Isolation and partial characterization of rat CNS axolemma enriched fractions

Abstract

Axolemma-enriched fractions were prepared from rat brain by osmotic shock of a purified preparation of myelinated axons and subsequent separation of myelin, two axolemma-enriched fractions and myelin-free axons by density gradient centrifugation. Compared with the starting whole homogenate, the fractions were enriched in specific activity of Na +K + ATPase, acetylcholinesterase, 5′nucleotidase as well as 2′,3′-cyclic nucleotide 3′phosphohydrolase. Compared with myelin, the axolemmal fractions are greatly enriched in high molecular weight proteins. The 1.0/1.2 fraction has a predominant peak of fucose-labeled glycoprotein with a molecular weight between that of the myelin associated glycoprotein and the Wolfgram protein which is absent from the myelin glycoprotein profile. Polyacrylamide gel electrophoresis showed that the protein profile of myelin isolated by this procedure was similar to that of myelin isolated by other procedures and that the myelin specific basic and proteolipid proteins were virtually absent in the axolemma-enriched fractions. Both axolemma fractions were enriched in higher MW proteins, some of which resembled proteins in the myelin protein profile. Both axolemma-enriched fractions specifically bind between 2 and 3 pmoles of [ 3Htetrodotoxin per mg protein. The axolemma-enriched fractions incorporated [ 3Hleucine and [ 14Cfucose exclusively into high molecular weight proteins and glycoproteins. In contrast myelin concomitantly isolated with the axolemma-enriched fractions had a significant amount of [ 3Hleucine labeled protein in myelin proteolipid and basic proteins. In addition to the myelin associated glycoprotein the [ 14Cfucose labeled a glycoprotein of slightly larger apparent molecular weight than proteolipid protein was found in the myelin fraction while the comparable labeled glycoprotein was absent in the axolemma-enriched fractions. The possible extent of contamination of these fractions by myelin or myelin subfractions and relationship of these axolemma-enriched fractions to other axolemma preparations are discussed.