Isolation and characterization of a novel membrane glycoprotein of 85,000 molecular weight from rat liver lysosomes.
We have purified and characterized a novel glycoprotein (r-lamp-3) with an apparent molecular weight (Mr) of 85,000 from membranes of triton-filled lysosomes (tritosomes) by the use of immunoaffinity chromatography on a column of monoclonal antibody-Sepharose 4B. r-lamp-3 accounted for approximately 4% of the total proteins in tritosomal membranes. The isoelectric point (pI) of r-lamp-3 was 4.5 and it was shifted to 6.5 after neuraminidase treatment with its molecular weight decreased by about 7000. Pulse-chase experiments in cultured rat hepatocytes using [35S]methionine showed that r-lamp-3 was initially synthesized as a 77,000 polypeptide and processed to a mature protein with an Mr of 85,000. Upon treatment with endo-beta-N-acetylglucosaminidase H (Endo H), the precursor and mature forms were converted to 55,000 and 73,000 polypeptides, respectively. From the Mr reduction of the precursor form, we estimated the presence of 10--12 N-linked oligosaccharides/r-lamp-3 polypeptide. The data on enzymatic deglycosylation suggested that the mature form of r-lamp-3 contained the same numbers of high mannose-type and complex-type N-linked oligosaccharide chains.
Akasaki
K
K
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan.
Kinoshita
H
H
Fukuzawa
M
M
Maeda
M
M
Yamaguchi
Y
Y
Furuno
K
K
Tsuji
H
H
D000818:Animals; D008099:Liver / Q000737:chemistry*; D008247:Lysosomes / Q000737:chemistry*; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D008562:Membrane Glycoproteins