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Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.

The compositional complexity of the mycobacterial cell envelope differentiates Mycobacterium species from most other prokaryotes. Historically, research in this area has focused on the elucidation of the structure of the mycobacterial cell envelope with the result that the structures of the mycolic acid-arabinogalactan-peptidoglycan complex from M. tuberculosis are fairly well understood. However, the current impetus for studying M. tuberculosis and other pathogenic mycobacteria is the need to identify targets for the development of new drugs. Therefore, emphasis has been shifting to the study of cell envelope biosynthesis and the identification of enzymes that are essential to the viability of M. tuberculosis. The publication of the complete M. tuberculosis genome in 1998 has greatly aided these studies. To date, thirteen enzymes involved in the synthesis of the arabinogalactan-peptidoglycan complex of M. tuberculosis have been identified and at least partially characterized. Eleven of these enzymes were reported subsequent to the publication of the M. tuberculosis genome, a clear indication of the rapid evolution of knowledge stimulated by the sequencing of the genome. In this article we review the current understanding of M. tuberculosis arabinogalactan-peptidoglycan structure and biosynthesis.

We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).

In the peptidoglycan of Mycobacterium leprae, L-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:L-alanine ligase) of M. leprae showed K(m) and V(max) for L-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine.

The structural core of the cell walls of Mycobacterium spp. consists of peptidoglycan bound by a linker unit (-alpha-L-Rhap-(1-->3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikusová, K., Mikus, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820-7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galp and UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, the Mycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Araf donor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis.

Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.

Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsA conditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 degrees C), but ceased growing when placed at 42 degrees C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 degrees C was observed when PI and phosphatidylinositol dimannoside contents dropped to approximately 30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy.

We report the cloning, overexpression, and partial characterization of two unique Z-isoprenyl diphosphate synthase homologs from Mycobacterium tuberculosis. The first enzyme, Rv1086, adds one isoprene unit to omega,E-geranyl diphosphate. The product, omega,E, Z-farnesyl diphosphate, is the putative substrate of the second enzyme, Rv2361c. This enzyme adds seven more isoprene units to omega, E,Z-farnesyl diphosphate and releases decaprenyl diphosphate. Both open reading frames were cloned from the M. tuberculosis H37Rv genome and overexpressed in M. smegmatis. Membrane and cytosol fractions from wild type and the two recombinant strains were assayed for [(14)C]isopentenyl diphosphate incorporation into isoprenyl diphosphates in the presence of various allylic isoprenyl diphosphate acceptors. Membrane fractions of recombinant cells overexpressing Rv2361c incubated with farnesyl diphosphate showed a 10-fold increase of [(14)C]isopentenyl diphosphate incorporation into decaprenyl diphosphate. Membrane fractions of recombinant cells overexpressing Rv1086 incubated with geranyl diphosphate showed a 5-fold increase of [(14)C]isopentenyl diphosphate incorporation into farnesyl diphosphate. Analysis of the stereochemistry revealed that all of the overexpressed farnesyl diphosphate was in the omega,E, Z-configuration. This is the first description of a short chain isoprenyl diphosphate synthase that generates products with Z-stereochemistry. Previously, all known short chain isoprenyl diphosphate synthases catalyze the synthesis of products with E-stereochemistry.

It is well documented that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate-derived product. Recently, it has been shown that free geranylgeraniol (GG-OH) and farnesol (F-OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG-OH and F-OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the posttranslational modification of key regulatory proteins. In this study we report that GG-OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [3H]thymidine incorporation. The increase in cell number and [3H]thymidine incorporation were significantly lower when F-OH was added. Under these conditions [3H]mevalonate and [3H]GG-OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP-binding proteins (19-27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2 ',3'-cyclic nucleotide 3'-phosphodiesterase. Analysis of the proteins metabolically labeled by [3H]mevalonate and [3H]GG-OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG-OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG-OH was added to lovastatin-treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the "salvage" pathway for the utilization of the free isoprenols is physiologically significant in the CNS.

The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in cellular proteins which terminate in the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is most often methionine or serine. Substrates include the p21ras proteins, nuclear lamins, and a series of retinal proteins. To date, a limited number of substrates for the farnesyltransferase have been identified, predominantly by demonstration of the attachment of a farnesyl group to previously identified cDNA clones which encode proteins containing an appropriate carboxyl-terminal tetrapeptide. We describe here the use of a cDNA fusion protein expression library, together with enzymatic in vitro [3H]farnesyl radiolabeling, as a means of identifying novel farnesylated proteins. One candidate cDNA was fully cloned and found to be a homologue of the Escherichia coli heat shock gene dnaJ. The predicted amino acid sequence of this protein was found to terminate with the tetrapeptide Cys-Ala-His-Gln, which conforms to the consensus sequence for recognition by farnesyltransferase, and was shown to undergo in vivo farnesylation. This farnesylated protein, designated RDJ2 (rat DnaJ homologue 2), is a novel and ubiquitously expressed DnaJ homologue and is the newest member of the subfamily of DnaJ-related proteins which are posttranslationally modified by protein farnesylation.

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5-10 microM GG-OH. Under these growth conditions [3H]GG-OH is actively incorporated into UT-2 proteins. Prominent [3H]geranylgeranylated polypeptides in the size range (19-27 kDa) of the small GTP-binding proteins are observed by SDS-PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [3H]geranylgeranylated cysteine residues. Even though [3H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 microM did not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.

The mechanisms by which peroxisome proliferators are able to regulate metabolic processes such as fat metabolism, while at the same time creating an environment for the development of hepatocellular carcinomas, is a central issue in the non-genotoxic carcinogenesis field. The convergence of two members of the steroid receptor family (peroxisome proliferator-activated receptor, PPAR; and retinoid X receptor, RXR) has provided strong support for an oxidative stress component in this carcinogenesis process, but has yet to define clearly a pathway for the classical tumor promotion events associated with peroxisome proliferation. The findings presented here integrate a third member of the steroid receptor family into this process and suggest a novel autocrine loop and mechanism for creating both oxidative stress and tumor promotion. A central regulatory component in this pathway is farnesol which has recently been shown to induce transcription mediated by the steroid receptor family member, farnesoid X receptor (FXR). In this report, it is clearly demonstrated that farnesol can also upregulate the transcriptional events of PPAR, but most likely through a different farnesoid metabolite, resulting in the regulation of an entirely different set of genetic components. Deregulation of the activities of these receptors offers a provocative mechanism for explaining the hepatocarcinogenic effects of peroxisome proliferators in chronically treated rodents.

Since we have previously shown that farnesol (F-OH) and geranylgeraniol (GG-OH) can be utilized for protein isoprenylation, the ability of the free allylic isoprenols to overcome the lovastatin-induced block on rat C6 glial cell proliferation has been investigated. Lovastatin an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in mevalonate biosynthesis, inhibited the rate of cell growth in a concentration dependent manner. The addition of mevalonate and GG-OH to lovastatin treated cells was shown to overcome the drug induced inhibition of cell proliferation. This result indicates that geranylgeraniol is capable of providing the mevalonic acid-derived products necessary to support cell growth. These results suggest that isoprenols are converted to an "activated' form, possibly the corresponding allylic pyrophosphate intermediate by reactions that remain to be characterized, prior to being utilized for sterol biosynthesis and protein isoprenylation.

The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC50 = 37 nM), pig brain (IC50 = 21 nM), and C6 glioma cells (IC50 = 35 nM) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis-isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of [3H]F-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab 1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [3H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC50 = 3-5 microM) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence has been obtained indicating that free farnesol (F-OH) can be utilized for isoprenoid biosynthesis in mammalian cells. When rat C6 glial cells and an African green monkey kidney cell line (CV-1) were incubated with [3H]F-OH, radioactivity was incorporated into cholesterol, ubiquinone (CoQ) and isoprenylated proteins. The incorporation of label from [3H]F-OH into cholesterol in C6 and CV-1 cells was blocked by squalestatin 1 (SQ) which specifically inhibits the conversion of farnesyl pyrophosphate (F-P-P) to squalene. This result strongly suggests that cholesterol, and probably CoQ and protein, is metabolically labeled via F-P-P. SDS-PAGE analysis of the delipidated protein fractions from C6 and CV-1 cells revealed several labeled polypeptides. Consistent with these proteins being modified by isoprenylation of cysteine residues. Pronase E digestion released a major labeled product with the chromatographic mobility of [3H]farnesyl-cysteine (F-Cys). A different set of polypeptides was labeled when C6 and CV-1 cells were incubated with [3H]geranylgeraniol (GG-OH). Both sets of proteins appear to be metabolically labeled by [3H]mevalonolactone, and [3H]-labeled F-Cys and geranylgeranyl-cysteine (GG-Cys) were liberated from these proteins by Pronase E treatment. These cellular and biochemical studies indicate that F-OH can be used for isoprenoid biosynthesis and protein isoprenylation in mammalian cells after being converted to F-P-P by phosphorylation reactions that remain to be elucidated.

When rat C6 glial cells were incubated with [3H]geranylgeraniol (GGol), radioactivity was incorporated into a delipidated protein fraction. SDS-PAGE analysis of the protein fraction labeled by [3H]GGol revealed a 46 kDa polypeptide and a group of labeled polypeptides (19-27 kDa) in the same size range as the small GTP-binding proteins. A similar pattern of labeled polypeptides was seen when C6 cells were metabolically labeled with [14C]mevalonolactone. An isotopically labeled product, chromatographically identical to geranylgeranylcysteine (GG-Cys), was released by Pronase E digestion of the [3H]GGol-labeled protein. When the protein fraction from cells metabolically labeled with [3H]mevalonolactone was digested with Pronase E, two radiolabeled products were released with the chromatographic mobilities of farnesyl-cysteine (F-Cys) and GG-Cys. These studies suggest that C6 glial cells are capable of converting GGol to geranylgeranyl pyrophosphate (GG-P-P), or perhaps a novel "activated" form of the allylic isoprenol, that can be utilized for protein isoprenylation reactions.

There are large increases in the rates of Glc3-Man9GlcNAc2-P-P-Dol (Oligo-P-P-Dol) biosynthesis and protein N-glycosylation during the proliferative response of murine B lymphocytes (B cells) to bacterial lipopolysaccharide (LPS). To learn more about the regulation of dolichyl-saccharide biosynthesis, the possible relationships between developmental changes in specific steps in dolichyl phosphate (Dol-P) and N-acetyl-glucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol) biosynthesis and the induction of Oligo-P-P-Dol biosynthesis were investigated. These studies describe an impressive induction of long chain cis-isoprenyltransferase (cis-IPTase) activity, an enzyme system required for the chain elongation stage in de novo Dol-P synthesis, which corresponded to the striking increase in the rate of Oligo-P-P-Dol biosynthesis in LPS-activated B cells. The cellular level and specific activity of cis-IPTase increase 15-fold in LPS-treated cells with relatively unaltered affinity for isopentenyl pyrophosphate. The rates of Dol-P and Oligo-P-P-Dol synthesis increased substantially when cis-IPTase activity was induced, suggesting a regulatory relationship between the level of cis-IPTase activity and lipid intermediate synthesis. Distinctly different developmental patterns were observed for cis-IPTase and HMG-CoA reductase activity, and when sterol biosynthesis was drastically inhibited by lovastatin, the rate of synthesis of Dol-P was slightly higher in the presence of the drug. Modest elevations in the cellular levels of dolichol kinase, Dol-P phosphatase, and UDP-GlcNAc:Dol-P N-acetylglucosaminylphosphoryltransferase (L-G1PT) activities were also observed, but these changes were relatively small compared with the increases in cis-IPTase activity and the rates of Dol-P, Gl-cNAc-P-P-Dol, and Oligo-P-P-Dol synthesis. The expression of the L-G1PT gene is an early event in the developmental program for Oligo-P-P-Dol synthesis, but GlcNAc-P-P-Dol formation is apparently not rate-limiting. In summary, large increases in cis-IPTase activity and the rate of Dol-P biosynthesis appear to play a key regulatory role in the induction of Oligo-P-P-Dol biosynthesis during the proliferative response of B cells to LPS, and the biosynthetic pathways for Dol-P and cholesterol are regulated independently in dividing B cells.

A large developmental increase in Glc3Man9-GlcNAc2-P-P-dolichol (Oligo-P-P-Dol) synthesis and protein N-glycosylation in primary cultures of embryonic rat brain cells has been reported previously. In vitro enzyme studies and metabolic labeling experiments now show that there is a coordinate induction of long-chain cis-isoprenyltransferase (IPTase) activity, an activity required for the chain-elongation stage of dolichyl monophosphate (Dol-P) biosynthesis de novo, and Oligo-P-P-Dol biosynthesis in embryonic rat brain. Different developmental patterns were observed for IPTase and beta-hydroxy-beta-methyl-glutaryl-CoA (HMG-CoA) reductase activity as well as Dol-P and cholesterol biosynthesis, indicating that these pathways are regulated independently in rat brain. Three separate experimental approaches provide evidence that the amount of Dol-P available in the rough endoplasmic reticulum (RER) is a rate-limiting factor in the expression of the lipid intermediate pathway. First, metabolic labeling experiments show that the biosynthesis of Dol-P is induced at the same time or just prior to the induction of Oligo-P-P-Dol biosynthesis. Second, the time of induction and rate of Oligo-P-P-Dol synthesis are accelerated when Dol-P is supplemented in the culture medium. Third, in vitro assays of mannosylphosphoryldolichol synthase and N-acetylglucosaminylpyrophosphoryldolichol synthase indicate that there are only minor increases in the levels of these enzymes during development, but the amount of endogenous Dol-P in the RER that is accessible to the glycosyltransferases increases when IPTase activity is induced. In summary, the current studies with embryonic rat brain cells document the coordinate induction of IPTase activity and Oligo-P-P-Dol synthesis, support the hypothesis that the availability of Dol-P in the RER is one rate-limiting factor in Oligo-P-P-Dol synthesis, and strongly suggest that increases in IPTase activity and the rate of de novo Dol-P biosynthesis enhance the capacity of embryonic rat brain cells for lipid intermediate synthesis early in the developmental program for N-linked glycoprotein biosynthesis.

Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1-14C]isopentenyl pyrophosphate [( 1-14C]I-P-P) into long-chain polyisoprenyl pyrophosphates (Poly-P-P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly-P-P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 microM for I-P-P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (5) was stimulated by dithiothreitol. The major products were identified as C90 and C95 allylic Poly-P-P on the basis of the following chemical and chromatographic properties: (1) the intact product co-chromatographed with authentic Poly-P-P on silica-gel-impregnated paper; (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly-P) by alkaline hydrolysis; (3) treatment of the labeled Poly-P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly-P from lipophilic Sephadex LH-20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (C95) isoprene units when analyzed by reverse-phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long-chain Poly-P-P. The enzymatic reactions by which the Poly-P-P intermediate is converted to dolichyl phosphate remain to be elucidated.

Free erucic acid (EA) (C22:1) was blended with Cambra oil (CBO), sunflower oil (SFO), and animal lard (AL) at 45% (w/w). Six experimental diets were formulated to incorporate either CBO, SFO, AL, or EA blended oils (CBO plus EA, SFO plus EA), or fats (AL plus EA) at 8%. The adverse effects of dietary EA blended with various types of oils or fats were studied by feeding 180-day-old White Leghorn cockerels for a 4-week growing period. In general, adverse effects of dietary EA were clearly reflected in feed consumption, chick growth, and apparent digestibility of total lipids as well as individual fatty acids. The AL plus EA produced significantly greater adverse effects than with SFO plus EA. The diet containing CBO (low EA, 5.1% rapeseed oil) depressed chick growth and feed consumption, but no additive effect was manifested when EA was supplemented. The fatty acid profile of dietary SFO appears to counteract the metabolic burden of excess dietary EA. It was concluded that high linoleic acid content in SFO may be the major contributing factor in alleviating the adverse response to EA.

[1-14C]Dolichol was mixed in vitro with sunflower seed oil and intubated into rats. Radioactivity began to appear in the blood at 3 h and peaked after about 6 h. The absorbed radioactivity was rapidly cleared from the blood. At 7.5 h postintubation two thirds of the radioactivity in the serum was associated with chylomicrons and about one quarter with the high density lipoproteins. At 12 h the proportion of the radioactivity in the chylomicrons had fallen to one third and that in the high density lipoproteins had increased to one half of the total. Less than 0.02% of the dose was found in the tissues after 12 h. Liver and blood each contained about one third of the total, with smaller amounts in the lungs and spleen. The heart, testes, brain, and kidneys contained only traces of radioactivity. After 12 h most of the radioactivity in the tissues and feces was present as [1-14C]dolichol. The radioactive compounds in the urine (about 0.05% of the dose) were more polar than [1-14C]dolichol as determined by thin-layer chromatography.

A procedure is described for the determination of total cholesterol, dolichol and dolichyl phosphate (Dol-P) in mammalian liver. It is based on extraction of these compounds into diethyl ether after alkaline saponification of the tissue. Extractability is affected by the length of saponification and concentration of potassium hydroxide (KOH) in the saponification mixture. After extraction, total cholesterol and dolichol are quantitated directly by reverse-phase high pressure liquid chromatography (HPLC) on C18. Dol-P requires further purification before quantitation by HPLC, this is accomplished by chromatography on silicic acid. These methods gave recoveries of over 90% for cholesterol and dolichol and about 60% for Dol-P, using [4-14C]cholesterol, a polyprenol containing 15 isoprene units, and [1-14C]Dol-P as recovery standards. Concentrations of total cholesterol, dolichol and Dol-P in livers from one month-old-CBA mice were found to be 5.7 +/- 0.7 mg/g, 66.3 +/- 1.2 micrograms/g and 3.7 +/- 0.3 micrograms/g, respectively.

The total amounts of cholesterol, dolichol and dolichyl phosphate (Dol-P) in kidneys and liver from 1- and 24-month-old mice were measured after saponification of the tissues. The cholesterol content of kidneys decreased by about 4-fold per g of tissue between the two ages, while the amount of cholesterol/g liver remained constant. The dolichol content of both tissues increased by about 2-fold. The amount of Dol-P in kidneys increased by 7-fold over 23 months, while the concentration in liver decreased some 6-fold. Metabolic labelling experiments using tritiated water demonstrated that the rate of synthesis of total dolichol (dolichol + Dol-P) in the kidneys did not change as a function of age. However, the kidneys of young mice incorporated 3-fold more radioactivity into dolichol than Dol-P, whereas this distribution was reversed in old mice. In liver, the rate of dolichol synthesis decreased 2-fold over 23 months. It was not possible to compare the rates of Dol-P biosynthesis between ages as Dol-P was undetectable in liver of old mice. The relative abundance of the 19 isoprene unit homologs of both dolichol and Dol-P decreased approx. 5% while the 17 isoprene unit homologs increased by a similar amount in both tissues of the old mice.