STRUCTURAL STUDIES OF MANDUCA-SEXTA LIPID TRANSFER PARTICLE WITH APOLIPOPROTEIN-SPECIFIC ANTIBODIES

Studies have been conducted to characterize structural and functional properties of Manduca sexta lipid transfer particle (LTP). LTP is a hi gh molecular weight complex of three apolipoproteins and lipid that fa cilitates the transfer of lipids between lipoproteins and tissues and among lipoproteins in insect hemolymph. Rabbit polyclonal antibodies w ere raised against each of the three LTP apolipoproteins isolated by p reparative electrophoresis. Immunoblot experiments demonstrated that t hey are apolipoprotein-specific and that LTP apolipoproteins are immun ologically distinct polypeptides. Antibody-capture enzyme-linked immun osorbent assay characterization of apolipoprotein-specific IgG demonst rated that each of the three antibodies recognizes native LTP and prov ided information on the apparent affinity of the antibodies for LTP. A polipoprotein specific IgG were then compared in lipid transfer assays to examine the effect of antibody binding on LTP-mediated lipid trans fer. Although each of the antibodies inhibited transfer activity, anti -apoLTP-II was capable of nearly abolishing activity at low IgG concen trations (< 26.7 mu g IgG/mu g LTP). In contrast, anti-apoLTP-I and an ti-apoLTP-III IgG inhibited LTP activity only at much higher concentra tions (> 133.3 mu g IgG/mu g LTP). These results indicate that apoLTP- II is a catalytically important apolipoprotein. In immunoprecipitation experiments, using I-125-labeled LTP, anti-holoLTP, anti-apoLTP-I, an d anti-apoLTP-II were each able to immunoprecipitate all three LTP apo lipoproteins while anti-apoLTP-III was not. When immunoprecipitations were carried out in the presence of the nondenaturing detergent, Nonid et P-40, however, anti-apoLTP-I and anti-apoLTP-II IgG were able to im munoprecipitate only apoLTP-I and -II while apoLTP-III was not immunop recipitated. Only apoLTP-III was immunoprecipitated by anti-apoLTP-III under these conditions. As expected, anti-holoLTP immunoprecipitated all three substituents in the presence of Nonidet P-40. These data sug gest that apoLTP-III can dissociate from the LTP complex upon treatmen t with nondenaturing detergent while apoLTP-I and -II remain associate d. The results also suggest that apoLTP-III interacts with the lipid c omponent of the LTP complex. Solubilization with detergent appears to disrupt this interaction, allowing the dissociation of apoLTP-III.