Distribution And Metabolism Of Dolichol And Its Derivatives

The phosphorylated derivatives of dolichol are essential intermediates in the biosynthesis of asparagine-linked glycoproteins. The aim of the research in this thesis was to obtain detailed information about the distribution and metabolism of these polyisoprenoid lipids in eukaryotic organisms and especially in rat tissues and liver subcellular fractions. Methods utilizing reverse-phase high performance liquid chromatography were developed for measuring tissue levels of dolichol, dolichyl phosphate and dolichyl fatty acyl esters. All rat tissues including serum contained varying concentrations of dolichol ranging from a high of 165(mu)g/g in the spleen and 60(mu)g/g in the liver to 23(mu)g/g in the heart. Only about 5% of hepatic dolichol was found in the microsomal compartment of the cell where glycoprotein biosynthesis occurs, while 50% was in a lysosome-enriched fraction. Other subcellular fractions including nuclei, mitochondria, plasma membranes and cytosol contained significant but smaller pools of dolichol. Dolichol in rat and human serum was associated with the high density lipoproteins and our data suggested that there is a carrier protein for cytosolic dolichol as well. The total concentration of all known derivatives of dolichol in the liver was 66(mu)g/g fresh weight, of which about 10% was esterified to long chain fatty acids, 80% was in the free alcohol form, 4% was present as dolichyl phosphate (Dol-P) and 5% was in other phosphorylated and glycosylated forms. Therefore these data are consistent with the proposal that Dol-P levels may be a rate-limiting factor in glycoprotein biosynthesis. Most of the liver Dol-P was in the microsomes where it accounted for over 40% of the dolichol present. Rough and smooth endoplasmic reticulum and Golgi all contained substantial pools of Dol-P, supporting the idea that Dol-P may be involved in the transbilayer movement of sugars used in glycoprotein formation from the cytosolic to the luminal side. Dol-P levels in the liver rose markedly when the rate of glycoprotein synthesis was increased by administering turpentine to rats. The elevation in Dol-P levels was due to increased amount of microsomal dolichol available for phosphorylation and decreased dephosphorylation of preformed Dol-P