Maturation and secretion of lipoprotein lipase in cultured adipose cells. II. Effects of tunicamycin on activation and secretion of the enzyme.
The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC50 values of inhibition by tunicamycin (approx. 0.2 microgram/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tunicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable Mr 52 000 protein, whereas a single Mr 57 000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells.
D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D005944:Glucosamine / Q000031:analogs & derivatives*; D005947:Glucose / Q000494:pharmacology; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008970:Molecular Weight; D014415:Tunicamycin / Q000494:pharmacology*
D014415:Tunicamycin; D008071:Lipoprotein Lipase; D005947:Glucose; D005944:Glucosamine