The PO Protein of Chick Sciatic Nerve Myelin: Purification and Partial Characterization

Abstract

The PO protein of the myelin of chick sciatic nerve was isolated and purified by propanoic acid extraction of peripheral nervous system (PNS) myelin, delipidation, Sepharose CL-6B chromatography in the presence of sodium dodecyl sulfate (SDS), and preparative SDS-polyacrylamide gel electrophoresis (PAGE). Approximately 15% of the PO protein in the sciatic nerve myelin was recovered in a homogeneous state. The purified protein monomer has an apparent molecular weight of 32.1K as determined by gel electrophoresis. The PO protein undergoes extensive aggregation during exhaustive dialysis and freeze-drying and yields stable dimers, trimers, and tetramers. The aggregation of the PO protein after freeze-drying is independent of the presence of a reducing agent (2-mercaptoethanol) in the solubilizing medium. The PO protein is a glycoprotein. The amino acid composition of the chick PO protein indicates a definite species difference when compared with mammalian PO proteins although the NH2-terminal isoleucine residue seems to have been retained during evolution.

Regulation of Myelination: Biosynthesis of the Major Myelin Glycoprotein by Schwann Cells in the Presence and Absence of Myelin Assembly

“…, 1973). This 28,000-32,000 M, glycoprotein h a s been isolated (Brostoff et al, 1975; Kitamura e t al., 1976; Roomi et al, 1978;Mezei and Verpoorte, 1981) and found t o have a carbohydrate content of 6.3% by weight (Roomi e t al., 1978). The carbohydrate moieties exist as a single b r a n c h that is N-glycosidically linked to asparagine 14 (Ishaque et a]., 1980).…”

Solid‐Phase Immunoassay of PO Glycoprotein of Peripheral Nerve Myelin

“…Three New Zealand white rabbits were immunized with PO protein as follows. Approximately 6 mg of the partially purified PO protein fraction, obtained after Sepharose CL-6B column chromatography (Mezei and Verpoorte, 1981), was applied to nine 0.9 x 6.0 cm preparative gels of 6% (wthol) polyacrylamide prepared by the method of Weber and Osborn (1975). After electrophoresis the PO protein was located on the gels as described previously (Mezei and Verpoorte, 1981), and the protein band corresponding to the PO monomer was cut out.…”

“…Approximately 6 mg of the partially purified PO protein fraction, obtained after Sepharose CL-6B column chromatography (Mezei and Verpoorte, 1981), was applied to nine 0.9 x 6.0 cm preparative gels of 6% (wthol) polyacrylamide prepared by the method of Weber and Osborn (1975). After electrophoresis the PO protein was located on the gels as described previously (Mezei and Verpoorte, 1981), and the protein band corresponding to the PO monomer was cut out. TWO gel slices containing about 0.3 mg of the PO monomer were washed briefly with 0.9% (wthol) sterile sodium chloride (saline) solution and then homogenized with 1 ml of saline in a Sorvall Omni-Mixer (micro-attachment) for 0.3 min.…”

“…Myelin was prepared according to the method of Oulton and Mezei (1976) and chicken PO protein as described by Mezei and Verpoorte (1981). Nerve tissues were dissected as reported previously (Oulton and Mezei, 1976), homogenized in 20 volumes of 0.1 M Tris-HC1, pH 7.5, and further diluted with a solution of sodium deoxycholate to prepare a partially solubilized extract containing 0.04 M Tris-HC1, pH 7.5; 0.4% (wt/vol) sodium deoxycholate; and 1% (wet weighthol) of the original peripheral nerve tissue.…”

Myelin

“…The amino acid composition (Table II) indicates that the Po protein contains a high proportion of nonpolar amino acids, although not as high as that of the proteolipid protein that might be considered its structural counterpart in the CNS. Isoleucine is the N-terminal residue in several species (Kitamura et al, 1976;Mezei and Verpoorte, 1981;. The N-terminal sequence of 17 residues far rabbit Po has been reported .…”