Cellular distribution, levels, and function of the diazepam-binding inhibitor/acyl-CoA-binding protein in last instar...

The diazepam-binding inhibitor (DBI) was originally isolated as an endogenous competitor of diazepam for mammalian central nervous system binding sites. Later DBI was found to be identical with an intracellular medium-long chain acyl-CoA-ester-binding protein (ACBP). Its phylogenetic distribution was also extended outside vertebrates to insects and fungi. We studied DBI/ACBP biochemistry and ultrastructural distribution to learn more about the potential role(s) of the insect protein in lipid transport in the tobacco hornworm, Manduca sexta. We expressed and purfied the M. sexta DBI/ACBP from E. coli for the production of specific polyclonal antisera. We also showed specific binding of [14C]-oleoyl-CoA to the purified protein, supporting its evolutionarily conserved role as an acyl-CoA-binding protein. With the use of an enzyme-linked immunosorbant assay (ELISA) and Northern analysis, it was determined that the M. sexta ACBP is expressed highest during times of active feeding and lipid transport by the larval midgut. Study of the ultrastructural distribution, by immunocytochemistry and electron microscopy, of ACBP in larval midgut showed that acyl-CoA transport is localized throughout the M. sexta columnar cell.