Facilitated diacylglycerol exchange between insect hemolymph lipophorins. Properties of Manduca sexta lipid transfer particle.

Abstract

Manduca sexta hemolymph lipid transfer particle (LTP) is a very high density lipoprotein (d = 1.23 g/ml) containing 14% lipid and 5% carbohydrate. Each of three apoprotein components, apoLTP-I (Mr approximately 320,000), apoLTP-II (Mr = 85,000), and apoLTP-III (Mr = 55,000), is glycosylated. Carbohydrate analysis revealed the presence of mannose and N-acetylglucosamine in a ratio of 4.5:1. A native Mr greater than 670,000 was determined by pore limiting gradient gel electrophoresis. Lipid analysis of LTP revealed the presence of phospholipid, diacylglycerol (DAG), free fatty acid, and triacylglycerol. Rabbit polyclonal antibodies directed against LTP were obtained. Anti-LTP serum was employed in experiments which indicated the presence of LTP in larval and adult animals and confirmed that LTP was unrelated to other M. sexta hemolymph proteins and lipoproteins. A quantitative lipid transfer assay measuring facilitated DAG exchange between isolated M. sexta lipoproteins was established. The level of LTP-catalyzed exchange of DAG increased linearly with increasing time and protein during the initial phase of the reaction. Inclusion of anti-LTP serum in the assay inhibited facilitated DAG exchange. Experiments designed to determine if the LTP holoprotein is required for transfer or if a component of LTP is the active principle were performed. Incubation of [3H]DAG labeled high density lipophorin with substrate amounts of LTP resulted in incorporation of labeled DAG into LTP. Subsequent incubation of [3H]DAG-labeled LTP with unlabeled lipophorin resulted in exchange of DAG and the appearance of labeled DAG in lipophorin. Nitrocellulose-bound LTP apoproteins did not facilitate DAG exchange, and pretreatment of LTP with detergents resulted in loss of transfer activity. Extraction of LTP lipids with ethanol/ether also resulted in loss of activity. The results suggest that the lipid component of LTP may be important in the transfer reaction.