Purification and characterization of a lipid transfer particle in Rhodniusprolixus: phospholipid transfer

In this study we report the purification and characterization of a lipid tr ansfer particle (LTP) from Rhodnius prolixus hemolymph, and its participati on in phospholipid and diacylglycerol transfer processes. H-3-diacylglycero l labeled low density lipophorin from Manduca sexta (H-3-LDLp) was incubate d with R. prolixus lipophorin (Lp) in the presence of Rhodnius hemolymph. F ollowing incubation and isolation, both lipoproteins showed equivalent amou nts of H-3-labeled lipids. Hemolymph was subjected to KBr gradient ultracen trifugation. SDS-PAGE analysis of gradient fractions showed the enrichment of bands with molecular masses similar to the M. sexta LTP standard. LTP co ntaining fractions were assayed and lipid transfer activity was of observed . Purification of LTP was accomplished by (i) KBr density gradient ultracen trifugation, (ii) size exclusion, (iii) Cu++ affinity and (iv) ion exchange chromatographies. LTP molecular mass was estimated similar to 770 kDa, com prising three apoproteins, apoLTP-I (315 kDa). apoLTP-II (85 kDa) and apoLT P-III (58 kDa). Phospolipid content of P-32-LTP was determined after two-di mensional TLC. P-32-phospholipid-labeled and unlabeled lipophorins, purifie d from R. prolixus were incubated in the presence of LTP resulting in the t ime-dependent transfer of phospholipids. LTP-mediated phospholipid transfer was not a selective process. (C) 2001 Elsevier Science Ltd. All rights res erved.