DETECTION OF LIPOPHORIN AS THE MAJOR CYCLOSPORINE-BINDING PROTEIN IN THE HEMOLYMPH OF THE GREATER WAX MOTH GALLERIA-MELLONELLA

The body distribution and binding of Cyclosporin A (CsA) to hemolymph proteins have been studied in Galleria mellonella larvae. H-3 dihydroc yclosporin A ((3)HdCsA), a radiolabelled analogue of CsA, was used to determine the body distribution and clearance of CsA in the hemocoel o f treated larvae and to label hemolymph binding proteins in vitro. The experiments with (3)HdCsA showed that the compound is rapidly removed from the hemolymph, then accumulated and stored in the fat body. Only a limited amount of radioactivity was excreted through the feces. Fou r hemolymph (3)HdCsA binding proteins were determined by the analytica l isoelectric focusing method (IEF). The major binding protein with a pI of about 7.3 was isolated through a density gradient followed by fa st performance liquid chromatography (FPLC). Gel permeation chromatogr aphy of purified protein on superose 6 column (FPLC) revealed a single peak. Denaturating polyacrylamide gel electrophoresis (SDS PAGE) reve aled the presence of two bands with the molecular weight of about 200 and 80 kD in both reducing and non-reducing conditions. Amino acid ana lysis showed a high content of aspartic and glutamic acids, leucine an d lysine, respectively. The CsA-binding protein was identified as lipo phorin, a major insect lipoprotein. This study provides the first dire ct evidence for the influence of hemolymph proteins on the binding and body distribution of secondary metabolites released by entomopathogen ic fungi. (C) 1997 Elsevier Science Inc.