An improved method of preparing rat brain synaptic membranes. Elimination of a contaminating membrane containing 2',3'-cyclic nucleotide 3'-phosphohydrolase activity.
Synaptosomes were prepared from rat cortex by subjecting a washed crude mitochondrial pellet to centrifugation first on discontinuous Ficoll-isotonic sucrose gradients and then on discontinuous sucrose gradients. The synaptosome fraction, collected from the 7.5-14% Ficoll band (II), was further separated into two additional fractions, designated IIA and IIB, which bank at the 0.32-1.05 M and at the 1.05-1.6 M sucrose interfaces, respectively. Electron microscopic analysis showed that fraction IIB contained synaptosomes and extra terminal mitochondria and was essentially free of membrane fragments. Further characterization showed that IIB contained 69% of the protein and 83% of the lactic dehydrogenase activity of fraction II and had a specific activity of a 2',3'-cyclic nucleotide 3'-phosphohydrolase approximately 1% of that obtained with myelin. Fraction IIA had approximately 50% the specific activity of the 2',3'-cyclic nucleotide 3'-phosphohydrolase found in myelin. Synaptic plasma membranes were prepared by lysing fraction IIB in 1 mM sodium phosphate, 0.1 mM EDTA at pH 8.5 and subjecting this preparation to centrifugation on a discontinuous sucrose density gradient. Enzymatic analysis indicated that membranes banding at the 0.6-0.8 M sucrose interface had high specific activities of plasma membrane enzymes (e.g. acetylcholinesterase, ATPase, 5'-nucleotidase). The specific activity of the (Na+ + K+)-ATPase in the purified membrane preparation was 8-fold higher than that in the original homogenate. Specific activities of various marker enzymes indicated that the composition of these membrane preparations for the most part was synaptic plasma membranes, approximately 7% mitochondrial outer membranes and 3% a membrane containing 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. The polypeptide compositions of three possible contaminating membranes and of synaptic membranes were compared by electrophoresis in 6-20% gradient polyacrylamide gels in the presence of sodium dodecyl sulfate. Whereas mitochondrial and myelin membranes had distinct compositions, the compositions of the microsomal and synaptosomal plasma membranes were similar. Synaptic plasma membranes contained at least 27 polypeptides; the three major polypeptides had molecular weights of 103,000; 54,000; and 50,000. The major polypeptides of soluble synaptosomal proteins had molecular weights of 54,000 and 42,000.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000032:analysis; D000110:Acetylcholinesterase / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D002540:Cerebral Cortex / Q000201:enzymology / Q000648:ultrastructure*; D005260:Female; D008297:Male; D051381:Rats; D013570:Synaptic Membranes / Q000648:ultrastructure*; D013574:Synaptosomes / Q000648:ultrastructure
D000110:Acetylcholinesterase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases