Interactions of lyso 1-palmitoylphosphatidylcholine with phospholipids: a 13C and 31P NMR study.

13C and 31P NMR spectroscopy were used to monitor interactions of lyso 1-palmitoylphosphatidylcholine (LPPC) in the interfacial region of egg phosphatidylcholine (PC) bilayers and determine the effect of LPPC on the phospholipid bilayer structure. 13C NMR spectroscopy of small amounts (0.5-10 mol%) of 13C carbonyl-enriched LPPC cosonicated with egg PC to form small unilamellar vesicles (SUVs) revealed separate carbonyl signals for LPPC in the inner and outer leaflets of the vesicles. The ratio of LPPC in the outer leaflet to that in the inner leaflet was > or = 3/1. Exchange of LPPC between bilayer leaflets ("flip-flop") was too slow to be measured (t1/2 > 12 h). Albumin added to the external buffer of LPPC/PC vesicles was shown by 13C NMR to extract LPPC only from the outer leaflet. LPPC was a poor detergent in egg PC multilayers and SUVs. Stable SUVs were prepared by cosonicating egg PC with up to 30 mol% LPPC, and preformed SUVs incorporated up to 40 mol % of LPPC (added as an aqueous solution) without undergoing any morphological changes as evidenced by 31P NMR spectroscopy. The presence of oleic or palmitic acid did not have observable effects on properties of LPPC in SUVs, such as the localization of the LPPC carbonyl in the interface, and the transbilayer distribution and movement of LPPC. The apparent pKa of the fatty acid (FA) carboxyl at the membrane interface (7.7) measured by 13C NMR was not affected by LPPC, but the FA carboxyl carbon resonance showed linewidth changes near the apparent pKa that were dependent on the FA/LPPC ratio. These data suggest weak interactions in the interfacial region between FA and LPPC when both lipids are present at low levels in PC vesicles.

NMR studies of phospholipase C hydrolysis of phosphatidylcholine in model membranes.

Hydrolysis of phospholipids in biological membranes by phospholipase C (PLC) produces an important second messenger molecule, 1,2-diacylglycerol (DAG), that is essential for the activation of protein kinase C (PKC). While the effects of DAG on model membranes have been investigated earlier, studies on physical properties of DAG introduced into phospholipid bilayers by PLC have been lacking. We present an NMR approach for studying structural and kinetic aspects of PLC-mediated hydrolysis of 13 carbonyl-enriched phospholipids in model membranes (small unilamellar vesicles). The product DAG is readily detected by 13C NMR, and its structural properties as well as those of the model membrane can be monitored continuously. PLC hydrolysis was limited to a low proportion of the model membrane by incorporating a small amount of ester phospholipid into a nonhydrolyzable ether-linked phospholipid matrix. Under these conditions, PLC (Bacillus cereus) hydrolyzed only the monolayer of phosphatidylcholine to which it was exposed (the outer monolayer). The 1,2-DAG product remained associated with the membrane bilayer and did not alter bilayer structure in any detectable way. From the chemical shift data, it is inferred that the DAG has an interfacial conformation similar to that of phosphatidylcholine. These results show that DAG could activate PKC by direct interaction with the enzyme rather than by perturbation of the membrane bilayer.

Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin.

Binding of 13C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin--two complementary fragments corresponding to the two halves of albumin and one fragment corresponding to the carboxyl-terminal domain--yielded unique patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding of the first 5 mol of oleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). Two of these resonances were seen upon addition of 1 or 2 mol of oleic acid to fragments representing either the carboxyl-terminal half (residues 307-582) or the carboxyl-terminal domain (residues 377-582); the third resonance was seen upon addition of 1 mol of oleic acid to the fragment representing the amino-terminal half (residues 1-306). The resonance patterns for the fourth and fifth moles of oleic acid added to albumin (secondary sites) could not be duplicated by addition of more oleic acid to individual fragments. These resonance patterns were generated, however, when the two complementary fragments were mixed in equimolar proportions to form an albumin-like complex with a reconstituted middle domain. Thus, two primary fatty acid binding sites are assigned to the carboxyl-terminal domain, one primary site is assigned to the amino-terminal half, and the secondary sites are assigned to the middle domain. This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domains.

The interfacial conformation and transbilayer movement of diacylglycerols in phospholipid bilayers.

The interaction of diacylglycerols, primarily 1,2-dilauroyl-sn-glycerol (1,2-DLG), with egg phosphatidylcholine (PC) bilayers was studied by NMR spectroscopy and other physical techniques. In the low proportions used (less than or equal to 20 mol % with respect to total lipid), 1,2-DLG formed bilayers with PC with no hexagonal phase separation, as assessed by light, polarizing and electron microscopy, and 31P and 13C NMR spectroscopy. The 13C-carbonyl chemical shift of 90% [13C]carbonyl 1,2-DLG was monitored in small unilamellar vesicles as a function of relative DLG content (1.5-20%) and temperature (10-55 degrees C). The chemically inequivalent sn-1 and sn-2 carbonyls gave a single, narrow resonance in vesicles, in contrast to neat 1,2-DLG and 1,2-DLG in organic solvents, whose spectra showed two well-separated carbonyl resonances. The chemical shift of 1,2-DLG in PC shows that the carbonyl groups are proximal to the aqueous interface, necessitating orientation of the DLG molecule along the normal to the bilayer. Both carbonyl groups are H-bonded to H2O, but the secondary ester (sn-2) carbonyl is relatively more hydrated than the primary ester (sn-1) carbonyl. The 13C-carbonyl chemical shift data further suggest that the interfacial conformation resembles that of crystalline and liquid crystalline lamellar 1,2-dilauroyl-sn-glycero-3-phosphatidylethanolamine and certain PCs, in which the glycerol backbone is perpendicular to the bilayer plane. This conformation is different from that of crystalline 1,2-dilauroyl-sn-glycerol, in which the glycerol backbone is parallel to the bilayer plane. Between 1.5 and 8% DLG in vesicles, the chemical shift of the 1,2-DLG carbonyl at a given temperature was constant. However, above 8% DLG the chemical shift at each temperature increased with increasing DLG concentration, suggesting increased hydration at higher DLG content. At low temperatures 13C NMR spectra of vesicles with the highest proportions of 1,2-DLG studied (15 and 20%) showed two DLG carbonyl resonances, which most likely represent 1,2-DLG on outer and inner leaflets of the vesicle bilayer. The two peaks collapsed into a single resonance by 38 degrees C, at which temperature the two environments equilibrate with a rate constant of approximately 60 s-1 (t1/2 approximately 10 ms). Thus, transbilayer movement of DLG is extremely fast compared with phospholipids. In vesicles the 1,3-isomer of DLG exhibited a narrow carbonyl peak slightly downfield from that of 1,2-DLG. Acyl chain migration from 1,2-DLG to 1,3-DLG was monitored directly in the vesicle by time-dependent NMR measurements.

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.