Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. III. Isolation of large fragments of the cerebellar glomeruli.
By a combination of differential and sucrose density gradient (both discontinuous and linear) centrifugation, large fragments of the cerebellar glomeruli were isolated in high purity from hand homogenised tissue. The final preparation contained only about 1% of the tissue protein, but over 90% of its volume was accounted for by the glomerulus particles. The ultrastructure of the glomerulus particles was well preserved. The enzyme profile was characteristic: the glomerulus particles were enriched in glutamate decarboxylase (GAD) activity (relative specific activity (RSA), 2.54), but the RSA of choline acetyltransferase (ChAc) was only 1.05. These findings are consistent with the view that GAD activity is very high in the inhibitory Golgi terminals, which occupy only a small fraction of the total volume of the particles, and acetylcholine may be a transmitter only in a relatively small fraction of the mossy fibre terminals. The glomerulus particles also contained a high concentration of succinate dehydrogenase (SDH) activity (RSA, 1.91), whereas the RSA of glutamate dehydrogenase (GDH) was only 1.15. The great asset of this preparation for future investigations is that it is composed almost exclusively from pre- and postsynaptic neuronal structures. Fractions containing neuropil fragments of non-glomerular origin were also obtained, but the profile of the estimated enzymes did not indicate unique characteristics.
D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D002262:Carboxy-Lyases / Q000378:metabolism; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002531:Cerebellum / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D002794:Choline; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005971:Glutamates; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013569:Synapses / Q000201:enzymology / Q000648:ultrastructure*
D005971:Glutamates; D009419:Nerve Tissue Proteins; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D005969:Glutamate Dehydrogenase; D000123:Acetyltransferases; D002262:Carboxy-Lyases; D002794:Choline