Characterization of phospholipase A2 activity in serum of the American alligator (Alligator mississippiensis)

Abstract

PLA(2) is a diverse class of enzymes with a broad spectrum of physiological functions. Secretory PLA(2) isoforms have been reported to exhibit important innate immune function in higher vertebrates. This study was conducted to characterize PLA(2) activity in the serum of the American alligator (Alligator mississippiensis). We used a glycerophospholipid with a fatty acid in the sn-2 position labeled with a fluorescent probe (BODIPY) to detect and quantify alligator serum PLA(2) activity. Incubation of BODIPY-labeled bacteria with different concentrations of alligator serum resulted in a concentration-dependent detection of PLA(2) activity. Kinetic studies showed that product formation was rapid, with substantial activity within 5 min, and maximal activity at approximately 20 min. The alligator PLA(2) activity was temperature-dependent, with activity at lower temperatures (5-10 degrees C) approximately half of that observed at temperatures of 30-40 degrees C. In addition, the generation of fluorescent product was reduced by a specific inhibitor (p-bromophenacyl bromide) of secretory PLA(2) in a concentration-dependent manner, enforcing the idea that the observed activities are due to a secretory PLA(2) enzyme in alligator serum.