Rapid and simple procedure for purifying PAS-4 glycoprotein from bovine milk fat 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-4 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 pI 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.
Kanno
C
C
Department of Applied Biochemistry, Utsunomiya University, Japan.
Hwangbo
S
S
Azuma
N
N
D000818:Animals; D018955:CD36 Antigens; D002417:Cattle; D005223:Fats / Q000737:chemistry; D005260:Female; D037102:Lectins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry
D018955:CD36 Antigens; D005223:Fats; D037102:Lectins; D008562:Membrane Glycoproteins; D008894:Milk Proteins; C069283:PAS-4 protein, Bos taurus