DEVELOPMENTAL-CHANGES IN ENZYMES INVOLVED IN DOLICHYL PHOSPHATE-METABOLISM IN CULTURED EMBRYONIC RAT-BRAIN CELLS

The rates of synthesis of dolichol-linked oligosacharide intermediates and protein N-glycosylation increased substantially during a developmental period corresponding to glial differentiation in primary cultures of embryonic rat brain. In this study developmental changes in three enzymes involved in dolichyl phosphate (Dol-P) metabolism have been examined by in vitro assays and correlated with the induction pattern for lipid intermediate synthesis and protein N-glycosylation. Dolichyl pyrophosphate (Dol-P-P) phosphatase activity was relatively low during the first 9 days in culture, but it increased significantly between days 9 and 25. Dol-P-P phosphatase did not change appreciably between days 22 and 30 in culture. A kinetic analysis of the developmental change in Dol-P-P phosphatase activity revealed that the V(max) increased 10-fold between days 4 and 22, and there was also a significant change in the apparent K(m) for Dol-P-P. Dolichol kinase activity increased during the period (9-15 days) when there was a significant induction in oligosaccharide-lipid synthesis and protein N-glycosylation, and then declined in parallel with lipid intermediate synthesis and protein N-glycosylation. Dol-P phosphatase activity was present in relatively low levels for the first 9 days in culture, but it increased steadily between days 9 and 30. A kinetic comparison of the activity in membrane fractions from brain cells cultured for 9 and 25 days indicated that there was a 10-fold increase in enzyme protein with unaltered affinity for Dol-P. The results suggest that elevated dolichol kinase activity enhances the rate of lipid intermediate synthesis, and subsequent reciprocal changes in dolichol phosphorylation-dephosphorylation are a regulatory factor in the deactivation of oligosaccharide-lipid synthesis, and consequently of protein N-glycosylation, during the period following glial differentiation in primary cultures of embryonic rat brain cells.