Abstract
A number of N-linked membrane glycoproteins are induced during chick oviduct differentiation. We have purified a major estrogen-inducible glycoprotein (Mr = 91,000) to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of partial NH2-terminal sequence data with membrane glycoproteins having similar Mr showed a limited homology with human and murine transferrin receptors. We observed that oviduct membranes contain estrogen-inducible transferrin receptor activity (Kd = 2-8 x 10(-8) M). Analytical purification of the putative receptor on an ovotransferrin-Affi-Gel affinity column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a protein of Mr, 180,000, which contains two disulfide-linked subunits of Mr 91,000. The receptor reacts very strongly with antibodies prepared against the 91-kDa glycoprotein on Western blots. Western blot analysis confirms that the 91-kDa glycoprotein is induced by estrogen. The protein has 2% total carbohydrate with Man, GlcNAc, Gal, GalNAc, and NeuAc in a molar ratio of 6:4:2:1:1. The protein contains at least one O-linked moiety. Analysis of the O-linked moiety by glycosidase digestions and gel filtration indicates there are sialo tetra- and trisaccharides and a neutral disaccharide(s). Labeled N-linked glycopeptides were prepared by pronase digestion, beta-elimination, and 3H-acetylation. The N-linked oligosaccharides include high mannose and complex neutral nonbisected biantennary types in an approximate ratio of 3:1 as determined by serial lectin affinity chromatography.
Highlights
From the Department of Biochemistn, and Molecular Biology, State University of New York Health Science Center, Syracuse, Nhw York 13210
A number of N-linked membrane glycoproteins are except lysozyme, areN-linked glycoproteins andthusare induced during chick oviduct differentiation
We observed that oviduct membranes contain estrogen-inducible transferrin receptor activity
Summary
The bumin (ovotransferrin) to membranes was carried outina 200-p1 conalbumin-Affi-Gelwas added to a column, washed, and thebound reaction volume containing 20mM Hepes, pH 7.4,0.1% bovine serum receptor was eluted with the SDS buffer, as described in the previous albumin, washed oviduct membranes, and various amounts of "'I- section. The column was washed with 100 ml of PBS, and the bound antibodies were subsequently eluted with 0.1 M glycine-HC1,pH 3.5,and dialyzed tube gels as described under "Experimental Procedures." Preparations of the 91-kDa protein obtained in this manner were homogeneous. MM Tris-HC1, pH 7.4, 5% Nonidet P-40 (v/v), and 1 mM PMSF by Amino acid analysis of the purified protein (Table I) remild sonication, centrifuged at 40,000 X g for 30 min, and the clear vealed a high proportion of acidic residues and approximately supernatant (50-100 pl) was applied to a conalbumin-Affi-Gel(2 ml) column.
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