Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A2: a 13C NMR study.
A new approach to study phospholipase A2 mediated hydrolysis of phospholipid vesicles, using 13C NMR spectroscopy, is described. [13C]Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A2 (Crotalus adamanteus). The 13C-labeled carboxyl/carbonyl peaks from the products [lyso-1-palmitoylphosphatidylcholine (LPPC) and palmitic acid (PA)] were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was approximately 50% ionized at pH approximately 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of 13C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.
Bhamidipati
S P
SP
Department of Biophysics, Boston University School of Medicine, Massachusetts 02118-2394.
Hamilton
J A
JA
D015060:1,2-Dipalmitoylphosphatidylcholine; D002247:Carbon Isotopes; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D010743:Phospholipids
D002247:Carbon Isotopes; D010743:Phospholipids; D015060:1,2-Dipalmitoylphosphatidylcholine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2