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 197 000 g aqueous supernatant by sodium dodecyl sulphate pore-gradient electrophoresis (SDS-p.g.e.) revealed a release of the major glycoprotein (P0) (29 000 Mr) 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 gamma-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 X-114 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 gamma-globulin by centrifugation at 197000g 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.
J F
JF
Poduslo
J K
JK
Yao
D000818:Animals; D011232:Chemical Precipitation; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D018993:Myelin P0 Protein; D009185:Myelin Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D009995:Osmosis; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012584:Sciatic Nerve / Q000378:metabolism
D008565:Membrane Proteins; D018993:Myelin P0 Protein; D009185:Myelin Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40