Isolation and characterization of unmyelinated axolemma from bovine splenic nerve

Abstract

Bovine splenic nerve was used as a source of axolemma-enriched fractions derived from mammalian unmyelinated axons. By electron microscopy, splenic nerve consisted entirely of fascicles of unmyelinated axons and associated Schwann cells. The epineurium and blood vessels were stripped from the dissected nerve, which was then homogenized followed by preparation of a microsomal fraction by differential centrifugation. The microsomes were fractionated on a 10% to 40% continuous sucrose gradient. The individual fractions were combined into six fractions based on sucrose concentration and each fraction was analyzed for membrane markers. The 20% to 23% region of the sucrose gradient was enriched approximately sevenfold in acetylcholinesterase activity and twofold enrichment in saxitoxin binding activity was noted in the same fraction. Relative to other microsomal fractions, this same fraction was less enriched in a microsomal marker (cytochrome c reductase) and only moderately enriched in the activity of a myelin membrane marker (2′,3′ cyclic nucleotide 3′ phosphohydrolase, CNPase). Polyacrylamide electrophoresis of the axolemma-enriched fraction revealed five prominent peptides ranging in molecular weight from 40 kDa to 130 kDa. Lipids, comprising 59.4% of the dry weight, were enriched in cholesterol and sphingomyelin, consistent with the origin from a peripheral nervous system (PNS) plasma membrane. On a molar basis, the major gangliosides were GT1b, GD1a, and GM1. As a whole, these molecular characteristics are consistent with the origin of the axolemma-enriched fraction in the unmyelinated splenic nerve axons. This membrane preparation should prove useful in future studies of the myelinogenic potential of mammalian unmyelinated axolemma. J. Neurosci. Res. 57:670–679, 1999. © 1999 Wiley-Liss, Inc.