Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A2: a carbon-13 NMR study

Abstract

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.

Interactions of Lyso 1-Palmitoylphosphatidylcholine with Phospholipids: A 13C and 31P NMR Study

“…Similar results were obtained by 3'P NMR spectroscopy of lysoPC in 1-palmitoyl-2-oleoyl-PC (POPC) vesicles and explained on the basis of the wedge shape of lysoPC which allows a better fit into the outer leaflet (Kumar et al, 1989). A preference of long-chain acyl CoA for the outer leaflet of SUVs has also been found (Boylan & Hamilton, 1992); in this case both size and charge of the CoA moiety may limit the incorporation of acyl CoA into the inner leaflet.…”

“…The chemical shift remained constant, and the intensity increased with increasing concentration of added ['3C]LPPC, as illustrated by the spectra corresponding to 0.6, 1.9, 2.8, and 4.0 mol %. The chemical shift is close to that for LPPC generated by PLA2 hydrolysis of the outer leaflet of palmitoyl-PC vesicles (Bhamidipati & Hamilton, 1989). The observation of this single signal confirms its assignment to LPPC in the outer monolayer of egg PC vesicles.…”

“…3'P NMR spectra at 121.0 MHz were acquired with a 90" pulse (14.0 ps), 8K data points, a 1.5 s pulse interval, and composite pulse 'H decoupling. 31P NMR spectra of vesicle samples at 8 1 .O MHz were recorded on a Bruker WP-200 NMR instrument as described elsewhere (Bhamidipati & Hamilton, 1993b). All spectra were acquired at 30-32 "C unless mentioned otherwise.…”

“…For example, in erythrocyte membranes lysophospholipid and FA generated by PLAr action did not destabilize the membrane even after extensive hydrolysis (Haest et al, 1981). LysoPC forms lamellar structures with long-chain FA as well as cholesterol when mixed in equimolar ratio (Jain et al, 1980;Klopfenstein et al, 1974;Ramsammy & Brockerhoff, 1982;Kumar et al, 1986;Ramsammy et al, 1984;Bhamidipati & Hamilton, 1993a). Jain and co-workers (1981) proposed that, in equimolar mixtures of lysoPC and FA, the acyl chains of the two lipid components pack parallel to one another as in diacyl phospholipid, and the free hydroxyl group of lysoPC and the carboxyl group of FA are in close proximity.…”

“…LPPC (lyso 1-palmitoyl-PC) represents the major species of lysoPC that would be generated from PC in membranes. The carbonyl carbon was chosen for I3C enrichment because of the high sensitivity of the carbonyl I3C chemical shift to changes in the local magnetic environment (e.g., H-bonding and proximity of polar groups) and the usefulness of I3C NMR for monitoring small amounts of I3C-enriched lipids in model membranes (Bhamidipati & Hamilton, 1989;Boylan & Hamilton, 1992;Cabral et al, 1986). I3C NMR studies presented here examined low proportions of LPPC in phospholipid bilayers, in the presence and absence of FA.…”