Lipoprotein lipase activity in cultured mesenchymal rat heart cells Part 5. effect on enzyme activity of the glycosylation inhibitors, 2-deoxyglucose and tunicamycin

Abstract

Lipoprotein lipase activity was studied in rat heart cells cultures grown for 6–8 days. Addition of 5 mM 2-deoxyglucose to the culture medium resulted in a marked reduction in lipoprotein lipase activity within 4 h. At lower concentrations (1.25–2.5 mM) of 2-deoxyglucose, enzyme activity decreased 20% in 2 h and 75% in 4 h. At the same time there was no change in the incorporation of labeled leucine into cellular proteins. The decrease in enzyme activity was accompanied by a 60–80% inhibition of glycoprotein synthesis as determined by the reduction in the incorporation of labeled glucosamine, mannose and galactose. Inhibition of glycosylation and lipoprotein lipase activity was also studied with tunicamycin. Incubation with tunicamycin (0.5–5 μg/ml) had no effect on lipoprotein lipase activity during the first 90 min, the decrease was measurable at 3 h and was 40–68% by 14 h. Exposure of the cultures to tunicamycin for 24 h resulted in a 9% decrease in protein synthesis and a 50% reduction in incorporation of labeled glucosamine and mannose into glycoproteins. Neither 2-deoxyglucose nor tunicamycin interfered with the transport of the active enzyme to the cell surface. The treatment with the inhibitors used in the present study results in the synthesis of glycoproteins deficient in asparagine-linked oligosaccharides and caused a marked reduction in lipoprotein lipase activity. Thus, is seems that interference with glycosylation affects the expression of enzyme activity. The possibility that interference with glycoslation resulted in enhanced degradation of the enzyme molecule was not ruled out by the present experiments.