Isolation and characterization of periaxolemmal and axolemmal enriched membrane fractions from the rat central nervous system

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Abstract
In this report, we describe the fractionation of crude axolemmal fractions from rat lower brainstem into subfractions enriched in markers for either periaxolemmal myelin or axolemma. These subfractions were isolated on density gradients as bands layering on 0.8M and 1.0M sucrose. Both subfractions consisted of unilamellar vesicles. Relative to myelin purified from the same starting material, the 0.8M subfraction was enriched in MAG, CNPase, carbonic anhydrase and Na+, K+ ATPase but was extremely low in PLP and MBP. In addition, this fraction exhibited a protein profile distinct from myelin. The 1.0M fraction was also highly enriched in Na+, K+ ATPase and had an overall composition similar to the 0.8M subfraction. However, it differed from the 0.8M subfraction by being low in MAG, CNPase, and carbonic anhydrase, but enriched in voltage‐dependent Na+ channel, axon‐specific fodrin, and MAP‐1B. Based on these characteristics we concluded that the 0.8M and 1.0M subfractions were highly enriched in periaxolemmal myelin and axolemmal membrane, respectively. Plasmolipin10 was unique with equally high levels in myelin and in the 0.8M and 1.0M subfractions. Both subfractions were enriched, relative to myelin, in the alpha subunit of the GTP binding protein, Go, and the alpha subunit common to all G proteins, GA/1. Electrophysiology with membrane subfractions fused to lipid bilayers showed that both membranes contained sets of K+ and Cl− channels, which based on channel sizes and open times, are largely distinct from one another.