglioside sialidase is lysosomal, although bound to lysosomes of low density. (3) The enzyme occurs mainly in lysosomes primarily located in the nerve endings, being trapped under the formation of the synaptosomes.     

ACYL GROUP COMPOSITION OF METABOLICALLY ACTIVE LIPIDS IN BRAIN: VARIANCES AMONG SUBCELLULAR FRACTIONS AND DURING POSTNATAL DEVELOPMENT

Using a combination of preparative TLC and GLC technique, the content and acyl group composition of diacyl-glycerophosphoinositols, diacyl-glycerophosphates, diacylglycerols and triacyl-glycerols in brain tissue were determined. The level of diacyl-glycerophosphoinositols in 40 day-old mouse brain was 2.7 μmol/g tissue as compared to 40–170 nmol/g for other minor lipids. The acyl groups of diacyl-glycerophosphoinositols were enriched in 18:0 and 20:4 (n-6). This characteristic acyl group profile was found in microsomes, synaptosomes, and in myelin. The acyl groups of diacyl-glycerophosphates and diacylglycerols were comprised mainly of 16:0, 18:0, 18:1 and 20:4 (n-6). In rat brain subcellular fractions, the acyl groups of diacylglycerols and diacyl-glycerophosphates in the microsomal fraction had a higher proportion of 22:6 (n-3) than those in the myelin and synaptosomal fractions. The acyl groups of the myelin lipids were higher in 18:l and lower in 20:4 (n-6) as compared to those in the microsomal and synaptosomal fractions. The triacylglycerols in brain exhibited an unusual acyl group profile which included small proportions of 14:0, 16:1, 20:4 (n-6), 22:4 (n-6) and 22:6 (n-3). Except for an increase in 18:1 and a corresponding decrease in 16:0 which was found in diacyl-glycerophosphoinositols, no apparent acyl group change was observed in other metabolically active lipids during postnatal brain development.     

FRACTIONATION OF RNA FROM BRAIN SYNAPTOSOMES AND CYTOPLASMIC SUBCELLULAR FRACTIONS

—RNA from rat brain synaptosomes, mitochondria and microsomes was analysed by gel electrophoresis under conditions allowing good resolution in three different molecular weight ranges: 4s-16s, 16s-28s and >28s. Two synaptosome specific RNA bands were found, one with comparatively low molecular weight (8-9 × 10⁴ Daltons) and another very large (s_E > 60s). RNA species with electrophoretic characteristics similar to those reported for liver mitochondrial RNA were found in brain mitochondria. From the electrophoretic data their mean geometric radii were determined.     

THE RELATIONSHIP BETWEEN ACETYLCHOLINE RELEASE FROM BRAIN SLICES AND THE ACETYLCHOLINE CONTENT OF SUBCELLULAR FRACTIONS PREPARED FROM BRAIN

Abstract— Acetylcholine (ACh) release from sliced cerebral cortex of rats was measured when the tissue was incubated in a high K⁺ (46 m m ) medium containing eserine. In the absence of hemicholinium (HC-3), ACh release was well maintained, but in the presence of HC-3, ACh release declined within 15–20 min. Subcellular fractions representing nerve-ending free (cytoplasmic) ACh and nerve-ending bound ACh were prepared from slices that had been stimulated to release ACh in the presence of HC-3. Both nerve-ending stores of ACh were depleted when their content was compared to tissue that had not been stimulated and there was no demonstrable difference in the rate of depletion of either of the two fractions. Stimulating slices with K⁺ in the absence of HC-3 also depleted cytoplasmic and vesicle-bound ACh. It is concluded that, under these experimental conditions, both nerve ending stores of ACh are available for release and that, in the absence of HC-3, ACh synthesis can maintain ACh release, but cannot maintain tissue ACh content.     

CALCIUM METABOLISM IN ISOLATED BRAIN CELLS AND SUBCELLULAR FRACTIONS

Abstract— The accumulation of calcium ions by brain mitochondria and microsomes and by fractions containing neuronal or glial cells has been studied in vitro with techniques involving ⁴⁵Ca and ultramicro-flame photometry. ATP and substrate-supported calcium accumulation by brain mitochondria was of the same magnitude as for mitochondria from other organs. Brain microsomes accumulated calcium approximately 15 times less than brain mitochondria. Variations in Na⁺/K⁺ ratios and in ATP/ADP ratios had a more marked influence on microsomal uptake than on mitochondrial uptake. The passive Ca²⁺ binding by glial cells was higher than neuronal perikarya and synaptosomes. Also the calcium accumulation ability in cell suspensions was slightly higher for glial cells as compared to neuronal perikarya. The calcium uptake by glial cells was stimulated by high external K⁺ concentration, which also was the case for nerve endings. The uptake in neuronal perikarya was unaffected by variations in K⁺ concentration. A comparison between neuronal and glial mitochondria showed that both reach a steady state level of similar magnitude, but that the rate of initial accumulation was greater for glial mitochondria. A high glial calcium accumulation was also observed for the microsomal fraction.     

TRIPHOSPHOINOSITIDE PHOSPHODIESTERASE IN DEVELOPING BRAIN OF THE RAT AND IN SUBCELLULAR FRACTIONS OF BRAIN

Abstract— An assay system for the measurement of triphosphoinositide phosphodiesterase in homogenates of rat brain is described. With triphosphoinositide (TPI) as substrate, and in the presence of 0·1 m -KCI and saturating amounts of diethyl ether, the activity of phosphodiesterase in myelinated brain was 400–500 μmoles of TPI hydrolysed per g wet wt. per hr. One quarter of the adult level of the enzyme was present in rat brain one day after birth, with the remainder being added prior to and during the early stages of myelination. On subfractionation of brain homogenates, substantial activity of the enzyme was located in the soluble portion and in the paniculate fractions enriched in myelin and synaptosomes. The enzyme associated with the particulate fractions could not be detached from the membranes by any of several methods employed. There was a rough correlation between distribution of phosphodiesterase and that of 5'-nucleotidase, an enzyme associated with plasma membrane in a number of tissues. Some implications of the results are discussed.     

GANGLIOSIDE COMPOSITION OF CHEMICALLY INDUCED RAT NEURAL TUMORS AND CHARACTERIZATION OF HEMATOSIDE FROM NEURINOMAS

Abstract– Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20mg/kg body wt, in the tail vein of the mother. The ganglioside content and pattern in these tumors and the normal tissues from which the tumors originated are described. The ganglioside content in tumors was reduced, on wet tissue weight basis, compared to normal control. However, there was no significant difference of ganglioside content on dry weight or protein basis. Altered ganglioside composition was found in most of the neural tumors. In central nervous system tumors, there was some increase in GM₃ and GT_1b′ (nomenclature according to Svennerholm , 1963), a marked decrease in GM₁ and some decrease in GD_1a, but no apparent loss in GD_1b. Extreme simplification of ganglioside pattern was seen in tumors originated from peripheral nervous system. Large accumulation of GM₃ with concomitant loss of all the higher gangliosides was seen. GM₃ from neurinomas as well as from normal gray matter was isolated and characterized. GM₃ from neurinomas separated into two bands on thin layer chromatographic plates. Both these GM₃ bands had identical sphingosine and carbohydrate composition but differed in their fatty acid composition. The fast moving band had 77% of the total fatty acids as C_20:0 or longer chain while the slow moving band had only 22% of the long chain fatty acids. Normal gray matter GM₃ had one major band containing 82% of and only 17% of the fatty acids as C_20:0 or higher. It is suggested that in the tumor cells either the specificity of the enzyme cytidine monophosphate-N-acetyl neuraminic acid: ceramide dihexoside sialyltransferase for C_18.0 fatty acid containing glycolipid was altered or that the compartmentation of precursor pools for the simpler glycolipids present in normal tissue did not exist in transformed cells.     

COMPARISON OF THE FATTY ACIDS OF LIPIDS OF SUBCELLULAR BRAIN FRACTIONS

Abstract— Rat brain grey and white matter were fractionated to yield myelin, nerve terminal, synaptic vesicle, nerve terminal 'ghost', and microsomal fractions of white and grey matter. Ester-type glycolipids were found in all fractions except myelin, while cerebrosides occurred in significant concentrations only in myelin and white microsomes. Comparison of the fatty acid profile of the ethanolamine- and serine-containing phospholipids showed marked differences between myelin and the particles from grey matter, while the microsomes of white matter were of intermediate composition. Docosahexaenoic acid, a minor acid in myelin, was a major fatty acid in microsomes of grey and white matter. The fatty acid composition of sphingomyelin was distinctly different in the fractions derived from grey and white matter, clustering about stearate and nervonate in the latter, but only about stearate in the grey. Marked differences in the positional distribution of fatty acids were seen within phosphatidyl choline from myelin and nerve terminals. Ribonucleic acid was found in nerve terminal and synaptic vesicle fractions. The sphingosine found in the ganglioside from microsomes of both grey and white matter was similar with respect to distribution of the C₁₈ and C₂₀ homologues.
The possibility is discussed that microsomes furnish characteristic lipids for the synthesis or renewal of specific membranes, and that these lipids are accumulated somewhat before being released.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

METABOLISM OF ARACHIDONOYL PHOSPHOGLYCERIDES IN MOUSE BRAIN SUBCELLULAR FRACTIONS

Abstract— Mouse brain subcellular fractions were prepared at 1, 12, and 24 h and 3 and 8 days after intracerebral injections of [1-¹⁴C]arachidonate. Initially, radioactivity was mainly distributed in the microsomal and synaptosomal fractions, but the proportion of radioactivity in the myelin increased from 5 to 16% within 8 days. Radioactivity of the microsomal lipids started to decline at 1 h after injection, and the decay was represented by two pools with half-lives of 19 h and 10 days, respectively. Radioactivity in the synaptosomal and myelin fractions did not reach a maximum until 24 h after injections. The half-life for turnover of synaptosomal lipids was 9 days.
The decline of radioactivity measured in the microsomal fraction was due mainly to diacyl-GPC and diacyl-GPI, since radioactivity of other phosphoglycerides (diacyl-GPS, diacyl-GPE and alkenyl-acyl-GPE) continued to increase for 12-24 h. In this fraction, half-lives of 10-14 h were obtained for the fast turnover pools of diacyl-GPC and diacyl-GPI, and slow turnover pools with half-lives of 7 days for diacyl-GPI and 10-14 days for other phosphoglycerides were also present. Among the synaptosomal phosphoglycerides, radioactivity of diacyl-GPI declined in a biphasic mode, thus exhibiting half-lives of 5 h and 5 days. Incorporation of labelled arachidonate into diacyl-GPE and diacyl-GPS in the synaptosomal fractions was observed for a period of 24 h. The half-lives for these phosphoglycerides ranged from 8 to 12 days. Results of the study have demonstrated the presence of small pools of arachidonoyl-GPI in synaptosomal and microsomal fractions which were metabolically more active than other arachidonoyl containing phosphoglycerides.     

EFFECT OF DELTA-9-TETRAHYDROCANNABINOL ON GANGLIOSIDES AND SIALOGLYCOPROTEINS IN SUBCELLULAR FRACTIONS OF RAT BRAIN

—The present paper reports the result of studies undertaken to determine the effects of the in vivo administration of Δ⁹-THC on the ganglioside and sialoglycoprotein contents of rat brain subcellular fractions. Results indicate that the administration of the drug under both acute and chronic conditions brings about characteristic changes in the sialoglycoproteins and ganglioside content in all the subcellular fractions. Both sialoglycoproteins and ganglioside contents were markedly increased in microsomal and synaptosomal fractions and decreased in the mitochondrial fractions although the increase in the synaptosomal fractions has been found to be most striking. After chronic treatment, both ganglioside and sialoglycoprotein content did not change substantially in all the fractions except for a small increase in case of synaptosomal fractions.     

GLYCOPROTEINS AND GLYCOLIPIDS OF SUBCELLULAR FRACTIONS FROM BOVINE RETINA. INCORPORATION OF MANNOSE AND SIALIC ACID1

Abstract— Endogenous lipids and proteins of bovine retina subcellular fractions were labelled from CMP-[³H]NeuNAc and GDP-[¹⁴C]mannose. The bulk of NeuNAc and mannose transfer activity was in membranes other than those from the rod outer segment (ROS). Lighter and heavier membranes, obtained from ROS free membranes by density gradient centrifugation, were the most active for the incorporation of NeuNAc and mannose, respectively. NeuNAc bound to a lipid indistinguishable from gangliosides, and a lipid that contains mannose (mannolipid-I) were found in the fraction extractable with chloroform-methanol (2:1, v/v). Mannose was also incorporated into a lipid fraction extractable with chloroform-methanol-water (1:1:0.3, by vol) (mannolipid-II). Mannolipid-I and mannolipid-II were labile to mild acid hydrolysis. In the presence of ROS free membranes, radioactivity of mannoli-pid-I was transferred to mannolipid-II and from this to proteins. Analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the proteins labelled from GDP-mannose migrated as a broad peak covering the range of molecular weights 20,000–30,000 and including the zone of rhodopsin migration. The proteins labelled from CMP-NeuNAc showed four radioactive peaks that were coincident with three out of four periodic acid-Schiff (PAS) positive bands.     

FATTY ACIDS OF LECITHIN IN SUBCELLULAR FRACTIONS DURING MATURATION OF BRAIN

Abstract— A study has been made of the fatty acyl profiles of lecithin in subcellular fractions of the brain in rat, guinea pig and rabbit. It was found that cerebral lecithins consisted of at least two groups with dissimilar fatty acyl profiles. The group obtained from myelin showed little variation with age of the rats or among two other species examined. The changes in lecithin fatty acyl composition of brain homogenates were in agreement with a progressively greater contribution of myelin lecithins to brain homogenate lecithins with increasing age.     

THE ASSOCIATION OF ACETYLCHOLINESTERASE AND MEMBRANE IN SUBCELLULAR FRACTIONS OF THE ELECTRIC TISSUE OF ELECTROPHORUS

Subcellular fractions of the electric tissue of the main organ of the eel Electrophorus electricus were prepared in sucrose media by differential centrifugation and differential discontinuous gradient centrifugation. The distributions of acetylcholinesterase, cytochrome oxidase, DNA, and protein were determined. The appearance of the fractions was determined by phase contrast microscopy and by electron microscopy. A fraction prepared by differectial centrifugation at 30,000 g for 20 minutes in 0.89 M sucrose contained 63 per cent of the total acetylcholinesterase activity at 4 times the specific activity of that of the tissue homogenate. A subfraction prepared by centrifugation in a discontinuous density gradient showed a peak of total and relative specific acetylcholinesterase activity of 35 per cent and 1.9, respectively. The average over-all purification was 7 times. The acetylcholinesterase peak was below the cytochrome oxidase peak and above the DNA peak in the density gradient. The presence of acetylcholinesterase in the fractions was correlated with the presence of large fragments of the cell membrane; however, the presence of other tissue components was noted. The acetylcholinesterase associated with membrane was found to be activated by incubation with sodium deoxycholate. The possible use of the peak fraction containing membranes rich in acetylcholinesterase for the investigation of other components of the acetylcholine system and of other properties of the membrane is discussed.     

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, G_M3, G_M2, G_M1 and G_D1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which G_D1a comprised two-thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly G_M3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [¹⁴C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; G_D1a was more extensively labeled in N18 cells and G_M3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP- N -acetylgalactosamine: G_M3 N -acetylgalactosaminyltransferase activity, which catalyzes the synthesis of G_M2 from G_M3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.     

DISC ELECTROPHORETIC SEPARATION OF PROTEINS IN NEURONAL, GLIAL AND SUBCELLULAR FRACTIONS FROM CEREBRAL CORTEX

Abstract— Soluble proteins were studied in preparations from rabbit brain cortex enriched in neuronal or glial cells and in subcellular cortical fractions. Analytical polyacrylamide gels were used for acidic (pH 9-5) and basic (pH 4-3) proteins and qualitative and quantitative differences are described. The isozymes of lactic dehydrogenase, brain specific proteins and radioactive labelling patterns were used to characterize some soluble proteins.     

家蚕后丝腺亚细胞组分中核酸和蛋白质的分布

丝心蛋白是家蚕后丝腺的分泌产物。在家蚕五龄早期,后丝腺细胞中迅速形成丝心蛋白合成所必需的一些亚细胞结构,在后丝腺细胞质中粗糙内质网迅速增多,平滑内质网迅速形成空泡状或管状。而且生化测定表明,在五岭早期后丝腺中DNA,RNA、蛋白质和脂肪的含量皆迅速增加,这些为在五龄后期丝心院的合成,即为丝心蛋白信息的转录和翻译,提供亚细胞结构和生化代谢的物质基础(Tashiro等1968;赤井弘1973)。因此有必要对家蚕后丝腺亚细胞组分如细胞核、线粒休、微粒体和105,000g上清液中核酸和蛋白质代谢进行研究。本文初步建立一种分离家蚕后丝腺亚细胞组分的差级离心方法,并测定各亚细胞组分中核酸和蛋白质的含量。