Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A2.
A high-affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA2-I binds to a single polypeptide with a mass of 190-200 kDa. The PLA2-I binding protein in the membranes was solubilized in an active form with n-octyl beta-D-thioglucoside, and then purified approx. 16,000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA2-I-affinity gel chromatography and gel-filtration high-performance liquid chromatography on a TSKgel G3,000SWXL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA2-I binding protein for receptor-ligand interactions.
Hanasaki
K
K
Shionogi Research Laboratories, Shionogi & Co., Osaka, Japan.
Arita
H
H
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003338:Corpus Luteum / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011956:Receptors, Cell Surface; D054507:Receptors, Phospholipase A2
D002352:Carrier Proteins; D006023:Glycoproteins; C516360:PLA2R1 protein, human; D011956:Receptors, Cell Surface; D054507:Receptors, Phospholipase A2; D010741:Phospholipases A; D054467:Phospholipases A2