Purification and characterization of an 85 kDa sialoglycoprotein in rat liver lysosomal membranes.
Sialoglycoprotein with a molecular mass of 85 kDa (LGP85) was purified from rat liver lysosomal membranes with a 0.9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included: preparation of lysosomal membranes, elimination of LGP107 and LGP96 with immunoaffinity columns, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. LGP85 contains about 22.8% carbohydrate and the carbohydrate moiety is composed of mannose, galactose, fucose, glucosamine, galactosamine, and neuraminic acid, in a molar ratio of 40:20:2:23:3:13. Susceptibility to neuraminidase and immunoreactivity of the protein in intact tritosomes were examined to study the topology of the protein in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the protein were not observed in intact tritosomes until the tritosomes had been disrupted by osmotic shock. These observations suggest that both oligosaccharide chains and the main protein portion of the protein are located on the interior surface of the tritosomal membranes. Subcellular localization of LGP85 was determined using enzyme immunoassay. The lysosomes seem to be the major location. LGP85 in the lysosomes was divided into the membrane bound form (90%) and the soluble form (10%). Immunoelectron microscopy clearly confirmed that the localization of LGP85 is mainly confined to lysosomes.
Okazaki
I
I
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Himeno
M
M
Ezaki
J
J
Ishikawa
T
T
Kato
K
K
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012795:Sialoglycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D002241:Carbohydrates; D008565:Membrane Proteins; D012795:Sialoglycoproteins