Composition of axolemma-enriched fractions isolated from bovine CNS myelinated axons.

Abstract

Axolemma-enriched fractions were isolated from the white matter of bovine corpus callosum via a purified preparation of myelinated axons which were osmotically shocked and fractionated on a discontinuous density gradient. Two membrane fractions of differing density were obtained: both were somewhat enriched over white matter whole homogenate in specific activity of acetylcholinesterase and 5'-nucleotidase and maximal binding capacity for saxitoxin. Both membrane fractions contained appreciable amounts of 2', 3'-cyclic nucleotide 3'-phosphohydrolase; the specific activity of antimycin-sensitive NAPH-cytochrome c reductase and cytochrome c oxidase indicated low levels of contamination by microsomal and mitochondrial membrane. The myelin which is concomitantly isolated with the axolemma-enriched fractions has a lipid and protein composition comparable to that of myelin isolated by other procedures. Both axolemma-enriched fractions contain about one half of their dry weight as lipid comprised of approximately 25% cholesterol, 25% galactolipid (cerebrosides and sulfatides in a molar ratio of about 4:1) and 50% phospholipid, mostly choline phosphatides and ethanolamine phospholes in an equimolar ratio. The axolemma fractions are also enriched in ganglioside content relative to the myelin fraction. The polypeptides of the axolemma-enriched fractions range from 20,000 to over 200,000 in molecular weight; the predominant proteins are in the range from 50,000 to 69,000. The most dense axolemma-enriched fraction is over fourfold enriched in glycoprotein content compared with myelin, with at least 10 different molecular-weight classes of glycoproteins as identified by Schiff stain of polyacrylamide gel protein profiles. The differences and similarities in the molecular composition of axolemma-enriched preparations which have been characterized to date are discussed.