Association and release of the major intrinsic membrane glycoprotein from peripheral nerve myelin

Hypo-osmotic homogenization of the endoneurium from the adult-rat sciatic nerve and subsequent evaluation of the 197000 g aqueous supernatant by sodium dodecyl sulphate pore-gradient electrophoresis (SDS-p.g.e.) revealed a release of the major glycoprotein (P₀) (29000 M(r)) from peripheral nerve myelin. Immunological verification of the presence of this asparagine-linked glycoprotein in the aqueous supernatant was obtained by immune overlay after SDS-p.g.e. and electrophoretic transfer to nitrocellulose using anti-P₀ γ-globulin followed by autoradiographic detection with ¹²⁵I-protein A. A comparison of successive hypo- and iso-osmotic extractions of the endoneurium revealed that the hypo-osmotic extraction released increasing amounts of P₀ into the supernatant fraction, whereas the iso-osmotic treatment revealed lower levels of P₀ extracted from the myelin and lesser amounts with each successive extraction. Three successive hypo-osmotic extractions resulted in a 2.0-, 2.9-, and 9.5-fold increase in the amount of P₀ released compared with the successive iso-osmotic extractions. Although these results suggest that this major myelin glycoprotein has properties similar to those of extrinsic membrane proteins, temperature-dependent phase-partitioning experiments with Triton-X114 revealed that this glycoprotein is recovered in the detergent-enriched lower phase. These results indicate that this major myelin glycoprotein is an amphipathic integral membrane protein with a distinct hydrophobic domain and yet has solubility characteristics typical of an extrinsic membrane protein. P₀ labelled in vitro with [³H]mannose could be immunoprecipitated from the aqueous supernatant with anti-P₀ γ-globulin by centrifugation at 197000 g without the addition of second antibody or protein A. Analysis of such an immune precipitate after incorporation in vitro with [¹⁴C]acetate to label endoneurial lipids revealed that all major endoneurial lipid classes contained radioactive label, as determined by fluorography after high-performance t.l.c. The mechanism of release of this intrinsic glycoprotein from the myelin membrane, therefore, involves the osmotic-dependent formation of mixed micelles or membrane vesicles with endogenous membrane lipids.