COMPARATIVE STUDIES OF TYROSINE MODIFICATION IN PANCREATIC PHOSPHOLIPASES .1. REACTION OF TETRANITROMETHANE WITH PIG, HORSE, AND OX PHOSPHOLIPASES-A2 AND THEIR ZYMOGENS

Reaction of horse, pig, and ox pancreatic phospholipases A2 and their zymogens with a 10-fold molar excess of tetranitromethane at pH 8 resulted in a considerable loss of enzymatic activity. In the presence of egg yolk lysolecithin micelles and sufficient Ca2+ ions a higher rate of inactivation of the active enzymes is observed. This effect is due to the incorporation of the reagent into the lysolecithin micelles, thus enhancing the nitration of those tyrosine residues involved in the micellar binding site of the phospholipases A2. In order to remove polymerized protein, we subjected the reaction mixture to gel filtration on Sephadex G-75. The elution patterns obtained revealed that nitration in the absence of lysolecithin resulted in the formation of approximately 30% of dimerized protein while in the presence of lysolecithin almost no dimerization occurred. This protecting effect of lysolecithin micelles against covalent dimerization does not occur for the zymogens; the effect is specific for the active enzymes and is due to the fact that the enzymes bind to the micelles and the zymogens do not. Further purification of the nitrated mo-nomeric protein fractions was performed by chromatography on QAE-A 25 Sephadex at pH 8, and the fractions obtained were found by analytical gel electrophoresis to be pure. Analysis of the various nitrated proteins by amino acid analyses, spectroscopic methods, and peptide mapping revealed that the invariant tyrosine-69 in all proteins as well as the variable tyrosine-123 in the porcine proteins was nitrated both in the presence and in the absence of lysolecithin micelles. It was also found that the variable tyrosine-19 was nitrated only in the active horse phospholipase A2 when lysolecithin micelles were present. Furthermore, the presence of lipids gives rise to a considerable increase in the amount of dinitrated species in the porcine and horse phospholipases with a corresponding decrease in the amount of unmodified and mononitrated enzymes. All the nitrotyrosine-69 phospholipases exhibited considerably decreased enzymatic activity on a micellar substrate (Vmax value of about 16% of that of the native enzymes). Upon conversion into the aminotyrosine-69 phospholipases, their enzymatic activities were restored to 55-80%. In contrast, the activity of the nitrotyrosine-19 horse phospholipase (43%) and the nitrotyrosine-123 porcine phospholipase (54%) decreased to 23 and 25%, respectively, upon conversion into their aminotyrosyl derivatives. From these results it can be concluded that the modified residues are not active site residues, but they could be involved in the binding of the enzyme to lipid-water interfaces. © 1979, American Chemical Society. All rights reserved.