CHARACTERIZATION OF RABBIT MYOCARDIAL PHOSPHOLIPASE A(2) ACTIVITY USING ENDOGENOUS PHOSPHOLIPID SUBSTRATES

We have developed an assay for studying myocardial phospholipase A(2) activity by measuring accumulation of lysophospholipids resulting from hydrolysis of the endogenous choline glycerophospholipid pool. This a ssay was used to characterize phospholipase A(2) activity in rabbit my ocardium. Lyophilized rabbit myocardium was incubated at 37 degrees C in Tris-HCl buffer containing either ethylene glycol bis(beta-aminoeth yl ether) N,N'-tetraacetic acid (EGTA)/EDTA or calcium, and palmitoyl- lysophosphatidylcholine (P-LPC), oleoyl-LPC, stearoyl-LPC, and 16:0-ly soplasmenylcholine were measured using a recently developed HPLC metho d. The identity of the individual species was confirmed by ion-spray L C-MS-MS. In the presence of EGTA/EDTA, incubation for up to 30 min cau sed a linear increase in all lysophospholipids. The main increases wer e found in P-LPC and 16:0-lysoplasmenylcholine, which increased by 37 +/- 3 (mean +/- SE, N = 8) and 48 +/- 3 nmol/g dry wt X min, respectiv ely. The apparent phospholipase A(2) activity was found to be calcium, temperature, and pH sensitive. The pH optimum was between 6.5 and 8.0 , and incubation at room temperature and 45 degrees C decreased the ac tivity by 80 and 40%, respectively. Studies of the metabolism of the f ormed lysophospholipids showed a substantial metabolism of the lysopho spholipids that accounted for about 40% of the total phospholipase A(2 ) activity. This method offers a novel approach to study phospholipase A(2) activities by measuring accumulation of products resulting from hydrolysis of endogenous phospholipid pools. (C) 1984 Academic Press,I nc.