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A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. 1988

P R Dunkley, and J W Heath, and S M Harrison, and P E Jarvie, and P J Glenfield, and J A Rostas
Neuroscience Group, Faculty of Medicine, University of Newcastle, New South Wales, Australia.

A method for preparation of synaptosomes from rat cerebral cortex, on a discontinuous Percoll gradient, was previously developed for use with a P2 pellet (Brain Research, 372 (1986) 115-129). Here the Percoll method has been adapted for use with an S1-supernatant which eliminates a potentially damaging resuspension step and saves over 30 min, representing a third of the total preparation time. The homogeneity of the synaptosomes in each of the 5 subcellular fractions obtained with the S1-Percoll method was determined biochemically by analysis of the distribution of total protein, myelin basic protein, synapsin I and pyruvate dehydrogenase across the gradient. Electron microscopy was also used to determine the homogeneity of the synaptosomes, as well as to determine their morphological characteristics. Fraction 4 was the most enriched in synaptosomes and contained the lowest level of contamination by myelin, extrasynaptosomal mitochondria and plasma membranes. The yield of synaptosomes in fraction 4 with the S1-Percoll method was 1.4-fold greater than with the P2-Percoll method. While all other fractions contained some synaptosomes the major additional content in fractions 1-3 and 5 was, respectively, unidentified small membranes, myelin, synaptic plasma membranes and extrasynaptosomal mitochondria. Fraction 1 was enriched for very small synaptosomes (0.34 micron mean diameter) only 8% of which contained mitochondria, while fractions 2-4 progressively included larger synaptosomes containing more mitochondria. Fraction 5 synaptosomes were approximately the same size as those in fraction 4 (0.63 micron mean diameter), but 83% contained mitochondria, significantly more than in fraction 4. The synaptosomes in fraction 5 were found to be relatively resistant to hypotonic lysis, explaining a previously observed lack of phosphorylation of synapsin I in this fraction. The differences in homogeneity and morphological characteristics of the synaptosomes in fractions 1-5 suggest that the basis for their fractionation on Percoll gradients is different from that achieved with the more traditional procedures for isolating synaptosomes and that unique synaptosomal fractions are obtained with the S1-Percoll procedure.

UI MeSH Term Description Entries
D008854 Microscopy, Electron Microscopy using an electron beam, instead of light, to visualize the sample, thereby allowing much greater magnification. The interactions of ELECTRONS with specimens are used to provide information about the fine structure of that specimen. In TRANSMISSION ELECTRON MICROSCOPY the reactions of the electrons that are transmitted through the specimen are imaged. In SCANNING ELECTRON MICROSCOPY an electron beam falls at a non-normal angle on the specimen and the image is derived from the reactions occurring above the plane of the specimen. Electron Microscopy
D009419 Nerve Tissue Proteins Proteins, Nerve Tissue,Tissue Proteins, Nerve
D010750 Phosphoproteins Phosphoprotein
D011205 Povidone A polyvinyl polymer of variable molecular weight; used as suspending and dispersing agent and vehicle for pharmaceuticals; also used as blood volume expander. Polvidone,Polyvidon,Polyvinylpyrrolidone,Arufil,Bolinan,Bolinan 40,Crospovidone,Dulcilarmes,Duratears Free,Enterodes,Enterodez,Hypotears,Kollidon,Lacophtal,Lacri-Stulln,Lagrifilm,Liquifilm Lagrimas,Nutrivisc,Oculotect,PVP 40,Periston,Plasdone,Polyplasdone XL,Povidone, Unspecified,Protagens,Protagent,Unifluid,Vidirakt S mit PVP,Vidisic PVP Ophtiole,Wet-Comod,Dulcilarme,Enterode,Hypotear,Polyvidons,Protagen,Unspecified Povidone
D011919 Rats, Inbred Strains Genetically identical individuals developed from brother and sister matings which have been carried out for twenty or more generations or by parent x offspring matings carried out with certain restrictions. This also includes animals with a long history of closed colony breeding. August Rats,Inbred Rat Strains,Inbred Strain of Rat,Inbred Strain of Rats,Inbred Strains of Rats,Rat, Inbred Strain,August Rat,Inbred Rat Strain,Inbred Strain Rat,Inbred Strain Rats,Inbred Strains Rat,Inbred Strains Rats,Rat Inbred Strain,Rat Inbred Strains,Rat Strain, Inbred,Rat Strains, Inbred,Rat, August,Rat, Inbred Strains,Rats Inbred Strain,Rats Inbred Strains,Rats, August,Rats, Inbred Strain,Strain Rat, Inbred,Strain Rats, Inbred,Strain, Inbred Rat,Strains, Inbred Rat
D002458 Cell Fractionation Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS. Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D002499 Centrifugation, Density Gradient Separation of particles according to density by employing a gradient of varying densities. At equilibrium each particle settles in the gradient at a point equal to its density. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Centrifugations, Density Gradient,Density Gradient Centrifugation,Density Gradient Centrifugations,Gradient Centrifugation, Density,Gradient Centrifugations, Density
D002540 Cerebral Cortex The thin layer of GRAY MATTER on the surface of the CEREBRAL HEMISPHERES that develops from the TELENCEPHALON and folds into gyri and sulci. It reaches its highest development in humans and is responsible for intellectual faculties and higher mental functions. Allocortex,Archipallium,Cortex Cerebri,Cortical Plate,Paleocortex,Periallocortex,Allocortices,Archipalliums,Cerebral Cortices,Cortex Cerebrus,Cortex, Cerebral,Cortical Plates,Paleocortices,Periallocortices,Plate, Cortical
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012822 Silicon Dioxide Transparent, tasteless crystals found in nature as agate, amethyst, chalcedony, cristobalite, flint, sand, QUARTZ, and tridymite. The compound is insoluble in water or acids except hydrofluoric acid. Silica,Aerosil,Aerosil 380,Cristobalite,Quso G-32,Quso G32,Tridymite,380, Aerosil,Dioxide, Silicon,G32, Quso,Quso G 32

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