Use of short-chain cyclopentano-phosphatidylcholines to probe the mode of activation of phospholipase A2 from bovine pancreas and bee venom.
A great mystery in the mechanism of phospholipase A2 (PLA2) and many other lipolytic enzymes is the "interfacial activation" induced by micellar but not monomeric substrates. Equally mysterious is the lack of interfacial activation in bee venom PLA2, as opposed to PLA2s from pancreas and other sources. We have probed these problems using the conformationally restricted short-chain cyclopentano-analogues of diacylphosphatidylcholine (Cp-DCnPC, all-trans isomer). In the reaction catalyzed by bovine pancreatic PLA2, Cp-DC8PC behaved differently from DC8PC in that its monomers and micelles showed comparable activities (but lower than the activity of DC8PC). This result suggests that the activity of PLA2 can be regulated by substrate conformation and supports the "substrate conformation model" (Wells, M. A. (1974) Biochemistry 13, 2248-2257), but raises a question as to whether Cp-DC8PC mimics monomers or micelles of DC8PC. Conformational analysis by 1H NMR revealed that monomeric Cp-DC8PC was conformationally restricted near the carbonyl region, a property characteristic of micelles. Thus, monomeric CP-DC8PC can be considered as a conformational analogue of micelles, but the important structural feature lies in the CH2COO region instead of the glycerol backbone. CP-DC8PC was then used to test a previous proposal that the bee venom PLA2 hydrolyzes monomers but not micelles (which would predict little or no activity for Cp-DC8PC since its conformation is micelle-like whether below or above its critical micelle concentration). The results showed that Cp-DC8PC is a relatively good substrate for the bee venom PLA2 in comparison with the pancreatic PLA2. This and other evidence together suggest that the bee venom PLA2 is not sensitive to the conformation of monomeric and micellar substrates and hydrolyzes both monomers and micelles. The results in both PLA2s demonstrate the usefulness of cyclopentano-phospholipids in probing the mechanism of phospholipases and the roles of substrate conformation in the catalysis of PLA2.
Lin
G
G
Department of Chemistry, Ohio State University, Columbus 43210.
Noel
J
J
Loffredo
W
W
Stable
H Z
HZ
Tsai
M D
MD
D000818:Animals; D001514:Bee Venoms / Q000032:analysis*; D002417:Cattle; D004789:Enzyme Activation; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2
D001514:Bee Venoms; D010713:Phosphatidylcholines; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2