Purification and characterization of the apically secreted 80 KDa glycoprotein from Madin-Darby canine kidney (MDCK) cells.
An 80 KDa glycoprotein (gp 80), known to be released predominantly from the apical surface by filter-grown Madin-Darby canine kidney cells, was purified to electrophoretic homogeneity. Purified gp 80 was found to have a disulfide-bonded dimeric structure, and appeared to exist in two molecular forms, a major (high-molecular weight) form consisting of a 46 KDa subunit and a 39 KDa subunit and a minor (low-molecular weight) form consisting of a 46 KDa subunit and a 33 KDa subunit. Upon de-glycosylation by N-glycanase treatment, the 46 KDa subunit was converted to a 25.6 KDa form, whereas both the 39 KDa and the 33 KDa subunit gave rise to a 21.1 KDa form. V8 protease mapping of deglycosylated polypeptides revealed the 39 KDa and the 33 KDa subunit to have nearly identical band patterns, which also exhibited a high degree of homology to that derived from the 46 KDa subunit. Radioimmunoassays revealed that the binding of the purified gp 80 to fibrinogen (or heparin) was dependent on both pH and divalent cations. Furthermore, binding of gp 80 to immobilized fibrinogen (or heparin) was inhibited in the presence of free fibrinogen (or heparin) added in the assay mixture.
Liu
J W
JW
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Lu
R L
RL
Han
J R
JR
Liu
M C
MC
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011863:Radioimmunoassay
D002241:Carbohydrates; D002413:Cations, Divalent; D008562:Membrane Glycoproteins; D005340:Fibrinogen; D006493:Heparin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase