Subcellular fractionation of rat cerebellum: separation of synaptosomal populations and heterogeneity of mitochondria.
A crude mitochondrial fraction (M) derived from manually disrupted cerebellar tissue and enriched in choline acetyltransferase (ChAT) activity was fractionated by centrifugation in discontinuous and continuous sucrose gradients. Further purification of 'cholinergic' synaptosomes was achieved (relative specific activity (RSA) of ChAT greater than 3), but the overlap with other synaptosomal populations was still considerable. Hand-homogenized cerebella processed through the full fractionation procedure described here and in previous papers yielded preparations enriched in certain neuronal structures and a fraction in which 'heavy' free mitochondria was concentrated. To characterize these preparations the activities of two transmitter enzymes (CHAT and glutamate decarboxylase, GAD) and 6 mitochondrial enzymes (succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), monoamine oxidase, citrate synthase, fumarase and GABA-aminotransferase) were determined. The distribution of the transmitter enzymes was clearly different in the preparations containing various neuronal structures. The GAD:ChAT RSA ratio was 2.4 for the glomerulus particles, 1.3 for the molecular layer fragments, 0.6 for the myelinated axon segments, and 0.2 for the 'cholinergic' synaptosomes. The mitochondrial enzyme profile of the preparations comprising mainly neuronal structures differed markedly from that of the 'free' mitochondrial fraction. Notably the latter was greatly enriched in GDH (RSA 5.6), whereas the SDH:GDH RSA ratio was relatively high in the former preparations. Nevertheless there were notable differences in the enzyme profile of the fractions of predominantly neuronal origin indicating that the enzyme composition of mitochondria of neuronal processes is not uniform.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002531:Cerebellum / Q000166:cytology* / Q000201:enzymology; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000378:metabolism; D005649:Fumarate Hydratase / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008995:Monoamine Oxidase / Q000378:metabolism; D051381:Rats; D013385:Succinate Dehydrogenase / Q000378:metabolism; D009435:Synaptic Transmission; D013574:Synaptosomes / Q000201:enzymology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D005680:gamma-Aminobutyric Acid; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D005969:Glutamate Dehydrogenase; D008995:Monoamine Oxidase; D002795:Choline O-Acetyltransferase; D002950:Citrate (si)-Synthase; D005968:Glutamate Decarboxylase; D005649:Fumarate Hydratase