Purification and characterization of a major glycoprotein in rat liver lysosomal membrane.
A major lysosomal membrane glycoprotein (LGP107) which has an apparent molecular weight (Mr) of 107 kilodaltons (kDa) was purified from rat liver by a simple method with a yield of 1 mg/87 g wet weight of liver. The purification procedures include; preparation of tritosomal membranes of triton-filled lysosomes (tritosomes), extraction of tritosomal membranes by Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. The quantitative immunoblot analysis indicated that LGP107 represents 6.2% of the total protein of tritosomal membranes. The isoelectric point of the purified glycoprotein was 2.7, and it moved toward neutral pH after sialidase treatment, with its molecular weight decreased by about 10 kDa. LGP107 contained 52% carbohydrates, and the carbohydrate moiety was compared of Fuc, Man, Gal, GlcNAc and sialic acid in a molar ratio of 7.2:68.2:40.6:63.0:32.3, respectively, indicating that LGP107 was highly glycosylated with N-linked complex-type olgosaccharide chains. Out of the N-linked glycans released from the glycoprotein by hydrazinolysis/N-reacetylation, about 70% was sialylated. Anion exchange and reverse-phase high performance liquid chromatography analysis on the structure of N-glycans revealed that a disialyl biantennary form is a major component in the oligosaccharide chains of LGP107.
Akasaki
K
K
Faculty of Pharmacy & Pharmaceutical Science, Fukuyama University, Hiroshima, Japan.
Yamaguchi
Y
Y
Ohta
M
M
Matsuura
F
F
Furuno
K
K
Tsuji
H
H
D000818:Animals; D007425:Intracellular Membranes / Q000737:chemistry*; D008247:Lysosomes / Q000737:chemistry*; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011919:Rats, Inbred Strains
D008562:Membrane Glycoproteins