The biological role of dolichol.
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Animals↗
Biomedical subjects
T Chojnacki
Publications and source records attributed to T Chojnacki.
Esterification of dolichol in rat liver.
In the various subcellular fractions of rat liver 45-75% of the total dolichol was esterified with a fatty acid. The esterification reaction was localized exclusively in the microsomes, and the transferase activity is 3-fold higher in the cation-insensitive smooth microsomes than in other microsomal subfractions. Although fatty acyl-CoAs tested served as substrates, palmitoyl-CoA was the most rapidly utilized. None of the phosphatidylcholine or phosphatidylethanolamine species tested could be utilized to esterify dolichol with a fatty acid, indicating the absence of transacylation. alpha-Saturated dolichols were esterified at a higher rate than their alpha-unsaturated counterparts. Albumin and low concentrations of Triton X-100 activated the esterification reaction, which was not dependent on mono- or divalent cations, ATP, or CoA. The sensitivity of the transferase activity to trypsin indicates localization of the enzyme(s) involved on the outer surface of microsomes (i.e. the cytoplasmic surface of the endoplasmic reticulum), as is also the case for enzymes of dolichol biosynthesis. Transferase activity was detected in all tissues examined but at a much lower level than in liver and testis. The patterns of fatty acids in dolichol esters of different organelles exhibited some specificity. Labeling in vivo indicated that esterification of dolichol may play a role in targeting this lipid from the endoplasmic reticulum to lysosomes.
Acyltransferases↗
The half-lives of dolichol and dolichyl phosphate in rat liver.
Rat liver dolichol and dolichyl-P were labeled by injection of [3H]mevalonate into the portal vein and their rates of synthesis and breakdown determined. In the initial phase the radioactivity appeared in alpha-unsaturated polyprenols. Subsequent saturation required 90 min. The half-lives of dolichols in microsomes were between 80 and 118 h, and shorter dolichols had shorter values of T1/2. The half-lives of dolichols in lysosomes were between 115 and 137 h, while microsomal dolichyl-P exhibited a T1/2 of 32 h. Injected dolichol was recovered in the lysosomes of hepatocytes and exhibited a rate of breakdown which was slower than that of the endogenous compound. These results indicate differences in the catabolism of dolichol at different subcellular locations, as well as differences between the catabolism of dolichol and dolichyl-P.
Animals↗
The terminal reactions in polyisoprenoid biosynthesis.
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Animals↗
Long-chain polyprenols in gymnosperm plants.
Over 100 species of gymnosperm plants were checked for the presence of long chain polyprenols. Poly-cis long chain prenols, mainly as acetates, were found in green needles of about 60 species. In Cycadopsida either prenol-18 or prenol-20 were the main components of the natural polyprenol mixture. In Coniferopsida either a single polyprenol family was present like in all species of Pinaceae, or polyprenols consisted of two families differing in the size of polyprenol molecules: one family in which prenol-17 was the dominating component, and the other family of prenol-23. These complex mixtures of polyprenols were present in Araucariaceae, Cupressaceae, Taxodiaceae and also in Taxopsida. Seasonal variations were observed in the polyprenol content in green leaves.
Plants↗
The alpha-saturation and terminal events in dolichol biosynthesis.
Incubations of 10,000 X g supernatant from rat liver with [3H]mevalonate were performed and the labeling of polyprenols was studied. It was demonstrated that factors like pH, substrate concentration, and presence of detergent not only greatly influence the total incorporation but also the relative distribution of radioactivity among the isoprenologues. The synthesis was shown to be extremely sensitive to Triton X-100. Substrate concentrations of 1 and 100 microM mostly gave polyprenols with 18 and 20 isoprenes, respectively. At a given substrate concentration, pH 6.5 resulted in shorter polyprenols than did pH 7.5. Ozonolytic fragmentation demonstrated that in the initial phase of incubation, polyprenols are elongated by 1 isoprene residue and saturated to give dolichols. No substantial dephosphorylation of polyprenyl phosphates to the free alcohol occurred. The production of dolichol in vitro was shown to utilize NADH for the saturation event. This seemed to occur concomitantly with the synthesis. alpha-Saturation of polyprenyl-P could not be achieved with the procedures employed. It is proposed that the synthesis of dolichol and dolichyl-P do not share the same terminal steps; saturation and terminal isoprene condensation occur in cooperation; and substrate concentration and pH influence the terminal enzyme(s) and the nature of the final product in the polyprenol biosynthesis.
Animals↗
Separation and quantitation of dolichyl esters by high-performance liquid chromatography.
An HPLC procedure for the isolation and quantitation of total and individual dolichyl esters in tissues has been developed. The purified lipid extracts are subjected to sequential reversed-phase, straight-phase, and reversed-phase HPLC, which yield complete resolution and high recovery of the individual dolichyl esters. The isoprenoid distribution in the esterified fraction was similar to that of the free alcohol fraction in liver, kidney, and spleen. All fatty acids present in the total fraction were also recovered in all the individual polyisoprenoids. Dolichyl esters thus appear to differ from other lipid esters in tissues in containing a broader range of fatty acids.
Animals↗
Dolichyl phosphate induces non-bilayer structures, vesicle fusion and transbilayer movement of lipids: a model membrane study.
The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles comprised of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was studied using a fluorescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilayer movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the phosphatidylcholine-specific transfer protein. 31P-NMR and freeze-fracture electron microscopy were employed to study the macroscopic organization of DOPC and DOPE containing model membranes in the absence or presence of dolichyl phosphate. The results indicate that both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; the amount of exchangeable PC from MLVs is increased by dolichyl phosphate, probably as a result of fusion processes; dolichyl phosphate destabilizes the bilayer organization in MLVs comprised of DOPE and DOPC, resulting in the formation of hexagonal (HII) phase and 'lipidic' particles.
Diterpenes↗
Inhibition of dolichyl phosphate biosynthesis by compactin in cultured rat hepatocytes.
Isolated rat hepatocytes were cultured in monolayer for about 24 h. During this period the cells exhibited constant protein and lipid synthesis. When the culture medium contained compactin, a competitive inhibitor of the 3-hydroxyl-3-methylglutary-coenzyme-A reductase, dolichyl-P synthesis was inhibited by 91% at the end of the incubation, as estimated by the incorporation of [3H]acetate and by 77% as estimated by the incorporation of 32Pi. These results indicate that in primary cultures of rat hepatocytes dolichyl monophosphate is mainly synthesized through a de novo process, while phosphorylation through the CTP-mediated kinase is of limited functional importance.
Acetates↗
Effectivity of dolichyl phosphates with different chain lengths as acceptors of nucleotide activated sugars.
Chemical synthesis of different S-forms of dolichyl-P was performed in order to investigate the use of these polyprenes in mannosyl, glucosyl and glucosaminyl transferase reactions. Determination of the Vmax values for a series of dolichyl-P demonstrated that the velocities of transferase reactions with all those dolichyl-P derivatives present in animal tissues are largely the same. The apparent Km values for the various dolichyl-P in the transferase system studied differed, but this property does not appear to have physiological importance.
Animals↗
Reaction of optically active S- and R-forms of dolichyl phosphates with activated sugars.
Chemical synthesis was used to produce optically active isomers of dolichol (S- and R-forms) with 18 and 19 isoprene residues. The phosphorylated polyprene was studied in rat liver microsomal GDP-mannosyl and UDP-N-acetylglucosaminyl transferase systems. The two dolichol-P forms in both transferase systems gave Vmax values which for the S-form exceeded 4-6 times what was obtained with the R-form. The Km values were also higher for the S-form. The hepatocyte appears to contain a large excess of dolichyl-P, by 100 times exceeding that of the Km values. For this reason the S-form of dolichyl-P seems to be one of the requirements for the normal establishment of the N-glycosidically linked oligosaccharide chain.
Animals↗
The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes.
The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P NMR, small-angle x-ray scattering, differential scanning calorimetry, and freeze-fracture electron microscopy. These studies suggest that dolichol and dolichol derivatives destabilize unsaturated phosphatidylethanolamine containing bilayer structures and promote hexagonal II phase formation; high concentrations of dolichol induce lipid structures characterized by "isotropic" 31P NMR and particulate fracture faces; dolichol, contrary to cholesterol, has no effect on the thermotropic behavior of membranes consisting of phosphatidylcholine, while dolichyl-P incorporation abolishes the transition from the gel to liquid crystalline phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine; both dolichol and dolichyl-P increase the fatty acid fluidity in phosphatidylethanolamine mixtures; the effect of dolichol on bilayer structure and fluidity is more pronounced with increasing number of isoprene residues; dolichol esters are only soluble to a limited extent in the bilayer and segregates into domains at low concentrations; the results are consistent with a localization of dolichyl-P in which the phosphate group is oriented to the water interphase. The induction of hexagonal II phase by dolichyl-P may elicit the transmembrane movement of glycosylated lipid intermediate.
Calorimetry, Differential Scanning↗
The presence of dolichol in liver supernatant.
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Animals↗
Dolichols and enzymatic formation of dolichyl phosphate sugars in the thymus of mice on neoplastic transformation.
The content of dolichol in thymus gland of mice increased up to four times upon malignant transformation evoked by X-ray irradiation of animals or/and in the case of spontaneous tumours as compared with normal thymus. Dolichyl phosphate mannose and dolichyl pyrophosphate N-acetylglucosamine were formed in homogenates of transformed thymus at a higher rate than in normal gland.
Animals↗
Metabolic labeling of dolichol and dolichyl phosphate in isolate hepatocytes.
Isolated hepatocytes from rat liver were incubated with [3H]mevalonate, and the labeling of polyprenols in the microsomal fraction was followed. After a 1-min incubation the alpha-unsaturated forms of polyprenyl-P2, -P, and polyprenol were mainly labeled and at this time point only 2, 8, and 17%, respectively, of the label was associated with the saturated forms. In the case of the free alcohol 2 h of incubation was required before all the labeling was recovered in the saturated form. After 1 min polyprenols and polyprenyl-P with 20 and 21 isoprene residues demonstrated much higher specific labeling than the shorter compounds, but after 5 min these differences were greatly reduced. In experiments utilizing short incubation times and chasing no evidence has been obtained that the phosphorylated form is a precursor of the free alcohol or vice versa, that the free alcohol is a precursor of the phosphorylated form. In human liver about 1% of the dolichol is present in the alpha-unsaturated form. These experiments suggest that: 1) the alpha-unsaturated form is the precursor of the alpha-saturated free alcohol, 2) dolichol does not necessarily arise directly from dephosphorylation of the phosphorylated form, and 3) the free alcohol is for the most part not phosphorylated under in vivo conditions in rat liver.
Animals↗
Role of dolichyl phosphate in regulation of protein glycosylation in 2-acetylaminofluorene-induced carcinogenesis in rat liver.
Hyperplastic nodules and hepatocarcinomas were produced in rat liver by 2-acetylaminofluorene-containing diet. The homogenates and isolated microsomes were analyzed for the content of lipid intermediates and glycosylation reactions. The dolichol content of hyperplastic nodules increases four times in the homogenate and six times in the microsomes. In developed hepatocarcinoma, the amount of dolichol was doubled. Concerning the distribution pattern of the polyprenols, there is a change in the relative amounts of dolichols with 18 and 19 residues. In contrast to the free alcohol, dolichyl phosphate was greatly decreased in nodules, a finding which might be explained by a decreased dolichol kinase and an increased dolichol monophosphatase activity. The percentage of total phosphorylated dolichol was related to the glycosylating capacity. In microsomes, mitochondria, and homogenate from normal liver and in homogenate from hyperplastic liver nodules, the percentages of dolichyl phosphate were 23, 2, 16, and 4, respectively. At maximal glycosylation in vitro, only part of the total dolichyl phosphate was glycosylated. Dolichol-mediated protein glycosylation exhibited a general decrease in the microsomes from nodules and cancer tissue; it is suggested that the main cause of the decrease is a shortage of the available dolichyl phosphate which is rate limiting and which also contributes to the synthesis of the modified oligosaccharide chain.
2-Acetylaminofluorene↗
The occurrence and seasonal distribution of C50-C60-polyprenols and of C100-and similar long-chain polyprenols in leaves of plants.
Large amounts of fully unsaturated, mainly-cis, higher isoprenoid alcohols consisting of 17-30 isoprene units were found in several plants of the Rosaceae family. They occur as mixtures of several prenologues with either C85 - or C100 - prenol dominating in the form of acetates. The highest level of these polyprenols (0.5-1.0% of wet weight) were found in Crataegus crus- galli , Cotonoaster lucida, Prunus serotina and Sorbus suecica (intermedia). Their content increased with increasing age of the leaves. The dynamics of this rise is different from that observed in the case of accumulation of free C50- C60 - prenols (up to 0.5% of wet weight) in leaves of various plant species.
Plant Physiological Phenomena↗
Effect of ethionine on dolichyl phosphate-dependent transglycosylation reactions in rat liver.
Dolichyl phosphates of different chain length (C35, C55 , C75 , Dol-mixture of C90 , 95, 100, 105 and C110 ) were tested as lipid acceptors in transglycosylation reactions. In the absence of exogenously added dolichyl phosphates there were no differences in the rate of synthesis in liver of dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate N-acetylglucosamine between normal and ethionine-treated animals. Addition of exogenous dolichyl phosphates of different chain length stimulated the synthesis of dolichyl phosphate mannose and dolichyl pyrophosphate N-acetyl-glucosamine 2 to 4 times depending on the chain length of dolichols , both in normal and ethionine-treated animals. In liver of ethionine-treated rats the formation of dolichyl phosphate glucose was not stimulated. Following ethionine treatment the concentration of free and esterified with fatty acids dolichols was increased.
Animals↗