Dolichol Synthesis Research Articles

Induction of the synthesis of an additional family of long-chain dolichols in the yeast Saccharomyces cerevisiae. Effect of starvation and ageing.

The yeast Saccharomyces cerevisiae strain W303 synthesizes in the early logarithmic phase of growth dolichols of 14-18 isoprene residues. The analysis of the polyisoprenoids present in the stationary phase revealed an additional family which proved to be also dolichols but of 19-24 isoprene residues, constituting 39% of the total dolichols. The transfer of early logarithmic phase cells to a starvation medium lacking glucose or nitrogen resulted in the synthesis of the longer chain dolichols. The additional family of dolichols represented 13.8% and 10.3% of total dolichols in the glucose and nitrogen deficient media, respectively. The level of dolichols in yeast cells increased with the age of the cultures. Since both families of dolichols are present in stationary phase cells we postulate that the longer chain dolichols may be responsible for the physico-chemical changes in cellular membranes allowing yeast cells to adapt to nutrient deficient conditions to maintain long-term viability.

Yeast Saccharomyces cerevisiae has two cis-prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis.

Dolichol is a family of long-chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis-prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1. It encodes a protein similar to Rer2p but its function has not been established. The cis-prenyltransferase activity of Srt1p has been proved biochemically in the lysate of yeast cells lacking Rer2p. The polyprenol product of Srt1p is longer in chain length than that of Rer2p and is not sufficiently converted to dolichol and dolichyl phosphate, unlike that of Rer2p. The subcellular localization of these two isozymes has been examined by immunofluorescence microscopy and by the use of GFP fusion proteins. Whereas GFP-Rer2p is localized to the continuous ER and some dots associated with the ER, GFP-Srt1p shows only punctate localization patterns. Immunofluorescence double staining with Erg6p, a marker of lipid particles in yeast, indicates that Srt1p is mainly localized to lipid particles (lipid bodies). RER2 is mainly expressed in the early logarithmic phase, while the expression of SRT1 is induced in the stationary phase. We have shown that yeast has two active cis-prenyltransferases with different properties. This result implies that the two isozymes have different physiological roles during the life cycle of the yeast.

Study on the biosynthesis of dolichol in yeast: recognition of the prenyl chain length in polyprenol reduction.

We synthesized three water-soluble biotin-tagged compounds with different prenyl chain lengths, biotinylated farnesal (BF), biotinylated C(55)-polyprenal (BP55), and biotinylated C(80)-polyprenal (BP80), and examined their effects on in vitro dolichol synthesis from farnesyl diphosphate. BF and BP55 did not affect the dolichol synthesis, whereas BP80 inhibited the reduction pathway from polyprenol to dolichol, accompanied by a decrease in the entire polyprenol and dolichol synthesis. Comparison of BP80 with eighteen detergents, including Triton X-100, CHAPS, octylglucoside, deoxycholate, and Tween 80, revealed the specific effect of BP80 on the reduction pathway. On SDS-polyacrylamide gel electrophoresis, BP80 was detected in an associated form with a 50 kDa protein. These results suggest that the reduction of polyprenol to dolichol in the dolichol biosynthetic pathway proceeds with the recognition of the polyprenol chain length by a 50 kDa protein.

Suppression of dolichol synthesis with isoprenoids and statins may potentiate the cancer-retardant efficacy of IGF-I down-regulation

Agents that inhibit the synthesis of mevalonate or of downstream isoprenoids block the G1-S transition and induce apoptosis in many cell lines; these agents include statins, phenylacetate, and a range of cyclic and acyclic isoprenoids. This cytostatic effect is mediated primarily by decreased availability of dolichol; this deficit impedes the glycosylation of nascent IGF-I receptors, preventing their transfer to the cell surface. In most tissues as well as transformed cell lines, IGF-I activity is crucial for transition to S phase, and also prevents apoptosis. Thus, down-regulation of serum levels of free IGF-I – as may be achieved by caloric restriction, low-fat vegan diets, and various estrogen agonists/antagonists – may represent a useful strategy for preventing and controlling cancer; however, a compensatory up-regulation of tissue expression of IGF-I receptors limits the efficacy of such an approach. Concurrent use of agents that inhibit dolichol synthesis can be expected to prevent an increase in plasma membrane IGF-I receptors, thus potentiating the cancer-retardant efficacy of IGF-I down-regulation. Since dolichol and IGF-I appear to be essential for angiogenesis, these measures may also prove useful for control of pathogenic neovascularization.

Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes.

Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.

Changes in isoprenoid lipid synthesis by gemfibrozil and clofibric acid in rat hepatocytes

We studied whether gemfibrozil and clofibric acid alter isoprenoid lipid synthesis in rat hepatocytes. After incubation of the cells with the agent for 74 hr, [ 14C]acetate or [ 3H]mevalonate was added, and the cells were further incubated for 4 hr. Gemfibrozil and clofibric acid increased ubiquinone synthesis from [ 14C]acetate and [ 3H]mevalonate. The effect of gemfibrozil was greater than that of clofibric acid. Also, gemfibrozil decreased dolichol synthesis from [ 14C]acetate and [ 3H]mevalonate. However, clofibric acid increased dolichol synthesis from [ 3H]mevalonate. Gemfibrozil decreased cholesterol synthesis from [ 14C]acetate and [ 3H]mevalonate. Clofibric acid decreased cholesterol synthesis from [ 14C]acetate, but did not affect synthesis from [ 3H]mevalonate. These results suggest that both agents, at different rates, activate the synthetic pathway of ubiquinone, at least from mevalonate. Gemfibrozil may inhibit the synthetic pathway of dolichol, at least from mevalonate. Contrary to gemfibrozil, clofibric acid may activate the synthetic pathway of dolichol from mevalonate. Gemfibrozil may inhibit the synthetic pathway of cholesterol from mevalonate in addition to the pathway from acetate to mevalonate inhibited by both agents.

Synthesis of mevalonate pathway lipids in fibroblasts from Zellweger and X-linked ALD patients.

Fibroblasts were cultured to determine the involvement of peroxisomes in cholesterol and dolichol synthesis. For this purpose, the behavior of cells from patients with Zellweger syndrome, with X-linked adrenoleukodystrophy, and from nondiseased control subjects was studied. Cells both after pretreatment with mevinolin and without pretreatment were incubated in a medium containing [3H]-mevalonate. In fibroblasts from patients with peroxisomal defects, the cholesterol content and mevalonate incorporation into cholesterol were decreased by 10-20% in comparison with control cells. Mevinolin pretreatment decreased the incorporation rate of [3H]-mevalonate into cholesterol but increased the labeling of ubiquinone and dolichol both in diseased and control cells. Squalene synthase activity was unchanged, whereas the activity of farnesyl-pyrophosphate synthase was increased in the diseased states. The results show that in patients with peroxisomal deficiency neither the amount nor the rate of synthesis of cholesterol and dolichol is reduced to any greater extent.

The Escherichia coli homologue of yeast RER2, a key enzyme of dolichol synthesis, is essential for carrier lipid formation in bacterial cell wall synthesis.

We found in the Escherichia coli genome sequence a homologue of RER2, a Saccharomyces cerevisiae gene required for proper localization of an endoplasmic reticulum protein, and designated it rth (RER2 homologue). The disruption of this gene was lethal for E. coli. To reveal its biological function, we isolated temperature-sensitive mutants of the rth gene. The mutant cells became swollen and burst at the nonpermissive temperature, indicating that their cell wall integrity was defective. Further analysis showed that the mutant cells were deficient in the activity of cis-prenyltransferase, namely, undecaprenyl diphosphate synthase, a key enzyme of the carrier lipid formation of peptidoglycan synthesis. The cellular level of undecaprenyl phosphate was in fact markedly decreased in the mutants. These results are consistent with the fact that the Rer2 homologue of Micrococcus luteus shows undecaprenyl diphosphate synthase activity (N. Shimizu, T. Koyama, and K. Ogura, J. Biol. Chem. 273:19476-19481, 1998) and demonstrate that E. coli Rth is indeed responsible for the maintenance of cell wall rigidity. Our work on the yeast rer2 mutants shows that they are defective in the activity of cis-prenyltransferase, namely, dehydrodolichyl diphosphate synthase, a key enzyme of dolichol synthesis. Taking these data together, we conclude that the RER2 gene family encodes cis-prenyltransferase, which plays an essential role in cell wall biosynthesis in bacteria and in dolichol synthesis in eukaryotic cells and has been well conserved during evolution.

The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis.

As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfalpha1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. USA 90:8179-8183, 1993). The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general. The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes. RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal. RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution. Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis. This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself. The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol.

Effect of Ethanol Administration on the Level of Dolichol in Rat Liver Microsomes and Golgi Apparatus

Data obtained in our laboratory had suggested that acute ethanol administration (6 g/kg body weight) selectively and rapidly affects the intracellular system of protein glycosylation at the level of the Golgi apparatus. Dolichols are important membrane components, and dolichyl phosphate is a glycosyl sugar carrier for N-glycosylation of proteins in endoplasmic reticulum and is considered rate-limiting for this process. In this study, modifications in the concentration and distribution of liver microsomal dolichols after acute ethanol administration were investigated. Between 3 and 24 hr after ethanol administration, the microsomal dolichyl phosphate concentration was significantly lower than in control animals. The highest reduction was observed at 12 hr (-52%). An earlier and more marked reduction of total dolichol was observed in the Golgi apparatus, and, in particular, in the secretory fraction F1 (-70% at 6 hr). Ethanol treatment of isolated hepatocytes led to a significant reduction of the de novo synthesis of both dolichyl phosphate and free dolichol. Moreover, in vitro experiments have demonstrated that pro-oxidant agents lead to a significant decrease of both free dolichol and dolichyl phosphate. Our results suggest that acute ethanol administration induces a marked decrease of dolichols, probably by increasing the degradation and impairing the biosynthetic pathway of these molecules.

Reduced utilization of Man5GlcNAc2-P-P-lipid in a Lec9 mutant of Chinese hamster ovary cells: Analysis of the steps in oligosaccharide-lipid assembly

Recently we reported that CHB11-1-3, a Chinese hamster ovary cell mutant defective in glycosylation of asparagine-linked proteins, is defective in the synthesis of dolichol [Quellhorst et al., 343:19-26, 1997: Arch Biochem Biophys]. CHB11-1-3 was found to be in the Lec9 complementation group, which synthesizes polyprenol rather than dolichol. In this paper, levels of various polyprenyl derivatives in CHB11-1-3 are compared to levels of the corresponding dolichyl derivatives in parental cells. CHB11-1-3 was found to maintain near normal levels of Man5GlcNAc2-P-P-polyprenol and mannosylphosphorylpolyprenol, despite reduced rates of synthesis, by utilizing those intermediates at a reduced rate. The Man5GlcNAc2 oligosaccharide attached to prenol in CHB11-1-3 cells and to dolichol in parental cells is the same structure, as determined by acetolysis. Man5GlcNAc2-P-P-polyprenol and Man5GlcNAc5-P-P-dolichol both appeared to be translocated efficiently in an in vitro reaction. Glycosylation of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was normally glycosylated in CHB11-1-3 cells, a large portion of G was underglycosylated, resulting in the addition of either one or no oligosaccharide to G. Addition of a single oligosaccharide occurred randomly rather than preferentially at one of the two sites.

Synthesis of Dolichol in a Polyprenol Reductase Mutant Is Restored by Elevation ofcis-Prenyl Transferase Activity

CHB11-1-3 is a glycosylation mutant of Chinese hamster ovary (CHO) cells, isolated by screening mutagenized cells for those with decreased intracellular lysosomal enzyme activity [C. W. Hallet al.(1986)Mol. Cell. Biochem.72, 35–45]. CHB11-1-3 synthesizes the lipid polyprenol, the metabolic precursor of dolichol, rather than dolichol, indicating a defect in polyprenol reductase. This defect was demonstrated previously in Lec9 CHO mutants, and cell fusion experiments confirmed that CHB11-1-3 is a member of this complementation group. A revertant of CHB11-1-3, CHBREV, isolated for its ability to grow at 39°C, synthesizes dolichol at near-normal levels. CHBREV is probably a second-site revertant, because it synthesizes three to four times as much polyprenol as CHB11-1-3 and exhibits a similar elevation in the specific activity ofcis-prenyl transferase. This higher activity appears to reflect an increase in enzyme molecules rather than the presence of an activator or absence of an inhibitor. These results suggest that CHB11-1-3 is a “Km” mutant, because synthesis of higher amounts of the substrate of polyprenol reductase obviates the defect.

Polyprenol formation in the yeast Saccharomyces cerevisiae: effect of farnesyl diphosphate synthase overexpression

Biosynthesis of polyprenols was followed in the erg mutants of Saccharomyces cerevisiae impaired in various steps of the mevalonate pathway. The end products of the enzymatic reaction carried out in vitro, in the wild type yeast and all mutants tested, were identified as dehydrodolichols (alpha-unsaturated polyprenols) whereas in vivo, yeast synthesize dolichols (alpha-saturated polyprenols) (Biochimie, 1996.78:111-112.) The strain defective in the farnesyl diphosphate (FPP) synthase, (coded by the erg20-2 gene) required the presence of exogenous FPP for synthesis of dehydrodolichols to occur in vitro. Overexpression of the ERG20 gene restored synthesis of polyprenols in vitro indicating that FPP is the allylic "starter" for cis-prenyltransferase in yeast. Overexpression of the ERG20 gene in the erg 9 mutant, defective in squalene synthase activity, not only restored synthesis of dehydrodolichols in vitro, but also increased the synthesis of dolichols in vivo, almost 10-fold in comparison with wild type yeast. On the other hand overexpression of the mutated FPP synthase, coded by the gene erg20-2 in the same genetic background, resulted in a 100-fold increase of the amount of dehydrodolichols. Interestingly, in addition to the family of typical for yeast C60-C80 compounds, dehydrodolichols of chain length up to C135 were synthesized both in vitro and in vivo.

Biochemistry of peroxisomes in health and disease.

The ubiquitous distribution of peroxisomes and the identification of a number of inherited diseases associated with peroxisomal dysfunction indicate that peroxisomes play an essential part in cellular metabolism. Some of the most important metabolic functions of peroxisomes include the synthesis of plasmalogens, bile acids, cholesterol and dolichol, and the oxidation of fatty acids (very long chain fatty acids > C22, branched chain fatty acids (e.g. phytanic acid), dicarboxylic acids, unsaturated fatty acids, prostaglandins, pipecolic acid and glutaric acid). Peroxisomes are also responsible for the metabolism of purines, polyamines, amino acids, glyoxylate and reactive oxygen species (e.g. O-2 and H2O2). Peroxisomal diseases result from the dysfunction of one or more peroxisomal metabolic functions, the majority of which manifest as neurological abnormalities. The quantitation of peroxisomal metabolic functions (e.g. levels of specific metabolites and/or enzyme activity) has become the basis of clinical diagnosis of diseases associated with the organelle. The study of peroxisomal diseases has also contributed towards the further elucidation of a number of metabolic functions of peroxisomes.

Inhibition of vitellogenin production by allatostatin in the German cockroach

Allatostatins with a typical YXFGL-amide C-terminus constitute a neuropeptide family, which was discovered because of its inhibitory action on insect juvenile hormone synthesis. In the search for possible new functions for allatostatins we focused our attention on the fat body. Our previous studies on the cockroach Blattella germanica suggested the occurrence of factors terminating vitellogenesis, and the hypothesis here was that allatostatins might be one of these factors. Our experiments have shown that allatostatin impaired vitellogenin release in fat bodies incubated in vitro, and that this effect appears to be mediated by the inhibition of vitellogenin glycosylation. Fluvastatin also inhibited vitellogenin release, and mevalonolactone counteracted the inhibitory effects of allatostatin. These results suggest that allatostatin acts upon the mevalonate pathway and synthesis of dolichol, which would explain the inhibition of vitellogenin glycosylation. We finally conclude that allatostatins may effectively contribute to the termination of the vitellogenic cycle in B. germanica.

Inhibition of squalene synthase of rat liver by novel 3′ substituted quinuclidines

Squalene synthase (SQS) is a key enzyme in the biosynthetic pathway for cholesterol and is a target for improved agents to lower plasma levels of low-density lipoprotein (LDL). A series of novel 3′ substituted quinuclidines have been discovered as inhibitors of the rat liver microsomal enzyme. In this study, we demonstrate the inhibitory effects in vitro and in vivo, of two examples of the series. When microsomes were preincubated with compounds, before addition of substrate, both 3-(biphenyl-4-yl)quinuclidine (BPQ) and 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH) were found to cause biphasic inhibition of the enzyme with apparent inhibition constants ( K′ i ) for the sensitive phases of 12 nM and 15 nM, respectively. The K′ i values for the insensitive phases were 1.8 μM and 2.9 μM, respectively. The two examples inhibited equally both steps of the SQS-catalysed reaction, as shown by parallel inhibition of 3H + release and labelled squalene formation from [1- 3H] farnesyl pyrophosphate (FPP). BPQ and BPQ-OH were shown to be inhibitors of hepatic sterol synthesis from mevalonate with ED 50 values of 10.6 and 7.1 mg/kg, respectively, after acute oral administration to the rat. BPQ-OH was chosen for further study and, to determine its selectivity of effect on the mevalonate pathway in vivo, the effect of a dose of 70 mg/kg on the pattern of labelled mevalonate incorporation into the various lipid fractions of the rat liver was examined. As expected, the incorporation into squalene and sterol products was inhibited by about 70%. An appearance of label in fractions corresponding to farnesyl and geranylgeranylpyrophosphates, as well as the corresponding alcohols, was observed in treated but not control animals. In addition, the administration of compound resulted in the appearance of peaks of mevalonate-derived radioactivity in an acidic fraction believed to represent metabolites of farnesol. Such results are consistent with inhibition of the mevalonate pathway at, and not before, SQS. In contrast, there was a significant increase in the incorporation of labelled mevalonate into ubiquinone 10, and the synthesis of dolichols was apparently unchanged. The results suggest a specific effect of BPQ-OH on rat liver SQS. The compound is, therefore, an interesting lead for further investigation of this class of compounds.

Long-term effect of cyclic AMP on N-glycosylation is caused by an increase in the activity of the cis-prenyltransferase.

Previously we have shown that long-term pretreatment of JEG-3 choriocarcinoma cells with 8-bromo-cAMP increases the capacity for N-glycosylation that was caused by an 8-10-fold enlargement of the dolichol pyrophosphoryl oligosaccharide (Dol-PP-oligosaccharide) pool [Konrad and Merz (1994) J. Biol. Chem. 269, 8659-8666]. The factors involved in the effect of cAMP on synthesis of Dol-PP-oligosaccharide are investigated here. The GlcNAc transfer to dolichol phosphate (Dol-P) was found to be unaffected by pretreatment with 8-bromo-cAMP. By measuring the uptake of [3H]mevalonate, a 20-fold increase in the incorporation of the label into Dol-P was observed in the cells treated with 8-bromo-cAMP. Under the same conditions, the synthesis of dolichol was enhanced 60-fold. However, the incorporation of the radioactivity into cholesterol was not increased in the JEG-3 cells pretreated with 8-bromo-cAMP, which suggests a specific stimulation of the dolichol/Dol-P pathway by cAMP. The cis-prenyltransferase activity was found to be increased 10-fold in cells pretreated with 8-bromo-cAMP. Dolichol kinase activity was unaffected by stimulation with 8-bromo-cAMP. The present study suggests that the larger glycosylation capacity in JEG-3 cells treated with 8-bromo-cAMP is caused by an increase in the microsomal cis-prenyltransferase activity.

Enzymes of the mevalonate pathway in rat liver nodules induced by 2-acetylaminofluorene treatment.

Certain enzymes of the mevalonate pathway have been investigated in persistent liver nodules induced in the rat by 2-acetylaminofluorene. In these nodules the dolichol level was increased 5-fold, the ubiquinone-9 content elevated 6-fold and the amount of cholesterol unchanged. Microsomal beta-hydroxy-beta-methylglutaryl-coenzyme A reductase activity was greatly increased compared to control liver tissue, which was also the case for the cytosolic farnesyl pyrophosphate synthase. A significant elevation of all-transgeranylgeranyl pyrophosphate synthase activity in the cytosol was also observed. The branch-point enzyme of microsomal dolichol synthesis, i.e. cis-prenyltransferase, was decreased in the nodules; whereas the activity of squalene synthase, the terminal regulating enzyme of cholesterol synthesis, remained unchanged. The dolichol species in nodular tissue were redistributed towards the longer chain length species. One factor regulating the chain length of the polyisoprene products formed in vitro was shown to be the ratio of the concentrations of isopentenyl pyrophosphate:farnesyl pyrophosphate employed. Other regulatory factors in the terminal steps of this biosynthetic pathway appear to determine the amounts and nature of the final isoprenoid compounds formed in vivo. In contrast to the microsomal trans-prenyltransferase activity, which was unchanged, the activity of nonaprenyl-4-hydroxybenzoate transferase, an enzyme participating in ubiquinone synthesis, was greatly elevated. The alterations observed in the activities of enzymes in the mevalonate pathway can at least partially explain the increased levels of dolichol and ubiquinone and the unchanged level of cholesterol found in liver nodules. It is reasonable to propose that this modified mevalonate metabolism will render nodular cells resistant to certain toxic factors and prone to cell proliferation.

Effect of squalestatin 1 on the biosynthesis of the mevalonate pathway lipids

The effects of squalestatin 1 on rat brain and liver homogenates and on Chinese hamster ovary tissue culture cells have been investigated. This compound effectively inhibits squalene biosynthesis in a highly selective manner. Cytoplasmic farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases are not affected, which is also the case for microsomal cis-prenyltransferase. In tissue culture cells, squalestatin 1 inhibits cholesterol biosynthesis completely, but does not alter dolichol synthesis or protein isoprenylation to a great extent. Incorporation of [ 3H]mevalonate into ubiquinone-9 and -10 increases 3–4-fold, probably as a result of increased synthesis of this lipid. Squalestatin 1 appears not only to be an effective inhibitor of cholesterol biosynthesis, but also to be more specific than other inhibitors used earlier in various in vitro and in vivo systems.

Dolichols and proliferating systems.

The results obtained on dolichol metabolism, in two in vivo model systems, the developing rat liver and the regenerating rat liver, which provide different timing and interplay of proliferation and differentiation processes, have been reported. The regenerating liver presents a marked increase of both synthesis and content of dolichol, a decreased cholesterol/dolichol ratio, unchanged synthesis and content of dolichyl phosphate, or dolichol-kinase and dolichyl phosphate-phosphatase activities; no significantly modified distribution of dolichol homologs, with respect to the control. Total content of dolichols is growing during perinatal development. At fetal stages only short chain dolichols are detectable, while the content of dolichyl phosphate is very low and the activity of dolichyl phosphate-phosphatase is high. The study of the role of liver in dolichol supply to the body in the partially hepatectomized rat shows an increased content of dolichol in the blood; blood dolichol is essentially provided by the release from liver and dolichol traffic in the blood is mediated by multiple carriers.