Isolation and characterization of lipophorin from the larval stalk borer Busseola Fusca

Abstract

In Kenya the most important and prevalent pests that attack maize and sorghum are the stalk-borers. There are three species of the stalk borer in Kenya namely, GhilQ which thrives in high altitude areas with cool climate, poses the greatest threat to the crops. Lipophorin acts as a reusable shuttle in the transportation of a variety of lipid classes from sites of storage, absorption and synthesis to sites of utilization (Chino, 1985). Lipids playa vital role in insect development as components of cell membranes and cuticle, source of metabolic energy and as hormones and pheromones. Interference with lipid transport would therefore be fatal to the insects. Studies of lipophorins thus hold potential for selective control of destructive insect pests. In this study, lipophorin, the principal haemolymph lipoprotein in most insects, was isolated and characterized from B. Lipophorin was isolated from larvae by density gradient ultracentrifugation. The apoproteins were isolated by electroellution following SDS-PAGE. The isolated lipophorin (Mr-700,000) was a high density lipoprotein (density = 1.13 g/ml), composed of 46% lipid, 50% protein and 4% carbohydrate. Analysis by SDS-PAGE revealed that it consisted of two apoproteins, apoLp-I (Mr-210,OOO) and apoLp-II (Mr-78,OOO), which were present in a molar ratio of 1:2 (apoLp-I: apoLp-II) in the intact lipophorin molecule. Both apoproteins were glycosylated and lipidated as shown by PAS and Sudan Black staining, respectively. The presence of high mannose containing oligosaccharide chains was demonstrated by the binding of lipophorin to concanavalin A-Sepharose column. Studies on the lipid lipophorin indicated predominance of phospholipids and diacylglycerol. Amino acid composition analysis of lipophorin showed predominance of glutamate (9%), aspartate (13%) and glycine (9%). Methionine was only present in trace amounts. Structural organization of the apoproteins was investigated by limited trypsin digestion and immunological studies. Limited trypsin digestion of the isolated lipophorin showed that apoLp-I was more susceptible to cleavage than apoLp-II, suggesting an interior location of apoLp-II .By immunoblotting,both apoproteins were shown to be immunoreactive towards antibodies to the isolated lipophorin .However,double radial immunodiffusion of the apoprotein against the antisera only showed precipitin line with apoLp-I. Thus in the intact lipophorin,apoLp-I is more exposed to the aqueos haemolymph enviroment than apoLp~II. An investigation was carried out to determine whether there was immunological cross-reactivity between lipophorins showed the same cross-reactivity. The results suggested that lipophorins from insects of the same order share antigenic determinants predominantly on apoLp-I.