RAPID AND SIMPLE PROCEDURE FOR PURIFYING PAS-4 GLYCOPROTEIN FROM BOVINE-MILK PAT GLOBULE-MEMBRANE

The isolation and partial characterization of PAS-4 glycoprotein (78 kDa) from bovine milk fat globule membrane (MFGM) is described, PAS-4 was selectively extracted with Triton X-114 nonionic detergent and then fractionated on DEAE-Sepharose at pH 7.5, The PAS-LI fraction that was not bound on DEAE-Sepharose gave a single band by SDS-PAGE, The recovery of PAS-4 was 57.4% from MFGM. An amino acid analysis found a high percentage of nonpolar residues, Approximately 7.2% of carbohydrate from PAS-4 was composed of mannose, galactose (Gal), N-acetylglucosamine, N-acetylgalactosamine (GalNAc), and sialic acid, most of the Gal and GalNAc in PAS-4 being released after mild alkaline hydrolysis, This indicated that PAS-4 contained both N- and O-linked sugar chains in concordance with the results of lectin affinity, PAS-4 had apparent isoelectric points of 7.45, 7.41, and 7.32, but these were shifted to pr 7.47 by a neuraminidase treatment, The apparent molecular weight of PAS-4 after deglycosylation with N-glycanase was approximately 57,000 by SDS-PAGE.