Glycoprotein synthesis in explants of developing rabbit mammary gland in culture

Abstract

Explants of mammary glands of pregnant rabbits cultured in the absence of insulin, prolactin and cortisol incorporated [2-3H]mannose into lipid-linked mono- and oligo-saccharide and protein. Inclusion of the hormones in the culture medium stimulated the incorporation of [2-3H]mannose into lipid-linked monosaccharide 4-fold, into lipid-linked oligosaccharide 4-fold and into protein 13-fold after 24 h in culture. Addition of tunicamycin to the incubation medium completely inhibited the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and protein after an initial lag period of about 2 h. Incorporation of this radiolabel into lipid-linked monosaccharide was increased 4-fold under these conditions. Incorporation of [4,5-3H]leucine into protein was unaffected by the presence of tunicamycin. Analysis of mannose-labeled proteins by polyacrylamide-gel electrophoresis indicated that a major radiolabled protein of apparent MW 65,000-70,000 was synthesized and approximately 70% of this protein appeared in the soluble fraction. Glycosylation of the protein but not synthesis of its peptide backbone was sensitive to tunicamycin. Possible origins of this glycoprotein synthesized when the tissue is stimulated to differentiate in culture are discussed.