The role of phospholipase A2 lysines in phospholipolysis of Escherichia coli killed by a membrane-active neutrophil protein.
Purified rabbit bactericidal/permeability-increasing protein at bactericidal concentrations is a membrane-perturbing agent that triggers hydrolysis of envelope phospholipids of a phospholipase A-less Escherichia coli (S17) mutant by a highly basic (pI greater than 10) phospholipase A2, purified from Agkistrodon halys blomhoffii snake venom. Most other purified phospholipases A2 do not degrade the phospholipids of E. coli killed by the bactericidal protein. To study the role of enzyme charge in bactericidal protein-dependent phospholipid hydrolysis, lysines of the Agkistrodon phospholipase A2 were modified, either by carbamylation (decreases net charge), or by reductive methylation (no delta charge). Incorporation of [14C]cyanate or [14C]formaldehyde and amino acid analysis served to monitor modification. Modification appears to be limited to epsilon-NH2 groups. Incorporation of up to 5 mol of cyanate or formaldehyde/mol of enzyme did not affect catalytic activity. In contrast, incorporation of, on average, 1 mol of either reagent/mol of protein reduced by 80% the activity of the enzyme toward E. coli S17 killed by the bactericidal protein. Since this loss is similar with carbamylation and reductive methylation, the role of the epsilon-NH2 group in the bactericidal protein-dependent hydrolysis seems independent of charge. Thus, the lysines in this phospholipase A2 are not essential for catalysis and substrate binding, but are essential for the action of this enzyme on E. coli killed by the bactericidal protein.
D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins / Q000494:pharmacology*; D003485:Cyanates / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism; D007700:Kinetics; D008066:Lipolysis; D008239:Lysine; D008565:Membrane Proteins; D009154:Mutation; D009504:Neutrophils / Q000502:physiology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011817:Rabbits; D012910:Snake Venoms
D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D003485:Cyanates; D008565:Membrane Proteins; D010743:Phospholipids; D012910:Snake Venoms; C021623:bactericidal permeability increasing protein; D005557:Formaldehyde; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine