Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.
Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.
Maloney
K M
KM
Department of Chemistry, Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, USA.
Grandbois
M
M
Grainger
D W
DW
Salesse
C
C
Lewis
K A
KA
Roberts
M F
MF
D000818:Animals; D001514:Bee Venoms / Q000201:enzymology; D002208:Caproates / Q000378:metabolism; D002417:Cattle; D004355:Drug Stability; D004546:Elapid Venoms / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate; D006868:Hydrolysis; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011312:Pressure; D013379:Substrate Specificity; D013499:Surface Properties
C093692:1-caproyl-2-palmitoylphosphatidylcholine; C093693:1-palmitoyl-2-caproylphosphatidylcholine; D001514:Bee Venoms; D002208:Caproates; D004546:Elapid Venoms; D008081:Liposomes; D010713:Phosphatidylcholines; D010741:Phospholipases A; D054467:Phospholipases A2; D016650:Fluorescein-5-isothiocyanate