Modification of arginine residues in porcine pancreatic phospholipase A2.
Although phenylglyoxal monohydrate reacts with Arg-6 in porcine pancreatic phospholipase A2, concomittantly the alpha-amino group of the N-terminal Ala-1 residue is quantitatively transaminated. Due to this latter reaction the enzymatic activity toward micellar substrate is lost irrespective of the Arg-6 modification. Upon reaction of [7-(14)C]phenylglyoxal monohydrate with alpha-amino-blocked phospholipase A2 analogs, two molecules of the reagent were incorporated per protein molecule, which were found to be present on Arg-6. Removal of alpha-amino-blocking groups after the modification reaction furnished the corresponding Arg-6-modified phospholipases possessing 30-38% of their original specific enzymatic activities in the egg-yolk assay. After reaction of 1,2-[1-(14)C]cyclohexanedione with porcine phospholipase A2 the crude reaction mixture was purified by chromatography on quaternary diethyl-(2-hydroxypropyl)aminoethyl-Sephadex in the presence of borate. A fraction was obtained containing a pure protein in which one molecule of 14C-labeled reagent per protein molecule was incorporated which was found to be localized almost exclusively on Arg-6. Cyclohexanedione modification of Arg-6 in phospholipase A2 does not significantly influence its catalytic activity when assayed toward monomeric and micellar substrates. The results of direct binding experiments using substrate analogs and of monolayer studies of the phospholipase modified at Arg-6 by cyclohexanedione are in agreement with previous findings that Arg-6 is involved in the interaction of the enzyme with lipid-water interfaces.
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001120:Arginine; D002250:Carbon Radioisotopes; D003488:Cyanogen Bromide; D003512:Cyclohexanones / Q000494:pharmacology; D007700:Kinetics; D008823:Micelles; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010658:Phenylglyoxal / Q000494:pharmacology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013552:Swine
D000447:Aldehydes; D002250:Carbon Radioisotopes; D003512:Cyclohexanones; D008823:Micelles; D010446:Peptide Fragments; C018031:1,2-cyclohexanedione; D001120:Arginine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D010658:Phenylglyoxal; D003488:Cyanogen Bromide