Large-scale preparation of synaptosomes from bovine brain using a zonal rotor technique

Abstract

A zonal rotor technique for the preparation of synaptosomes in bulk from bovine brain frontal cortex based on an impirical transformation of a small-volume discontinuous sucrose density gradient arrangement is presented in detail. The procedure yields new information concerning synaptosomes prepared in sucrose gradients. Cerebroside analysis and electron microscopy show myelin contamination to be restricted to the leading, less dense edge of the synaptosomal profile, free mitochondria to the trailing, more dense edge. Exclusion of fringe areas yields a highly purified synaptosome preparation which entirely enters the next dense layer beyond the 0.8 : 1.2 M sucrose interface. This interface collects most of the oubain-sensitive (Na+, K+) adenosine triphosphatase activity. The purified synaptosomes display very high intrinsic sialidase activity and are rich in di-, tri-, and tetrasialogangliosides, the preferred substrates for the enzyme. Up to 90% of the cholinesterase activity in the zonal rotor synaptosome preparation is specific acetylcholinesterase.