ISOLATION AND CHARACTERIZATION OF SOLUBLE ACIDIC LIPOPROTEINS FROM RAT BRAIN SYNAPTIC VESICLES
Authors: Soller M. , Koenig H. , Mylroie R. , Hughes C. , Lu C. Y. 
Published: October 2006

Abstract—Highly purified fractions of synaptic vesicles were prepared from rat cerebrum or cerebral cortex by density gradient centrifugation. Treatment of synaptic vesicle fractions by autoincubation, freeze‐thawing and sonication in an isotonic alkaline‐salt medium or in 0·1‐0·3% (v/v) Triton X‐100 released increasing quantities of synaptic vesicle protein and phospholipid into solution. When the soluble synaptic vesicle proteins were extracted with 0·1% (v/v) Triton X‐100, the insoluble residue consisted mostly of 5–8 nm‐thick membranes resembling the limiting membranes of intact synaptic vesicles. This finding, together with other considerations, suggested that the soluble proteins and accompanying phospholipids originated from the interior of the synaptic vesicles. A 0·3% (v/v) Triton X‐100 extract of synaptic vesicle was fractionated by ultracentrifugal flotation and dialysis into three lipoprotein fractions: a low density lipoprotein (d < 1·21 g/ml), a high density lipoprotein (d = 1·21–1·35 g/ml) and a very high density lipoprotein (d > 1·35 g/ml). The phospholipid contents of the low, high and very high density lipoprotein fractions were 0·74, 0·38 and 0·20 mg/mg of protein, respectively. All three apolipoproteins had a high ratio of acidic to basic, and of polar to nonpolar, amino acids, and were rich in glycine, alanine and serine. Polyacrylamide gel electrophoresis of the alkaline‐salt and Triton X‐100 extracts of synaptic vesicles at pH 8·8 resolved a single anionic component which stained for protein, lipid (Sudan black B; iodine) and anionic groups (acridine orange). Polyacrylamide gel electrophoresis of synaptic vesicle extracts at pH 2·7 in 5 m urea and 0·25% (v/v) Triton X‐100 resolved about 20 protein components. However, the protein profiles of electropherograms of the Triton X‐100 and alkaline‐salt extracts differed in certain respects, suggesting that these media to some extent solubilized different proteins. However, most of the protein bands in electropherograms of the Triton X‐100 and alkaline‐salt extracts also stained for lipid and anionic groups. In addition, two lipoprotein components in the alkaline‐salt extract and four in the Triton X‐100 extract contained carbohydrate. Isoelectric focusing of synaptic vesicle extracts resolved 6–8 protein fractions. The major fraction in Triton X‐100 and alkaline‐salt extracts had an apparent isoelectric point of approximately 4·2 and contained 0·24 mg of phospholipid per mg of protein. Soluble synaptic vesicle proteins released by incubating, freeze‐thawing and sonicating in the alkaline‐salt medium, and protein fractions of the latter obtained by electrofocusing had an absorption maximum of 260–265 nm which was enhanced in a cold 0·5 n perchloric acid extract, an observation suggesting the presence of a bound nucleotide. These findings demonstrate that rat brain synaptic vesicles contain a heterogenous array of soluble acidic lipoproteins which vary in buoyant density, lipid content, amino acid and carbohydrate composition and electrophoretic mobility in polyacrylamide gels. These acidic lipoproteins apparently comprise the bulk of the macromolecular contents of synaptic vesicles and probably serve as ‘carrier’ proteins for the binding and sequestration of the neurotransmitters.