Purification and characterization of a membrane-bound tyrosine-O-sulfate-binding protein from bovine liver.
A membrane-bound 175-kDa tyrosine-O-sulfate (TyrS)-binding protein from bovine liver was purified to electrophoretic homogeneity. The purified protein exhibited an apparent molecular weight of 175,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Upon SDS-PAGE under nonreducing conditions, the TyrS-binding protein migrated considerably faster than the dimeric fibronectin, indicating its presence in the monomeric form and therefore the absence of a disulfide-bonded subunit structure. The purified TyrS-binding protein was found to bind concanavalin A-Sepharose and yielded a positive reaction toward periodic acid-Schiff (PAS) staining, indicating its glycoprotein nature. The purified TyrS-binding protein displayed strong binding to TyrS, but not the unmodified tyrosine, covalently bonded to Sepharose. Using a tyrosine-sulfated cholecystokinin octapeptide (CCK-8) as the ligand in a radioimmunoassay, it was found that the binding of the TyrS-binding protein was pH-dependent, being strong from pH 8.0 down through 6.5, and becoming dramatically weaker at pH below 6.0. Divalent cations, added in the assay mixture, exerted significant promoting effects on the binding in the order Mn+2 greater than Ca2+ greater than Mg2+. Western blot analysis clearly showed that the purified TyrS-binding protein was capable of forming complexes with two tyrosine-sulfated proteins, fibronectin and fibrinogen, but not two non-tyrosine-sulfated proteins, transferrin and albumin. These results provided support to a role of the TyrS-binding protein being a putative receptor for tyrosine-sulfated proteins.
Lu
R L
RL
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Tang
X B
XB
Han
J R
JR
Suiko
M
M
Liu
M C
MC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002846:Chromatography, Affinity; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D006882:Hydroxyapatites; D036341:Intercellular Signaling Peptides and Proteins; C057501:tyrosine-O-sulfate-binding protein, Bos taurus; C005662:tyrosine O-sulfate; D014443:Tyrosine; D017886:Durapatite