Developmental Changes in Enzymes Involved in Dolichyl Phosphate Metabolism in Cultured Embryonic Rat Brain Cells

Abstract

The rates of synthesis of dolichol-linked oligosaccharide 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 Vmax increased 10-fold between days 4 and 22, and there was also a significant change in the apparent Km 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 at 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Long‐Chain os‐Isoprenyltransferase Activity Is Induced Early in the Developmental Program for Protein N‐Glycosylation in Embryonic Rat Brain Cells

“…This study extends earlier work on a large developmental increase in Oligo-P-P-Do1 biosynthesis in primary cultures of embryonic rat brain cells (Bhat and Waechter, 1988;Bhat et al, 1991) by showing that there is a coordinate induction of IPTase activity, polyisoprenoid biosynthesis, and Oligo-P-P-Do1 bio- synthesis. All of the present results support the hypothesis that increases in IPTase activity and polyisoprenoid metabolism enable rat brain cells to increase the rate of synthesis, and consequently the RER pool size, of Dol-P. Several lines of evidence suggest that expansion of the Dol-P pool in the RER results in an increased rate of synthesis of Oligo-P-P-Do1 and protein N-glycosylation.…”

“…Although the reduction of the a-isoprene unit of dolichol has been investigated (Vigo and Adair, 1982;Ekstrom et al, 1984Ekstrom et al, , 1987, the mechanistic details of this reaction are still incompletely understood. Until the polyprenyl substrate for the putative reductase is firmly established, it will not be possible to outline the precise reactions by which Poly-P-P is converted to Earlier work described a large developmental increase in Oligo-P-P-Do1 biosynthesis and protein N-glycosylation that correlated with glial differentiation in primary cultures of embryonic rat brain cells (Bhat and Waechter, 1988;Bhat et al, 1991). In this article, we have extended the previous studies on the control of Oligo-P-P-Do1 synthesis in embryonic rat brain cells by examining the regulatory relationship between developmental changes in IPTase activity, Dol-P synthesis, and the induction of Oligo-P-P-Do1 biosynthesis.…”

Human Dolichol Kinase, a Polytopic Endoplasmic Reticulum Membrane Protein with a Cytoplasmically Oriented CTP-binding Site

“…The enzymatic transfer of phosphoryl groups from CTP to dolichol catalyzed by microsomal fractions from mammalian cells was first detected more than 20 years ago (3,4). Developmental changes in dolichol kinase (DK) activity corresponding to an increased capacity for protein N-glycosylation have been reported for sea urchin embryos (5), estrogen-treated chick oviducts (6), Dictyostelium discoideum (7), and pig (8) and rat brain (9,10). If the enzymatic reduction of the ␣-isoprene unit in dolichol occurs at the free polyprenol level as proposed by Sagami et al (11), DK would catalyze the final step in the de novo pathway for Dol-P biosynthesis (1).…”

Inhibitors of N‐linked oligosaccharide processing glucosidases interfere with oligodendrocyte differentiation in culture

“…Differentiation of oligodendrocytes, the myelinforming cells of the CNS, depends on cell interactions probably mediated by developmentally expressed glycoproteins. Previous studies have demonstrated a striking induction of N-glycosylation activity in primary cell cultures of developing rat brain (Bhat and Waechter, 1988;Bhat et al, 1991) corresponding temporally to the onset of oligodendrocyte differentiation, suggesting thereby a possible relationship between the two processes. This suggestion has received further support from studies using inhibitors of glycoprotein processing enzymes.…”