Dolichol Derivatives Research Articles

A proteomic approach for identification of plasma biomarkers of hepatocellular carcinoma in the Gambia

2H-NMR investigation of polyisoprenols (PIs) in model membranes has revealed information about their motions, relative order, and locate within the membrane. Initial 2H-NMR studies of the organization of the shorter chain homologues geraniol (C10), farnesol (C15), and solanesol (C45) were carried out by incorporating 2H-acetyl esters of the alcohol or the di-perdeuterome-thylated derivatives of the omega-labeled phenols into multilamellar phosphatidylcholine (PC) vesicles. 2H-NMR powder patterns interpretable in terms of quadrupole splittings and spin-lattice relaxation times were obtained. Similar experiments have now been carried out with the labeled free alcohol, acetyl ester, and phosphate ester of dolichol (C95) and undecaprenol (C55). 2H-NMR results show that the head and tail 2H-labeled sites of C35 and C95 exhibit a fast motion isotropic signal only; no slower motion anisotropy, as exhibited by the short chain PIs, was observed. These data suggest that C55 and C95 either havesubstantially different (faster motions and/or conformations relative to the shorter chain PIs within the membrane, and that the longer PIs alter the membrane host packing matrix. This conclusion was supported by 31P-NMR studies of C55 and C95 derivatives in PC and PE/PC membranes, which showed new pronounced spectral changes relative to the results obtained with the shorter chain PIs. These spectral changes indicate that undecaprenol and dolichol derivatives appear to induce a non-bilayer (isotropic) organization of phospholipid molecules in PE/PC (2:1) vesicles. The possible physiological consequences of this perturbation remains to be determined.

Dolichol-dependent synthesis of chitobiosyl proteins and their further mannosylation. A second route for glycosylation of proteins in rat-spleen lymphocytes?

Incubation of whole lymphocytes with UDP-N-acetyl [3H]glucosamine used as the only precursor leads to the formation of dolichyl diphosphate [3H]chitobiose, DolPP-(GlcNAc)2, and dolichyl diphosphate N-acetyl-[3H]glucosamine, DolPP-GlcNAc. Although very few dolichyl diphosphate oligosaccharides are formed, a high level of radioactivity is recovered with proteins and has been characterized, using hydrazinolysis procedure, as [3H]chitobiosyl and N-acetyl[3H]glucosaminyl units. Addition of tunicamycin inhibits, to the same extent, both the synthesis of DolPP-(GlcNAc)1-2 and the incorporation of the N-acetyl[3H] glucosaminyl residues onto proteins, indicating that these carbohydrate units are transferred onto proteins acceptors from their dolichol derivatives. Chase experiments have indicated that, in fact, the DolPP-(GlcNAc)1-2 were utilized in two ways: either their transfer onto proteins or their degradation into water-soluble saccharidic material. Moreover, the transfer reaction appears to be a slow process compared to the degradation since the radioactivity chased from the DolPP-(GlcNAc)1-2 is not recovered on proteins. This fact allows to show that part of the [3H]chitobiose previously bound to proteins is further converted into oligomannosidic glycans in the presence of GDP-mannose. This direct mannosylation of chitobiosyl-proteins may represent a second route for the N-glycosylation of proteins.

Thyrotropin controls dolichol-linked sugar pools and oligosaccharyltransferase activity in thyroid cells

We previously showed that thyroglobulin (Tg) glycosylation is enhanced 1.5-fold under thyrotropin (TSH) stimulation, corresponding to an increased number of oligosaccharide chains per molecule of Tg. Now the steps involving dolichol components and oligosaccharyltransferase activity have been studied. Porcine thyroid cells were cultured on porous bottom filters with or without TSH and incubated with [ 14C]mevalonate. Under TSH regulation, the level of the whole of dolichol components was increased 1.25-fold without modifying their distribution. Dolichol, and free and monosaccharide-linked dolichyl-phosphate, represented respectively 40% and 45% of total dolichol components while dolichyl-pyrophosphate-oligosaccharide represented 3% only. A marked enhancement (4.2-fold) of oligosaccharyltransferase activity occurred in stimulated cells, which could correspond to the addition of the two TSH effects: stimulation of Tg synthesis (3-fold) and of Tg glycosylation (1.5-fold). The amount of lipid carriers appeared to be insufficiently increased but no component is a limiting step, suggesting that the turnover of dolichol derivatives may be increased under TSH control through their use by higher amounts of Tg.

Three enzymes involved in oligosaccharide-lipid assembly in Chinese hamster ovary cells differ in lipid substrate preference

Initial steps in N-linked glycosylation involve formation of a large oligosaccharide structure on a lipid carrier, dolichyl phosphate. We have previously characterized Chinese hamster ovary (CHO) glycosylation mutants (Lec9 cells) that utilize the polyisoprenoid lipid polyprenyl phosphate rather than dolichyl phosphate in these glycosylation reactions. Polyprenyl phosphate differs from dolichyl phosphate only in the degree of saturation of its terminal isoprenyl unit. Our goal was to determine whether the glycosylation defect of Lec9 cells could be explained simply by knowing lipid substrate preferences of the enzymes involved in the assembly of oligosaccharide-lipid (OSL) intermediates. In this study, we have used in vitro assay systems to compare the ability of dolichyl phosphate and polyprenyl phosphate to act as substrates for three glycosyl transferase enzymes involved in OSL assembly. In order to insure that we were only examining lipid substrate preferences of the enzymes and not other potential defects present in Lec9 cells, we used membranes prepared from wild-type cells in these in vitro reactions. Our results indicate that one of the enzymes, mannosylphosphoryldolichol (MPD) synthase, exhibited a significant preference for the dolichol substrate. Glucosylphosphoryldolichol (GPD) synthase, on the other hand, showed no binding specificity for the dolichol substrate, although the enzyme used the dolichol substrate at a twofold higher rate. N,N'-diacetyl-chitobiosylpyrophosphoryldolichol (CPD) synthase was able to use either lipid substrate with equal efficiency. These results suggest that not all glycosyl transferases in this pathway show a preference for dolichol derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and characterization of dehydrodolichyl diphosphate synthase from the yeast Saccharomyces carlsbergensis.

When the membrane fraction of Saccharomyces carlsbergensis was incubated with radiolabeled isopentenyl diphosphate in the presence of farnesyl diphosphate and Mg2+, phosphorylated and free long-chain polyprenols were formed. The reaction was inhibited by EDTA and heavy metal cations. A series of non-ionic detergents were studied for their efficacy to solubilize the prenyltransferase. The enzyme completely lost its activity in the presence of 0.1% of Triton X-100. n-Octyl-beta-(D)glucopyranoside at the concentration of 0.25-0.5% (10-15 mM) was used to solubilize the prenyltransferase. Both the membrane-bound enzyme and the solubilizate possessed a broad pH optimum shifted to alkaline pH values. The temperature optimum of the solubilizate was somewhat lower than that of the membrane preparation, owing to the significantly lower thermostability of the solubilized enzyme in comparison with the membrane-bound one. The phosphorylated reaction products formed in the presence of the membrane preparation had the same composition as the yeast dolichol synthesized in vivo. Non-phosphorylated polyprenols were formed during the incubation with membranes but not the solubilized enzyme. The composition of the polyprenols was also coincident with that of yeast dolichol, and the individual C80-homolog of the mixture was polyprenol but not dolichol as judged by adsorption HPLC. The results are discussed in relation to the terminal stages in the biosynthesis of dolichol derivatives.

Uptake and transport of fluorescent derivatives of dolichol in human fibroblasts

We are using fluorescent derivatives to visualize the endocytic transport of dolichol intermediates from the cell surface to the lysosome, and to estimate their rate of turnover within the lysosome. Anthroyl dolichol and anthroyl [l- 14C]dolichol were synthesized and purified by chromatography on silica and C 18 Sep-Paks followed by high-performance liquid chromatography on C 18. The successful synthesis of anthroyl polyisoprenoid alcohols was confirmed by the use of uv-visible spectrometry and by fluorescence spectrometry. The purified esters were taken up into Ham's media containing 10–30% fetal calf serum or alternatively reconstituted into phospholipid liposomes for delivery to human fibroblasts in culture. The uptake of fluorescent dolichol esters into the cell and into lysosomes was demonstrated using fluorescence microscopy. The localization of anthroyl dolichol in lysosomes was further documented by simultaneously labeling fibroblasts with anthroyl dolichol and FITC-dextran a recognized lysosomal marker. Fibroblasts generally showed several groupings (domains) of lysosomes, some were dually labeled while others were labeled exclusively with either anthroyl dolichol or FITC-dextran. Labeling with anthroyl dolichol was very slow relative to labeling of the same fibroblasts with FITC-dextran suggesting that anthroyl dolichol acts as a labeling agent for intracellular membranes, particularly those of the lysosome while the dextran fluorescence is presumably of lysosolic origin. Several types of experiments were done with anthroyl [1- 14C]dolichol to establish that the fluorescence seen in lysosomes represents anthroyl dolichol. Anthroyl dolichol appears to enter fibroblasts intact, since we were unable to recover any free [1- 14C]dolichol from total lipid extracts of (i) media used for the uptake of anthroyl dolichol or (ii) the media removed from cells labelled for 42 h. In addition, attempts to hydrolyze anthroyl [1- 14C]dolichol in vitro using whole fibroblast homogenates at pH 4.0 and 7.5 were unsuccessful, even though the fibroblasts expressed acid lipase activity using 4-methylumbelliferyl palmitate as substrate.

Isoprenoid biosynthesis in rat liver peroxisomes. Characterization of cis-prenyltransferase and squalene synthetase.

Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes.

Dolichol: a curriculum cognitionis.

Dolichols were first described about 30 years ago when animal tissues were being examined for the presence of a putative precursor to the polyisoprenoid side chain of ubiquinone. These long-chain 2,3-dihydro-polycis-isoprenoid alcohols are found in all eukaryotic organisms. In many plant tissues they are accompanied by families of other polyisoprenoid alcohols that are usually similar molecules and possess an unsaturated alpha-isoprene residue. Analogy with the role of bactoprenyl phosphates in the synthesis of bacterial wall glycans led to the discovery that the mono- and di-phosphates of dolichols function as cofactors in protein N-glycosylation, involving the formation of glycosylated derivatives of dolichol as intermediates. Variation of the concentration of dolichyl phosphate was shown to be one way of controlling protein N-glycosylation. This can be achieved by modification of the relative activities of dolichol kinase and dolichol phosphate phosphatase. Modulation of the biosynthetic pathway, still not fully understood, of dolichyl phosphate may also be an important factor. Several disease conditions involve aberrations in these pathways.

Dolichol metabolism in Chinese hamster ovary cells

The addition of oligosaccharide to asparagine residues of soluble and membrane-associated proteins in eukaryotic cells involves a polyisoprenoid lipid carrier, dolichol. In Chinese hamster ovary cells, the major isomer of this polyisoprenol has 19 isoprenyl units, the terminal one being saturated. Our laboratory has developed a procedure to analyze the levels and nature of the cell's dolichyl derivatives. Chinese hamster ovary cells contain predominately activated, anionic dolichol derivatives, such as oligosaccharyl pyrophosphoryldolichol, monoglycosylated phosphoryldolichols, and dolichyl phosphate. Our studies show that in growing cells there is continual synthesis of total dolichol. Also, preliminary data suggest there is no catabolism or secretion of this lipid. The level of dolichyl phosphate did not change significantly under a variety of conditions where the levels of enzyme activities utilizing dolichyl phosphate did change. These results suggested that these enzymes had access to the same pool of dolichyl phosphate and had similar Km values for this lipid.

Changes in murine tissue concentrations of dolichol and dolichol derivatives associated with age

The concentrations of the three major cellular forms of dolichol (free, esterified and phosphorylated) were determined in murine liver, kidney and heart. The tissue levels of these forms of dolichol were studied in detail as a function of age. Changes in the activities of dolichyl phosphate phosphatase and dolichol kinase were also determined. In liver, the concentration of unesterified dolichol, fatty acyl dolichol and dolichyl phosphate increased markedly over a period of 6 to 25 months (four-fold, 5.5-fold and nine-fold, respectively). In kidney only, free dolichol and phosphorylated dolichol increased (approximately four-fold in each case). However, this tissue consistently showed the highest concentrations of all forms of dolichol as compared to liver and heart. In heart, both free and esterified dolichol concentrations increased (approximately 3.25-fold in each case); dolichyl phosphate levels were not determined in this tissue. In all tissues studied, the activity of the dolichyl phosphate phosphatase enzyme was considerably higher than that of the dolichol kinase enzyme. In liver, there was no evidence to suggest that either enzyme was critical in determining the relative concentrations of dolichol and dolichyl phosphate. Evidence for such a role for the kinase in the kidney was stronger. Treatment of aging mice with meclofenoxate, a drug that is reported to cause dissolution of lipofuscin, failed to prevent accumulation of dolichol and dolichyl phosphate with age. These observations suggest that not all accumulated dolichol is associated with lipofuscin. Meclofenoxate treatment had no consistent effect on the activities of the enzymes studied.

Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo.

We examined changes in the levels of the dolichol forms in Chinese hamster ovary cells containing alterations in the levels of activity of two enzymes in the oligosaccharyl-P-P-dolichol biosynthetic pathway, namely UDP-GlcNAc:dolichyl phosphate:GlcNAc-phosphotransferase (GlcNAc-1-phosphotransferase) and mannosylphosphoryldolichol (Man-P-Dol) synthase. Under normal conditions in wild type cells, Glc3Man9GlcNAc2-pyrophosphoryldolichol was the most abundant form. Of the other anionic forms of dolichols, dolichyl phosphate, Man-P-Dol, glucosylphosphoryldolichol, and Man5GlcNAc2-pyrophosphoryl dolichol were approximately equally abundant. When 3E11 cells (a tunicamycin-resistant Chinese hamster ovary line containing 15 times more GlcNAc-1-phosphotransferase activity than wild type), B4-2-1 cells (a mutant lacking Man-P-Dol synthase activity), and wild type cells incubated with or without tunicamycin were utilized, significant changes in the levels of most of the anionic dolichol derivatives, with the exception of dolichyl phosphate, were found. Since changes in dolichyl phosphate levels were not detected under a variety of conditions where the levels of enzyme activity utilizing this substrate were varied, all three enzymes appear to have access to the same pool of dolichyl phosphate, and further, to have similar Km values for dolichyl phosphate.

Novel effect of serum on the incorporation of [2-3H]mannose into dolichol-bound carbohydrates and proteins

In the present work we report a serum induced decrease in the rate of [2-(3)H]mannose incorporation into dolichol derivatives and proteins in the embryonic chick optic lobe. This novel effect of serum is widely distributed in sera from different species including man, and it seems to be a unique response of the central nervous system cells. It also appears not to be a generalized toxic effect since it does not affect mannose uptake and deoxyglucose phosphate accumulation. These findings acquire more physiological significance since the in vivo conditions were approached by the preservation of the topological relationship among the cells in each tissue particle and the maintenance of steady state labeling conditions.

Nuclear magnetic resonance studies of polyisoprenols in model membranes

2H-NMR investigation of polyisoprenols (PIs) in model membranes has revealed information about their motions, relative order, and locate within the membrane. Initial 2H-NMR studies of the organization of the shorter chain homologues geraniol (C 10), farnesol (C 15), and solanesol (C 45) were carried out by incorporating 2H-acetyl esters of the alcohol or the di-perdeuterome-thylated derivatives of the omega-labeled phenols into multilamellar phosphatidylcholine (PC) vesicles. 2H-NMR powder patterns interpretable in terms of quadrupole splittings and spin-lattice relaxation times were obtained. Similar experiments have now been carried out with the labeled free alcohol, acetyl ester, and phosphate ester of dolichol (C 95) and undecaprenol (C 55). 2H-NMR results show that the head and tail 2H-labeled sites of C 35 and C 95 exhibit a fast motion isotropic signal only; no slower motion anisotropy, as exhibited by the short chain PIs, was observed. These data suggest that C 55 and C 95 either havesubstantially different (faster motions and/or conformations relative to the shorter chain PIs within the membrane, and that the longer PIs alter the membrane host packing matrix. This conclusion was supported by 31P-NMR studies of C 55 and C 95 derivatives in PC and PE/PC membranes, which showed new pronounced spectral changes relative to the results obtained with the shorter chain PIs. These spectral changes indicate that undecaprenol and dolichol derivatives appear to induce a non-bilayer (isotropic) organization of phospholipid molecules in PE/PC (2:1) vesicles. The possible physiological consequences of this perturbation remains to be determined.

The biosynthesis of dehydrodolichyl phosphates by rat liver microsomes

Using improved conditions with rat liver microsomes in the presence of 20% glycerol and 2% Triton X-100 at pH 8.5 it was shown that dehydrodolichyl diphosphate and dehydrodolichyl phosphate were synthesized from isopentenyl diphosphate and farnesyl diphosphate. Small amounts of geranylgeranyl diphosphate and geranylgeranyl phosphate were also formed. The carbon chain lengths of the dehydrodolichyl diphosphate and dehydrodolichyl phosphate were identical (C 80C 85). A kinetic study showed that dehydrodolichyl diphosphate formed from farnesyl diphosphate and isopentenyl diphosphate was subsequently hydrolyzed to dehydrodolichyl phosphate. As the concentration of isopentenyl diphosphate was increased from 1 to 50μ M, the chain-length distribution of dehydrodolichyl products shifted from C 75C 80 to C 80C 85. Addition of MgCl 2 into the assay mixture decreased product formation, but did not affect the chain-length distribution (C 80C 85). The shift of the chain-length distribution to the same as that observed in naturally occurring dolichol derivatives (C 90C 95) was observed when Triton X-100 was omitted from the assay mixture, although deletion of the detergent decreased the enzyme activity. These results, which provide insight into optimal conditions for enzymatic synthesis of the dolichol chain, are discussed in the context of the in vivo pathway for dolichol biosynthesis.

A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions.

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.

Synthesis of dolichol derivatives and protein glycosylation in trypanosomatids.

Synthesis of dolichol derivatives and protein glycosylation in trypanosomatids.

Glycoprotein synthesis in Drosophila Kc cells. Biosynthesis of dolichol-linked saccharides.

The biosynthesis of dolichol and dolichol-linked saccharide intermediates in glycoprotein synthesis was studied in an embryonic Drosophila cell line (Kc) that lacks the squalene-cholesterol branch of the polyisoprenoid biosynthetic pathway. Kc cells were labeled with [5-3H]mevalonic acid and the radioactive lipids formed were analyzed. Although the major labeled product was coenzyme Q, dolichol and a variety of dolichol derivatives could be readily detected. On the basis of their chromatographic and chemical properties, these derivatives were identified as dolichyl phosphate, glucosylphosphoryldolichol, mannosylphosphoryldolichol, and oligosaccharylpyrophosphoryldolichol. Both short term (4-h) and steady state (4-day) labeling experiments with mevalonate, rather than sugars as previously used, were performed to assess the level of these intermediates. The results of these studies, using a precursor common to all the intermediates, reveal that the early intermediates, N-acetylglucosaminylpyrophosphoryldolichol and N,N'-diacetylchitobiosylpyrophosphoryldolichol, are present at very low levels (less than 5%) relative to the other intermediates on the pathway to oligosaccharylpyrophosphoryldolichol. The total amount of dolichol intermediates remained essentially constant during the chase phase of pulse-chase experiments, indicating the absence of a major catabolic pathway for the polyisoprenoid backbone. As expected, however, the sugar moiety, studied with mannosylphosphoryldolichol, underwent rapid turnover. These results are discussed in the context of our current understanding of the pathway whereby dolichol derivatives participate in glycoprotein synthesis.

Synthesis of dolichol derivatives in trypanosomatids. Characterization of enzymatic patterns.

We have previously described that in certain parasitic protozoa, namely the trypanosomatids, the dolichol-P-P-linked oligosaccharides synthesized in vivo and transferred to protein are devoid of glucose residues and contain 6, 7, or 9 mannose units depending on the species. We have now conducted a cell-free characterization of the enzymatic patterns responsible for these phenotypes. Microsomes from Trypanosoma cruzi, Crithidia fasciculata, Leishmania enriettii, and Blastocrithidia culicis were found to synthesize dolichol-P-[14C]Man but not dolichol-P-[14C]Glc when incubated with rat liver dolichol-P and GDP-[14C]Man or UDP-[14C]Glc, thus providing for an explanation to the absence of glucosylated dolichol-P-P derivatives. Formation of dolichol-P-P-oligosaccharides was assayed in incubation mixtures containing rat liver dolichol-P, GDP-[14C]Man, microsomes, and unlabeled Man5-8GlcNAc2-P-P-dolichol from bovine liver. Membranes from species synthesizing dolichol-P-P-linked Man6GlcNAc2 or Man7GlcNAc2 in vivo were found to synthesize the same compounds but not the higher homologues in the cell-free assay. Species forming Man9GlcNAc2-P-P-dolichol in vivo were found to synthesize lipid-linked Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 in vitro. It is concluded that there are at least three and probably four different dolichol-P-Man-dependent enzymatic activities involved in the synthesis of dolichol-P-P-linked Man9GlcNAc2 and that microorganisms not forming that compound are devoid of all mannosyltransferases responsible for the addition of the missing residues and not only of the enzyme involved in the synthesis of the homologue higher than the oligosaccharide occurring in vivo by a single mannose unit.

Long-term distribution and metabolism of [1-14C]dolichol injected intravenously into rats.

We have previously shown that [1-14C]dolichol mixed in vitro with rat serum and injected intravenously is rapidly cleared from the circulation and appears primarily in the liver. One day after injection the liver accounted for 80% of the isotope in whole animals, whereas after 130 days it represented only 50%. During the 130 days the specific radioactivity (dpm/g liver) decreased by more than 20-fold. In contrast, the spleen retained at 130 days 85% of the radioactivity initially present and its specific radioactivity decreased by only a factor of two. At this time small amounts of isotope were also found in carcass (internal organs removed), gastrointestinal tract and contents, and lungs. Trace amounts of radioactivity were extractable from testes and kidneys, while the heart and brain were essentially free of radioactivity. At all times after injection nearly all the radioactivity present in all tissues was still associated with dolichol. Only trace amounts of [1-14C]dolichyl fatty acyl ester and no [1-14C]phosphorylated derivatives of dolichol were present in the liver and spleen removed 156 days postinjection. Fractionation of liver between 1h and 93 days after injection suggested that [1-14C]dolichol becomes associated primarily with a lysosome-enriched fraction. The accumulation of [1-14C]dolichol in this and other subcellular compartments involved both an inward and outward flow of radioactivity, suggesting that deposition of dolichol in lysosomes is not a one-way terminal process.

Differential regulation of the synthesis of mannosylphosphoryl derivatives of dolichol and retinol in HeLa cells

We have shown earlier that in HeLa S3G cells, glucocorticoids stimulate the synthesis of dolichyl phosphorylmannose (Dol- P-Man) with a concomitant increase in the glycosylation of proteins (Ramachandran C.K., Gray S.L. and Melnykovych G. (1982) Biochem. J. 208, 47–52). Although controversial, there have been several lines of evidence suggesting that the synthesis of retinyl phosphorylmannose (Ret- P-Man) and Dol- P-Man may be carried out by the same enzyme. We examined this possibility and conclude that in HeLa S3G cells the syntheses of Dol- P-Man and Ret- P-Man are catalyzed by two different enzymes located in the same microenvironment. Our conclusion is based on the following observations: (1) exogenously added dolichyl phosphate and retinyl phosphate did not compete with each other; (2) when the cells were grown in the presence of 1 μM dexamethasone, the microsomal synthesis of Dol- P-Man was stimulated, without affecting the Ret- P-Man synthesis; (3) Arrhenius plots on Ret- P-Man and Dol- P-Man synthesis showed breaks at 22 and 37.7°C.