Mechanism of cationic amphiphilic drug inhibition of purified lysosomal phospholipase A1

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Abstract
Cationic amphiphilic drugs like chlorpromazine, propranolol, and chloroquine inhibit lysosomal phospholipase A in vitro. Some workers have proposed that cationic amphiphilic drugs inhibit the activity of phospholipase A1 by forming substrate-drug complexes which cannot be degraded while others have reported competitive inhibition implying drug effects on the enzyme. To analyze the mechanism of inhibition, we examined the binding ability of these drugs to unilamellar vesicles of dioleoylphosphatidylcholine and correlated these results with a detailed kinetic analysis of phospholipase A. Chlorpromazine and propranolol bound to small unilamellar liposomes of dioleoylphosphatidylcholine substrate in a positive cooperative way consistent with two binding sites: a high-affinity site with low capacity and a low-affinity site with high capacity. The affinity of chlorpromazine for the high-affinity site was 2 times greater than that of propranolol (KA = 13807 .+-. 1722 vs 8481 .+-. 1078 M-1), and the saturation number for chlorpromazine was 3 times greater than for propranolol (N = 0.20 .+-. 0.004 vs. 0.07 .+-. 0.02 mol of drug/mol of phosphatidylcholine). Chloroquine did not bind to unilamellar liposomes of dioleoylphosphatidylcholine. We carried out detailed kinetic studies using purified lysosomal phospholipase A1 from rat liver. In the case of chloroquine inhibition, the Lineweaver-Burk double-reciprocal plots showed straight lines, but the slope replots were curved, indicating the formation of complexes having 2 mol of chloroquine/mol of enzyme (EI2 complexes). Thus, chloroquine is a competitive inhibitor which forms EI2 complexes with phospholipase A1. However, in the case of chlorpromazine and propranolol, the observed kinetic data do not fit to the same equilibrium used for the case of chloroquine. If the concepts of free drug, free substrate, and enzyme-substrate-drug complex (ESbIb) are introduced into the equilibrium developed for chloroquine, two general types of solutions can be obtained which fit to the observed data points. Values for free drug and free substrate were calculated from the results obtained from drug binding experiments carried out in parallel under conditions identical with the inhibition studies. In the first type of solution, the formation of ESbIb is insignificant, and in the other type of result, ESbIb formation occurs, but it is converted to product at a rate which is nearly identical with that of the enzyme-substrate complex.

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