Cell-free translation of avian adipose tissue lipoprotein lipase messenger RNA

Abstract

Poly(A) + mRNA isolated from chicken adipose tissue directed cell-free translation in a rabbit reticulocyte lysate system. Immunoadsorption with polyclonal antibodies against lipoprotein lipase detected a protein of 56 ± 2 kDa. Immunodetection of this protein was prevented by inclusion of purified lipoprotein lipase in the assay mixture. Identification of the 56 kDa protein as lipoprotein lipase was confirmed by immunoadsorption to the monoclonal antibody CAL 1–11. Inclusion of dog pancreatic microsomal membranes in the translation system resulted in isolation of an additional protein of 62 kDa. Treatment of the 62 kDa protein with endo- β- N-acetylglycosaminidase H or endo- β- N-acetylglucosaminidase F decreased the observed molecular mass to that of the primary translation product, indicating that the increase in molecular mass resulted from the addition of N-linked oligosaccharides. Starving and refeeding chickens prior to poly(A) + mRNA isolation resulted in a 3-fold increase in the amount of immunodetectable lipoprotein lipase synthesized.