Zonal density gradient electrophoresis of intracellular membranes of brain cortex

Rat cerebral cortex was homogenized in a sucrose-free medium and a number of intracellular membrane fractions were isolated therefrom by means of differential and density-gradient centrifugation. The mitochondrial and microsomal membrane fractions, as well as the separated rough and smooth endoplasmic reticulum components of the latter, were subjected to zonal density gradient electrophoresis. With this technique it was possible to determine the electrokinetic profile of the membranes contained in each fraction. The application of zonal density gradient electrophoresis to the membrane populations obtained by density-gradient centrifugation revealed marked differences in electrophoretic mobility between them which correlated directly with both their membrane-bound N-acetylneuraminic acid content and their acetyl-cholinesterase (EC 3.1.1.7) activity. The fraction containing membranes in which both of these components were highest and, hence, presumably synaptic in derivation, was isolated as a single, homogeneous electrophoretic peak.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33000/1/0000384.pd

The action of trypsin and neuraminidase on the synaptic membranes of brain cortex

The population of intracellular membranes of rat cerebral cortex which exhibited the highest relative specific concentration of bound N-acetylneuraminic acid (NANA) and the highest relative specific activity of acetylcholinesterase (EC 3.1.1.7) (synaptic membranes) was incubated with bacterial neuraminidase and with trypsin under a number of experimental conditions. The electrokinetic profile displayed by the membrane preparation upon zonal density gradient electrophoresis changed as a result of the removal of bound NANA by neuraminidase, while its acetylcholinesterase activity remained unaffected; conversely, the action of trypsin led to the inactivation of acetylcholinesterase before any significant alterations of the electrophoretic mobility of the membranes became apparent. Prolonged incubation alone or in the presence of either neuraminidase or trypsin, resulted in the loss of membrane electrophoretic homogeneity, a circumstance which, most likely, reflects a disaggregation of the structural matrix of the membrane.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33001/1/0000385.pd