Use of a resolving density gradient created with dextran and poly(ethylene glycol) to purify brain synaptosomes.
Poly(ethylene glycol) and dextran are exclusion agents which when mixed above critical concentrations account for phase separation. In this work, we have used a density gradient created by mixing dextran (denser) and PEG (lighter) solutions to achieve a further purification of synaptosomes by centrifugation. When a brain synaptosomal preparation, containing about 40% of contaminating material, is sedimented in such gradient, two bands of material were obtained. Glutamate decarboxylase activity and glucose-dependent DCIP reduction (an improved marker of the metabolic performance of synaptosomes) were preferentially located in the lower band. These results, together with the electron micrographs of the bands, proved the synaptosomal nature of this lower band. The upper band contained myelin, membranes, vesicles and some synaptosomes. The gradient used shows a high resolution for isopycnic separation of sedimenting material and could be of general interest for subcellular fractionation.
Muiño-Blanco
T
T
Departamento de Bioquímica, Biología Molecular y Celular, Facultad de Veterinaria, Zaragoza, Spain.
Enríquez
J A
JA
Laveaga
M J
MJ
López-Pérez
M J
MJ
D015086:2,6-Dichloroindophenol; D000110:Acetylcholinesterase / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003911:Dextrans; D005947:Glucose / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008854:Microscopy, Electron; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000378:metabolism / Q000648:ultrastructure*
D003911:Dextrans; D011092:Polyethylene Glycols; D015086:2,6-Dichloroindophenol; D007770:L-Lactate Dehydrogenase; D000110:Acetylcholinesterase; D005947:Glucose