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The cytosolic fractions from rat liver, brain, kidney, spleen and testis demonstrate the capacity to synthesize two products from [3H]isopentenyl diphosphate, i.e., farnesyl diphosphate and geranylgeranyl diphosphate. The highest rate of geranylgeranyl diphosphate synthesis was found in brain, testis and spleen, accounting for up to 30% of the total incorporation of radioactivity under optimal conditions. In all tissues examined the geranylgeranyl diphosphate formed was identified as the trans,trans,trans-isomer. The ratio of geranylgeranyl diphosphate to farnesyl diphosphate produced was specific for the tissue investigated and could be altered by the addition of divalent cations. The results in this study demonstrate the presence of a specific trans,trans,trans-geranylgeranyl diphosphate synthetase showing high affinity for farnesyl diphosphate.

Coenzyme Q (CoQ) deficiency occurs in genetic disorders, during aging and various diseases. Diagnosis requires skin fibroblasts in tissue culture. [3H]Mevalonate incorporation was appropriate to measure the rate of CoQ synthesis in fibroblasts and hepatoblastoma cells. [14C]p-Hydroxybenzoate had limited permeability, but it could be increased with Fugene and cyclodextrin. Inhibition of decaprenyl-4-hydroxybenzoate transferase results in the accumulation of decaprenyl diphosphate, an indicator of enzyme deficiency. Also, analysis of the corresponding mRNAs in this case is useful. In vitro assays to measure trans-prenyltransferase and decaprenyl-4-hydroxybenzoate transferase activities are not available. Neither measurement of methyltransferases is reliable in human cells. In vitro reconstruction of CoQ synthesis, in opposite to cholesterol synthesis, proved to be unsuccessful. Thus, the biochemical characterization of the CoQ biosynthetic system in human cells is restricted to a few reliable analytical procedures.

The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans -prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans -Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn 2+ . Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle.

The electrical measurements of phospholipid bilayers and the studies of phospholipid vesicles by using the transmission electron microscopy (TEM) showed that dotriacontaprenol (C160) isolated from leaves of Spermatophyta influences some properties of membranes. The current–voltage characteristics, the membrane conductance–temperature relationships, the membrane breakdown voltage and the membrane capacitance have been measured for different mixtures of C160/DOPC. The membrane conductance, the activation energy of ion migration across the membrane and the membrane thickness were determined. Dotriacontaprenol decreases the membrane breakdown voltage, the activation energy and the membrane capacitance, and increases the membrane conductance and the membrane hydrophobic thickness. The analysis of TEM micrographs shows several characteristic structures, which have been described. The results indicate that dotriacontaprenol increases the membrane elasticity and modulates the surface curvature of the membranes by the formation of fluid microdomains. We suggest that the long polyprenols facilitate the formation of transmembrane, ions-conductive pores.

The biosynthesis of cholesterol, dolichol and dolichyl-P were investigated in a murine model of Niemann-Pick type C disease using both in vitro and in vivo systems. In vivo incorporation of [3H]mevalonate into squalene, dolichol and dolichyl-P decreased. The amount of dolichyl-P was elevated due to a decrease in the rate of degradation. Labeling of squalene and cholesterol of liver homogenates in vitro was decreased in the diseased mice and a lowering of microsomal activities of both HMG-CoA reductase and squalene synthase were also observed. In experiments with brain homogenate, decreased [3H]mevalonate labeling of squalene, cholesterol and dolichol was found in vitro. The decreases in cis-prenyltransferase and squalene synthase activities were observed at a very early phase of the disease. In contrast to the decreased biosynthesis of cholesterol observed in vitro, the labeling of total liver cholesterol was found to be increased in Niemann-Pick type C liver upon in vivo investigation, possibly due to the accumulation of this lipid as a result of a deficient transport process. In the brain, where in vivo labeling reflects only biosynthesis, a decreased rate of cholesterol synthesis was demonstrated.

Three branch-point enzymes of the mevalonate pathway, farnesyl pyrophosphate synthase, cis-prenyltransferase and squalene synthase were characterized in rat hepatic cytosol, microsomes and peroxisomes isolated from rats after treatment with peroxisome proliferators, inducers of the endoplasmic reticulum or modulators of lipid metabolism. Cholestyramine and phenobarbital induced primarily the cytosolic farnesyl pyrophosphate synthase, whereas clofibrate and phthalates elevated the corresponding peroxisomal activity. cis-Prenyltransferase activities in microsomes were induced 4–5-fold after clofibrate, phthalate and phenobarbital administration, but these same treatments affected the peroxisomal activity to only a limited extent. Squalene synthase activity in microsomes was completely abolished, but the peroxisomal activity was unaffected after administration of cholesterol. On the other hand, clofibrate and phthalate induced only the microsomal activities. Mevinolin treatment greatly increased peroxisomal and cytosolic farnesyl pyrophosphate synthase activities, but not the mitochondrial activity, and the cis-prenyltransferase activities were elevated in peroxisomes, but not in microsomes. These results demonstrate that the branch-point enzymes in cholesterol and dolichol biosynthesis at various cellular locations are regulated differentially and that the capacities of peroxisomes and the endoplasmic reticulum to participate in the synthesis of polyisoprenoid lipids is affected profoundly by treatment with different xenobiotics.

The current-voltage steady-state characteristics, cyclic voltammograms and capacitance-voltage steady-state relationship of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl-12 phosphate have been studied. Sustained fluctuations of the capacitance of dolichyl phosphate modified bilayers under applied voltage were observed. The results suggest that the dynamics of dolichyl phosphate molecules in membranes can be regulated by transmembrane electrical potential.

Various polyprenyl phosphates were prepared by chemical phosphorylation of native and partially hydrogenated polyprenols. They were tested as lipid acceptors of sugars from nucleoside diphosphate sugars using a microsomal preparation from rat liver and membrane preparations from B. stearothermophilus, S. typhimurium, and Sh. flexneri. With the microsomal glycosyl transferase system, a demand for saturation of the α-isoprene residue of polyprenyl phosphate was observed; the chain length and cis/trans configuration of polyprenyl radical were less important. With bacterial glycosyl transferases, a demand for the unsaturated α-isoprene residue was observed. In B. stearothermophilus, the rate of synthesis of polyprenyl monophosphate glucose did not depend on the chain length of fully unsaturated polyprenyl phosphate. In S. typhimurium, C55-polyprenyl phosphate was the most effective precursor of polyprenyl diphosphate galactose.

Chemical synthesis was used to produce optically active isomeres 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-acetyl-glucosaminyl transferase systems. The two dolichol-P forms in both transferase systems gave V max values which for the S-form exceeded 4–6 times what was obtained with the R-form. The K m 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 K m 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.

Two types of bovine pituitary gland polyprenols were resolved by silica gel chromatography; i. e., the high molecular weight dolichols of 17 to 23 isoprene units with the OH-terminal isoprene residue saturated, and a fully unsaturated decaprenol. The latter compound was found to be a mixture of molecules differing in the proportion of cis - and trans - isoprene units.

The racemic, optically inactive mixture of C-3 enantiomeric forms of C 95 -dolichyl phosphate can be prepared chemically from fully unsaturated, di trans -polycis-C 95 -prenol isolated from leaves of Sorbus suecica . Purified mannosyl transferase from yeast was capable of catalysing the mannosylation of over 75% of this 1:1 R / S mixture, demonstrating that both enantiomers could function as acceptors. However, the R -form appears to accept mannose more slowly than the S -form. Both R - and S -forms of C 75 -dolichyl phosphate and C 55 -dolichyl phosphate prepared from the respective plant tri trans -poly cis -prenol were also active as lipid acceptors of mannose from GDPmannose.

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 α-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 lysomes 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.

Short and long dolichols and polyprenols in free form or esterified with fatty acids were incorporated into liposomes and administered to rats through a gastric tube. The free alcohols were taken up by the liver to different extents. While uptake in other organs was less, it also involved the fatty acid esters. The use of systems other than liposomes did not increase the efficiency of uptake. Most of the administered lipids were recovered in the lysosomes. Exogenous dolichols and polyprenols were both partly esterified in the liver and, to some extent, also phosphorylated; a portion of the polyprenols was also α-saturated. These results indicate that various polyisoprenes are taken up, to a small extent, from the diet by tissues under normal conditions and in liver these dietary lipids undergo terminal modifications.

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.

Voltammetric investigations of bilayer lipid membranes modified by polyisoprenols (PI) and phosphopolyisoprenols (phospho-PI) are described. The theoretical background of voltammetric analysis with application to a system is presented. It is shown that contrary to the behaviour of phospho-PI, PI increases membrane permeability and membrane deformability, decreases the activation energy of ionic transmembrane migration, decreases membrane stability and does not change membrane selectivity. The results also indicate that a transmembrane potential can facilitate the formation of local non-bilayer structures in dolichyl phosphate-phosphatidylcholine bilayer membrane and accelerate the transbilayer movement of the polar part of a dolichyl phosphate molecule. Our hypothesis of PI and phospho-PI ordering, dynamics and function in membranes is presented.

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.

Two procedures for quantitative determination of dolichol were studied and these were applied to analyze tissue and subcellular distribution. In the first procedure the dolichols were oxidized with Cr 2 O 3 and reduced with NaB 3 H 4 . The radioactivity in the individual dolichols was measured using reversed-phase thin-layer chromatography. In the second procedure, dolichols were analyzed by high-pressure liquid Chromatograph v. For determination of dolichyl phosphates the lipid extract was subjected to acid and alkaline hydrolysis, and after hydrolysis with acid phosphatase the distribution was determined by high-pressure liquid chromatography. Recovery was monitored by the addition of dolichol D15 and D23 phosphate to the homogenate. Rat spleen had the highest dolichol content (114 μg/g) followed by lower content in rat liver and brain. The distribution pattern was similar in all organs, with 18 and 19 isoprene residues as dominating components. Human organs contain considerably higher concentrations of dolichol, with the 19 and 20 isoprene residues as the main components. In rat liver, outer mitochondrial and Golgi membranes, lysosomes and plasma membranes contain considerable amounts of dolichol. A drastic increase in dolichol content was observed in rat liver hyperplastic nodules while human liver cirrhosis and hepatocarcinoma showed a marked decrease in dolichol. In the latter case, the distribution pattern was also changed. Of the total amount of dolichol present in the tissues, 2% was phosphorylated in human liver, 10% in human testis and 18% in rat liver. In rat liver mitochondria and in microsomes 4 and 31%, respectively, of the polyprenols were in activated form. The results demonstrated that dolichyl phosphate and dolichol concentrations were regulated by different mechanisms and that the two forms possessed an independent distribution.

Summary Partially hydrogenated polyprenols were prepared by chemical hydrogenation of plant undecaprenol and C 45 -solanesol. The resulting alcohols were phosphorylated and tested as lipid acceptors of glucose from UDPglucose using microsomal preparation from rat liver as the source of enzyme. Phosphates of partially hydrogenated polyprenols were better acceptors than the phosphates of fully unsaturated, undecaprenol and solanesol. The phosphates of alpha-dihydroundecaprenol and alpha-dihydrosolanesol were as effective as the phosphate of naturally occuring C 95 -dolichol.

A particulate preparation of thermophilic bacterium Bacillus stearothermophilus NCA 2184 contains an enzyme system synthesizing polyprenyl monophosphate glucose and unknown macromolecular material. In this strain, fully unsaturated C55-polyprenol with two internal trans-isoprene residues and an α-cis-residue is present. C55-Polyprenyl phosphate has also been isolated. Some properties of thermophilic glucosyltransferase are described.

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.

The role ,of polyprenyl !~hospha:tes i~ the biosyn!,he ,sis o f various sugar polymers has been flem.onstrat~fl in bacterial ,as well as e~hkaryotic ceils. Tanner I l l presenled evidence t h a t a tip~)philic mann'ose flefiva~;i'~e nfight be the Necursor o f yeast merman and later [2] :observed a mode,rote s t imulat ion .of the fo rmat ion o f ,the m a n n o l i N d on using chemical ly pht>sphoryaa~efl doHehols f~om pig liver (a n~xture of cis/trans C85--C] 1f) polyprenols wi~h a satmatefl ~-~-esidraes) and f,zom yeast (a mix ture ,of,gis]trans C70--C90 polypreno!s wi th sa tma ted a i e s i d u e s ) . The ~0rmafion of the mannol ip id by part iculate enzyme p.leparafions f rom yeast as ~epr~Ited by Nen~andlen and Immpen [3, 4] w, as t he in termediate s lep in the Mosyn~uesis ,of merman f,~om GDP-marmose. The p l e sem paper describes the fo rmat ion o f lipidl inked sx~gars by pal t icuIaie ,enzyme preparat ion froxn yeast us ing UDP-glucos e and ,UDP-galac~ose as t h e sugar d o n o r s : . . T h e r e a c t i , o n ofthese nuc!eosid,e d iphosphate sugars and of GDP-mannose wi th b o t h endogenous lipid acceptors f lora yeast ,and f rom Pig liver as well as wi th the m o n o p h o s p h a t e 'of the plant polypreno1--fical~ren0] ,{a m~xture .ofCs,0-~ C55an d C : 6 o c i s / * r a n s l~oly :prenols I5] is.'the subject o f this :_report. pr, e~,ous stui!es [7]. Unhb~lled nue]eoside diphosphate sugars were G'om Serve, HeidelbeT:g, G~rmany. A c o m m e : ~ yeast strain, cult ivated on a m e d i u m contai l f ing 2t4 ~-ucos,~, 2% pepton~ arid 2 ~ yeas t extract was co]!e,¢ted in early s ta t ionary pha~e (see o u r gen,emtien) and used routineay. Two wi]d strains f,~om o~r ,own eoEaction (S-288'C, haploid and SBTD, dip]old) were also tes ted. Enzyme prey,ma~on: F~esh yeast e e l s weie mechanical ly disrapted ,-~th BallofinJ be~ds, suspended. in Tris-m~tleate b~ffe~ (50 ruM), 13I-t 7 , c~n~tain~g ] n-xM meIcaptoe thano] and centr i fuged a,t 1000 g and lr~) 0 0 0 g fO]~ ]~) rain and the s e d i r l q r e n ~ w a s l ] ] $ e ~ f l e g ] , ,~.,e supernatan~: was centr i fuged at 105 000 g fo~ 1 tg mad the ~esMfing pellet ofpa_rfic~flate f~acfion was used as the source o f enzyme. L~pid a:cceptor £rom yeast (~.ra.de dol~¢hol phospha te p~eparadon) was obta~nefl by :the m e ~ f l ,of preparat~en of d,o,li~ho] plao~pha:te from pig liver [7, :8]. 'Lipid ext rac t .obtained f~om ] kg o f f lesh b~l~,er'~ yeas~ (partially dried and defa t ted w i th acetone) was subjec ted t o alkaline and acid hy.droly:sis and f lact i0na!ed on the col,mnn o f DEAE-~ellMose wi~h increasing con-_ C entrrations o f a m m o n i u m aceta,te ,~0--9 2 M) in ~¢Moro1%rm--me. ~'aanol~ 2::1 2nixtuN.;, ~lThe f ~ c l i o n s w e r e washed wi th wate~ t,o l emove a m m o n i u m acetate . I 9 ] and tes ted for the presence o f sugar-binding lipids a n d • . . f o r t o t a l p h e s p h o r u s ~ l 0 ] . " " :2. Materials :and mefhods " .The bio~cnthesis o f lipid-linked sugars ~was measured

Fractionation of unsaponifiable pig liver lipids on DEAE-cellulose yielded two lipids which stimulate the incorporation of labeled glucose from UDPglucose into lipids of rat liver microsomes. The main one appeared to be dolichol monophosphate. Synthetic ficaprenol phosphate also stimulated the formation of polyisoprenol monophosphate sugar from UDPglucose. Phytol phosphate had no stimulatory effect. No formation of lipid-linked sugars from labeled UDPgalactose was observed in rat liver and brain microsomes.

An enzyme synthesizing polyprenol monophosphate glucose from UDPglucose is present in the membrane fraction from Shigella flexneri 2a; UDPgalactose can also be used as the sugar donor. GDPmannose, UDPglucuronic acid and UDP-N-acetylglucosamine were ineffective. Triton X-100 or deoxycholate were required for enzymic activity. Ficaprenol (mainly C 55 ) phosphate was the most effective lipid acceptor of glucose. The phosphate esters of other polyprenols (phytol, betulaprenol, solanesol and dolichol) were much less effective. Lipid extracts from Sh. flexneri did not stimulate the formation of lipid-linked glucose. No polyprenol phosphate phosphatase was detected in membrane preparations from 2a and Y strains of Sh. flexneri .