Competitive, Reversible Inhibition of Cytosolic Phospholipase A2 at the Lipid-Water Interface by Choline Derivatives That Partially Partition into the Phospholipid Bilayer

Cytosolic phospholipase A 2 (cPLA 2 ) catalyzes the selective release of arachidonic acid from the sn -2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA 2 using membranes isolated from [ 3 H]arachid...

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Published in:The Journal of biological chemistry 1999-07, Vol.274 (27), p.18864-18871
Main Authors: Burke, J R, Witmer, M R, Zusi, F C, Gregor, K R, Davern, L B, Padmanabha, R, Swann, R T, Smith, D, Tredup, J A, Micanovic, R, Manly, S P, Villafranca, J J, Tramposch, K M
Format: Article
Language:English
Subjects:
Arachidonic Acid - metabolism
Calorimetry, Differential Scanning
Cholesterol - metabolism
Choline
Humans
Inositol 1,4,5-Trisphosphate - metabolism
Kinetics
Lasers
Lipid Bilayers - metabolism
Lipids
N-Formylmethionine Leucyl-Phenylalanine - pharmacology
Phospholipases A - antagonists & inhibitors
Phospholipases A2
Quaternary Ammonium Compounds - pharmacology
Scattering, Radiation
U937 Cells
Water
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Summary:Cytosolic phospholipase A 2 (cPLA 2 ) catalyzes the selective release of arachidonic acid from the sn -2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA 2 using membranes isolated from [ 3 H]arachidonate-labeled U937 cells as substrate, 2-(2′-benzyl-4-chlorophenoxy)ethyl-dimethyl- n -octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC 50 = 5 μ m ). It was over 70 times more selective for the cPLA 2 as compared with the human nonpancreatic secreted phospholipase A 2 , and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N -formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl- sn -glycero-3-phosphomethanol containing 6–10 mol % of 1-palmitoyl-2-[1- 14 C]arachidonoyl- sn -glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface ( K I * app ) was determined to be 0.097 ± 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 ± 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA 2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n -alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller K I * app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [ 3 H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [ 3 H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.274.27.18864