Isolation of a product from the trypsin-digested glycoprotein of sciatic nerve myelin.
When purified rabbit sciatic nerve myelin, whether lyophilized or not, is treated with low amounts of trypsin (25 microgram/ml) for 0.5, 3, or 24 h the resulting protein patterns viewed on sodium dodecyl sulfate (SDS) gel electrophoresis are similar. The most striking feature of the trypsinized myelin is the accumulation of a heavy band at the basic protein position, molecular weight 19 000, which is accounted for as a degradation product of the PO protein, referred to as the TPO protein. The PO protein, the major glycoprotein of sciatic nerve myelin, as well as the 23K and P2 proteins and albumin, an absorbed component, are all partially degraded; most high molecular weight bands are lost. The TPO protein, isolated by gel filtration in 2% SDS on an agarose column, like the PO protein, is highly insoluble in aqueous solvents. It is a glycoprotein (8% carbohydrate), staining with periodic acid-Schiff reagent; containing 3  mannose, 1 galactose, 3 N-acetylglucosamine, 1 sialic acid, and 1 fucose residues and is identical to the nonasaccharide of the parent PO protein. The amino acid composition of the TPO protein, is similar to the PO protein, but has a much higher content of hydrophobic residues and begins with NH2-methionine. This suggests that the PO protein is an amphipathic membrane protein in which its more polar character is confined to the first third of its NH2-terminus. This polar domain is probably positioned above the lipid leaflet where it is accessible to trypsin which cleaves a sensitive lysinyl (or argininyl)-methionine linkage. The more hydrophobic domain (the TPO protein) is buried in the myelin bilayer where it is protected from further tryptic attack. Thus trypsin can serve as a useful probe of myelin structure.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D006023:Glycoproteins / Q000302:isolation & purification; D008970:Molecular Weight; D009185:Myelin Proteins / Q000302:isolation & purification; D009186:Myelin Sheath / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D012584:Sciatic Nerve / Q000032:analysis*; D014357:Trypsin
D000596:Amino Acids; D002241:Carbohydrates; D006023:Glycoproteins; D009185:Myelin Proteins; D010446:Peptide Fragments; D014357:Trypsin