IDENTIFICATION AND LOCALIZATION OF 2 DISTINCT MICROENVIRONMENTS FOR THE DIACYLGLYCEROL COMPONENT OF LIPOPHORIN PARTICLES BY C-13 NMR

C-13 nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microen vironment of diacylglycerol (DG), their major neutral lipid component, Natural abundance C-13 NMR spectra of high density lipophorin exhibit ed several well-separated resonances derived from its lipid moiety, in cluding those for the carbonyl carbon atoms of phospholipid and DG fat ty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-C-13]oleoylglyce rol high density lipophorin was isolated after instilling a bolus of t ri[1-C-13]oleoylglycerol into the midgut of larvae fed a fat-free diet . C-13 NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm, Expansion of this reg ion revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from sp ontaneous acyl chain migration of 1,2-DG following lipoprotein isolati on. On the basis of compositional and structural analysis of this lipo protein, it is postulated that these DG species are localized predomin antly in the hydrophobic core of the particle. By contrast, natural ab undance C-13 NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surfa ce of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed. The data revealed that loss of the phospholi pid acyl chain carbonyl carbon resonance correlated with the appearanc e of two additional DG acyl chain carbonyl resonances (separated by 0. 2 ppm) possessing chemical shifts similar to those observed in LDLp, I nterestingly, the resonance assigned as 1,2-DG at the surface, predomi nanted immediately after phospholipase C hydrolysis, but its intensity decreased with time. Concomitantly, there was a corresponding increas e in the resonance assigned as 1,3-DG, consistent with an interconvers ion of these isomers through acyl chain migration, Taken together, the results provide the first direct experimental evidence that DG molecu les present at the surface monolayer and the particle core, respective ly, can be distinguished. Thus it should be possible to design experim ents to evaluate exchange between these two locations as well as the r ole of surface associated DG in binding of the exchangeable apolipopro tein, apolipophorin in.