We have previously demonstrated that when phospholipase A₂ is treated with either tissue tranaglutaminase or human plasma Factor XHIa, a striking increase of its catalytic activity is observed, due to the formation of an intramolecular e-(7-glutamyl)-lysine crosslink [Cordella-Miele et al. (1990) J. Biol. Chem. 265,17180–17188]. Here, we report the effect of transglutaminase substrates such as mono-, di-, and polyamines on this transglutaminasecatalyzed post-translational modification of phospholipase A₂. Incorporation of radioactively labeled polyamines into phospholipase A₂ was demonstrated by using porcine pancreatic phospholipase A₂ as a substrate in a conventional transglutaminase assay. These results were further confirmed by SDS-polyacrylamide gel electrophoresis followed by fluorography. Additionally, y-glutamyl-polyamine was detected and unequivocally identified in proteolytic digests of polyaminated phospholipase A₂. When phospholipase A₂ was incubated with transglutaminase in the presence of putrescine, spermine, spennidine, dansylcadaverine, or methylamine, a 2-3-fold increase in phospholipase A₂ activity was observed. The increase of phospholipase A₂ activity was found to be dependent upon the concentration of phospholipase A₂, preincubation time, and the duration of the reaction. Increase in phospholipase A₂ activity after transglutaminase treatment in the presence of polyamines was demonstrated using two different assay systems. Kinetic studies on phospholipase A₂ pretreated with spermidine and transglutaminase demonstrated a significant increase of the apparent Fmax but no significant change in the apparent K_m Unlike phospholipase A₂ pretreated with transglutaminase alone, polyaminated phospholipase A₂ does not undergo non-covalent dimerization in solution. Polyaminated phospholipase A₂ was further purified by chromatofocusing and was found to contain N-mono(γ-glutamyl)-spermidine in a molar ratio of about 1:1 to phospholipase A₂. Freshly purified, polyaminated phospholipase A₂ had a specific activity approximately 3-fold higher than that of control phospholipase A₂ treated in an identical way except for the absence of transglutaminase. To our knowledge, this is the first demonstration that transglutaminase catalyzes the incorporation of amines into a phospholipase, and that this post-translational modification increases phospholipase A₂ activity.