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The dolichol of rat liver was labelled by injecting [4S-3H]mevalonate, the precursor of cis-isoprene residues, into partially hepatectomized animals. The optimum conditions for labelling the dolichol were to inject the animals with radioactive mevalonate 48h after hepatectomy and to kill them 12h later. The concentration of radioactive dolichol was five times as great in regenerating rat liver as in normal liver. The highest concentration of radioactive dolichol was found in the crude mitochondrial and nuclear-debris fractions of the cell. The crude microsomal fractions also contained radioactive dolichol, but at a lower concentration.

Hen oviduct membranes catalyze the transfer of xylose-[ 14 C] from UDP-xylose-[ 14 C] into a xylosyl lipid; a product tentatively identified as an oligosaccharide lipid; and glycoprotein(s). The synthesis of the xylosyl lipid is stimulated by exogenous dolichol phosphate. This finding, along with studies on the chemical properties of the xylosyl lipid, suggests that it has the structure xylosyl phosphoryl polyisoprenol. When exogenous, partially purified [ 14 C]-xylosyl lipid is incubated with the membrane preparation, label is found in the oligosaccharide lipid and in glycoprotein(s).

Endogenous dolichol was shown to function as a natural acceptor of mannose residues by using regenerating rat liver containing [3H]dolichol. When subcellular fractions from this liver were incubated with GDP-[14C]mannose a double-labelled lipid, which represented 30% of the total [14C]mannolipid, could be isolated. This lipid was shown to be identical with the dolichol phosphate mannose formed from exogenous dolichol phosphate, by chromatography, stability to alkali and by chemical cleavage to mannose and dolichol derivatives. It was formed by the rough endoplasmic reticulum and mitochondria. If it is concerned in glycoprotein synthesis this would suggest that it functions in the formation of both secreted and mitochondrial glycoproteins. When both the dolichol and retinol of rat tissue were radioactive they made similar contributions to the synthesis of the lipid by liver microsomal fractions and intestinal epithelial cells.

original Fil: Behrens, Nicolás H.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina. Universidad de Buenos Aires. Facultad de Ciencias exactas y Naturales.; Argentina Fil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina. Universidad de Buenos Aires. Facultad de Ciencias exactas y Naturales.; Argentina Blanco y negro 7 páginas en pdf LFL-PI-O-ART. Artículos científicos Unidad documental simple AR-HYL-2018

When pig liver microsomal preparations were incubated with GDP-[(14)C]mannose, 10-40% of the (14)C was transferred to mannolipid and 1-3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [(14)C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [(14)C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [(14)C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [(3)H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [(14)C]mannose from GDP-[(14)C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[(14)C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.

Chloroplasts, concerned with photosynthesis, contain another quinone first discovered by Kofler (1946) (and often referred to as koflerquinone: see also Kofleret al. 1959a) and rediscovered by Crane (1959) and designated Q254or plastoquinone (II, figure 1). So far, only one plastoquinone has been fully characterized but there appear to be others in nature (Crane 1961; Kegel, Henninger & Crane 1962). Plastoquinone is 2,3-dimethyl-l,4-benzoquinone with a solanesyl side chain. Solanesol is a monohydric alcohol C45H73OH, discovered in tobacco by Rowland, Latimer & Giles (1956) and characterized (III, figure 1) later by Eriksonet al. (1959) and by Kofleret al. (1959b). Ubiquinones (Mortonet al. 1958, Wolfet al. 1958; Shunket al. 1958) are fully substituted 1,4-benzoquinones with two methoxyl substituents in positions 2 and 3, one methyl and one polyisoprenoid substituent. When the latter contains 30, 35, 40, 45 or 50 carbon atoms the compound is designated ubiquinone (30), ubiquinone (35), etc. (Morton et al. 1958; Gloor 1958). The group of workers led by D. E. Green at Madison refers to these compounds as coenzymesQand uses the notation coenzymeQ6if, for example, the polyisoprenoid side chain contains 6 five-carbon units or coenzymeQ10if there is a C50side chain made up of 10 five-carbon units (Lester, Crane & Hatefi 1958). According to the report of the International Commission on Enzymes (1961), ubiquinone (50) or coenzymeQ10should now be described as ubiquinone10orUQ10, the numerical suffix indicating the number of isoprene units. Two heterocyclic compounds, isomeric respectively with plastoquinone and ubiquinone, are also known. Solanachromene (IV, figure 1) (Rowland 1958) and ubichromenol (V, figure 1) have an ethylenic linkage in a heterocyclic ring and a phenolic hydroxyl group in addition to a polyisoprenoid chain. Ubichromenol (50), related to ubiquinone (50), was isolated from human kidney and fully characterized (Laidman, Morton, Paterson & Pennock 1959, 1960). In fractionating kidney unsaponifiable matter the first aim was to isolate and purify ubiquinone, a-toeopherol (VI, figure 1) and ubichromenol. Later, other fractions were studied, and from them a new polyisoprenoid alcohol with 100 carbon atoms was isolated and designated dolichol (Gr. dolichos) because of its very long aliphatic chain (Pennock, Hemming & Morton i960). Burgos, Hemming, Pennock & Morton (1962) have now shown that its formula is C100H163OH with 19 isoprene units. The hydrocarbon squalene, C30H50is now recognized to be an intermediate in the biosynthesis of sterols. The squalene molecule is symmetrical; two farnesyl (pyrophosphate) residues having been linked tail to tail. The carotenoid molecules (with 40 carbon atoms) are also capable of symmetry, thus both lycopene andβ-carotene are made up of identical halves. A 40-carbon analogue of squalene, namely, lycopersene (see table 1) has been synthesized but with one exception it has not been found to occur naturally (Davies 1962). Evidence of the presence of two related substances, phytoene C40H64and phytofluene C40H62, is however occasionally obtained. In all likelihood these compounds are biosynthetic precursors of carotenoids (Goodwin 1955).

Membranous particulate preparations of hen oviduct and bovine thyroid catalyze the transfer of mannose from GDP-mannose to endogenous acceptors. One of the mannosylated acceptors is a lipid, which has been partially purified and characterized. Mild acid hydrolysis of labeled mannosyl lipid yields [14C]mannose, whereas alkaline hydrolysis produced [14C]mannose phosphate. The mannosyl lipids formed in both enzyme preparations are chromatographically identical to synthetic mannosyl phosphoryl dolichol. Exogenous dolichol phosphate stimulates the synthesis of mannosyl lipid synthesis in oviduct preparations 7-fold. Synthesis of mannosyl lipid is freely reversible; in the presence of GDP the transfer of mannosyl groups from endogenously labeled mannosyl lipid to GDP-mannose is observed. These results indicate that the mannosyl lipid has the structure of mannosyl phosphoryl polyisoprenol; the precise chain length of the polyisoprenyl moiety remains to be established. A kinetic study of the transfer of mannose from GDP-mannose to endogenous acceptor in oviduct membranes revealed that, in addition to mannosyl phosphoryl polyisoprenol, two other labeled components are formed. One of these, termed soluble mannosylated endogenous acceptor (mannosyl s-acceptor), has the unique property of being insoluble in chloroform-methanol (2:1) and in water, but is soluble in chloroform-methanol-water (1.0:1.0:0.3). The structure of mannosyl s-acceptor is unknown. The other labeled component, termed residual mannosylated endogenous acceptor (mannosyl r-acceptor), is insoluble in all of the above named solvents. Based on gel filtration and electrophoresis studies performed on mannosyl r-acceptor before and after treatment with proteolytic enzymes it is concluded that this component consists of one or more mannosylated proteins. Several lines of evidence indicate that in oviduct membranes mannosyl phosphoryl polyisoprenol is an obligatory intermediate in the synthesis of mannosyl s-acceptor and the mannosylated protein(s) from GDP-mannose. (a) The formation and turnover of mannosyl phosphoryl polyisoprenol is rapid. Mannosyl s-acceptor is formed more slowly and turns over more slowly. The mannosylated protein(s) is formed even more slowly, and does not turn over. (b) The effect of a chase with unlabeled GDP-mannose on synthesis of labeled mannosyl phosphoryl polyisoprenol, mannosyl s-acceptor and the mannosylated protein(s) was immediate cessation of synthesis of labeled mannosyl phosphoryl polyisoprenol; in contrast, a marked delay was observed in cessation of synthesis of labeled mannosyl s-acceptor and of the mannosylated protein(s). (c) Exogenous, partially purified mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of mannosyl s-acceptor and the mannosylated protein(s). Because both the enzymes involved in these reactions and the mannosylated protein(s) produced are associated with membranous components of the cell-free preparations, it is possible that this enzyme system plays an important role in assembly of membrane glycoproteins.

The product formed by incubation of dolichol monophosphate glucose with liver microsomes was studied. It is insoluble in most solvents, but is soluble in a chloroform-methanol mixture with a high content of water. Treatment with ammonia gave rise to the formation of a water soluble, negatively charged compound of molecular weight 3550. The negative charge could be removed by treatment with phosphatase. Acid hydrolysis of the original compound led to the liberation of an uncharged, water-soluble compound (molecular weight 3550). Acetolysis of the latter gave rise to the formation of a series of products, which appeared to be oligosaccharides when chromatographed on paper or silica plates. The original substance behaved like a polyprenol pyrophosphate when chromatographed on DEAE-cellulose. Molecular weight measurements of the deoxycholate inclusion compound gave a value of 14,300, while dolichol monophosphate glucose under the same conditions gave 11,300. It is tentatively suggested that the compound is dolichol joined through a phosphate or pyrophosphate bridge to an oligosaccharide containing about 20 monosaccharide residues. original Fil: Behrens, Nicolás H.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Parodi, Armando José A.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Blanco y negro 4 páginas en pdf LFL-PI-O-ART. Artículos científicos Unidad documental simple AR-HYL-2018

The unsaponifiable lipid fraction of Saccharomyces cerevisiae contains a phosphorylated component which accepts a mannosyl residue from GDP-mannose and transfers it to growing mannan chains. Both reactions are catalyzed by membrane-bound enzymes. The lipid component has been purified more than 1000-fold (based on lipid-bound phosphate). After dephosphorylation with an orthophosphoric monoester phosphohydrolase this acceptor was shown by mass spectroscopy to be identical with authentic yeast dolichol. The “lipid” intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol. The “lipid” intermediate intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol monophosphate. Yeast dolichol is a mixture of five homologous polyprenols with saturated α-isoprene units, as shown by Dunphy et al. in 1967. All these homologues seem also to be present in the cells as monophosphates. As compared to free dolichols the phosphorylated forms only amount to about 10–20%.

A crude preparation of liver microsomes was shown to catalyze the transfer of glucose from UDP-Glc to Dol-P** to form Dol-P-Glc [ 1 ] . ~ub~quently it was observed that microsomes also transferred the glucose from Dol-P-Glc to an endogenous acceptor. This latter compound is probably another dolichol derivative containing about 20 monosaccharide units bound to dolichol through a phosphate or pyrophosphate bridge [2,3]. The abbreviation Glc-acceptor will be used for this dolichol derivative until the details of its structure are known with certainty, Liver microsomes also catalyze the transfer of mannose to Dol-P from GDP-mannose and of N-acetyl ~ucos~ine from UDP-GlcNAc to form what seems to be dolichol monophosphate mannose and dolichol monophosphate N-aeetyl glucosamine [4, S] . It is possible that the dolichol derivatives are intermediates involved in the synthesis of the sugar portions of the various glycoproteins and glycolipids occurring in smooth microsomes, the Golgi system and other cellular membranes. It is therefore necessary to establish the subcellular loc~ization of these derivatives.

IN 19561 a long-chain isoprenoid alcohol, solanesol, was isolated from tobacco and tobacco leaves. While searching for solanesol in animal tissues we have isolated an isoprenoid alcohol of greater chain-length for which we propose the name ‘dolichol’.

Incubation of mouse myeloma microsomes with GDP-[ 14 C]mannose results in the biosynthesis of [ 14 C]mannose phosphoryl dolichol [Baynes, J. W., Hsu, A.-F. & Heath, E. C. (1973) J. Biol. Chem. 248, 5693-5704] and a [ 14 C]mannose- and N -acetylglucosamine (GlcNAc)-containing oligosaccharide derivative of dolichol. Thus, [ 14 C]mannose phosphoryl dolichol and [ 14 C]mannose-labeled oligosaccharide pyrophosphoryl dolichol were isolated from incubation mixtures by solubilization in 2% (w/v) Triton X-100 and the lipids were separated from small molecules by gel filtration fractionation. After removal of radioactive protein from the preparation, the two lipid derivatives were separated quantitatively by fractionation on a concanavalin A-Sepharose column; [ 14 C]mannose phosphoryl dolichol was not retained by the affinity resin but [ 14 C]mannose-oligosaccharide pyrophosphoryl dolichol adsorbed to the gel and was eluted with α-methylmannoside.[ 14 C]Mannose-oligosaccharide pyrophosphoryl dolichol appeared to be homogeneous when fractionated on DEAE-cellulose and in several thin-layer chromatographic systems. Treatment of [ 14 C]mannose oligosaccharide pyrophosphoryl dolichol with 10% (w/v) NH 4 OH at 100° for 1 hr resulted in the formation of a water-soluble radioactive oligosaccharide phosphate which was isolated and characterized as [Man] 5 → [GlcNAc → GlcNAc → P. Incubation of [ 14 C]mannose-oligosaccharide pyrophosphoryl dolichol with myeloma microsomal preparations results in the transfer, presumably, of the entire oligosaccharide to endogenous protein. Kinetic studies indicate that the dolichol derivatives serve as intermediates in the glycosylation of protein as follows: [Formula: see text]

— Mannose was transferred from GDP-[14C]mannose by homogenates of embryonic chick and adult rat brain to mannolipids with properties identical to manriosyl-phosphoryl-dihydropolyisoprenols. Embryonic chick brain formed six-fold larger quantities of mannolipid than adult rat brain. The reaction was stimulated by Mn2+ ions and Triton X-100 but inhibited by EDTA. Phosphoenolpyruvic acid had no effect on the reaction. A crude mitochondrial fraction was two to three times more active than the microsomal fraction. All radioactivity in the mannolipid could be displaced by the addition of non-radioactive GDP-mannose. The endogenous lipid acceptor in brain was readily labelled in vivo by injection of [3H]mevalonate into the amniotic sac of 7-day-old embryos. The mannolipid formed had the properties of an acidic phospholipid on column and TLC, was stable to dilute alkali but readily cleaved by dilute acid. Synthesis was markedly stimulated by the addition of pig liver or calf brain dolichol phosphate in the presence of Triton X-100 and Mn2+. The mannolipid so formed displayed chemical characteristics identical to the endogenous lipid acceptor. Incubation of the purified radioactive mannolipid with the ‘post-nuclear’ fraction from 14-day-old embryonic chick brain in the presence of EDTA and Triton X-100 resulted in the transfer of 40-50 per cent of the radioactive mannose to protein and 40-45 per cent to water soluble compounds. The efficiency of transfer of radioactivity from endogenously formed mannolipid with or without the addition of dolichol phosphate was similar to exogenously added highly purified mannolipid. These results are compatible with the hypothesis that synthesis of the mannose core of brain glycoproteins involves the synthesis first of mannosyl-phosphoryl-dolichols followed by transfer of the mannose to glycoprotein.

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 .

A particulate enzyme fraction isolated from yeast (Hansenula holstii) catalyzes the transfer of mannose from GDPmannose to endogenous lipid acceptors. Kinetic studies are presented which suggest that one of the mannolipids is a precursor to cell wall mannan. The solubility and chromatographic properties, the stability to mild alkali, and the release of mannose by mild acid hydrolysis are characteristic of polyisoprenyl phosphoryl mannose. Addition of dolichol phosphate to the enzyme system stimulates the synthesis of a mannolipid with properties similar to that synthesized from endogenous lipid. That the exogenous dolichol phosphate was acting as a mannosyl acceptor was demonstrated by showing that dolichol [32P]phosphate was converted to dolichol [32P]phosphate mannose.

Incubation of liver microsomes with dolichyl- d -glucosyl- 14 C monophosphate led to the labelling of an endogenous acceptor. This compound seems to be also a dolichol derivative. It contains a high-molecular weight oligosaccharide bound to dolichol through a phosphate or pyrophosphate bond. Various treatments of the labelled oligosaccharide afforded further information on its structure: Reduction with sodium borohydride, followed by acid hydrolysis gave only radioactive d -glucose indicating that the labelled d -glucose is not incorporated at the reducing end of the oligosaccharide. The percentage of radioactivity, liberated as formic acid after periodate oxidation, indicates that more than one molecule of d -glucose is incorporated and that at least one of them is inside the oligosaccharide chain. Alkaline treatment of the otherwise neutral oligosaccharide gave two positively charged derivatives which could be neutralized by N -acetylation, indicating the presence of two hexosamine residues. The oligosaccharides isolated from different tissues by the same method as that used for rat liver, were similar as judged by their migration in paper chromatography and by the pattern of products liberated by acetolysis.

It has been reported that liver microsomes catalyze the transfer of glucose from uridine diphosphate glucose to dolichol monophosphate so as to produce dolichol monophosphate glucose. Dolichol is a polyprenol containing about 20 isoprene units. The glucosyl residue of dolichol monophosphate glucose is transferred to an endogenous acceptor on further incubation with liver microsomes. The glucosylated endogenous acceptor appears to be an oligosaccharide of about 20 monosaccharide units bound to dolichol through a phosphate or pyrophosphate bridge. In this paper it is reported that liver microsomes catalyze the transfer of the oligosaccharide from the glucosylated endogenous acceptor to an endogenous protein. This transfer reaction requires the presence of bivalent cations, manganese being more effective than magnesium. The presence of deoxycholate is also required. Besides the glycoprotein, several water-soluble products are also formed. Preliminary evidence indicates that they are glucose, iligosaccharides of different size, and possibly oligosaccharides bound to amino acids. original Fil: Parodi, Armando José A.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Behrens, Nicolás H.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Carminatti, Hector. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina 5 páginas en pdf LFL-PI-O-ART. Artículos científicos Unidad documental simple AR-HYL-2018

An unknown lipid previously isolated in our laboratory from beef and human pituitaries was shown to have the same structure as dolichol from pig liver. Beef pituitaries contained ca. 130 mg/kg wet tissue, of which ca. 25% was esterified to fatty acids. Human pituitaries contained ca. 1.4 g/kg, mainly as the free alcohol.

original Fil: Behrens, Nicolás H.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Carminatti, Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Staneloni, Roberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Leloir, Luis Federico. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina Fil: Cantarella, Ana I.. Instituto de Investigaciones Bioquímicas Fundación Campomar; Argentina blanco y negro 1 ejemplar LFL-PI-O-ART. Artículos científicos Unidad documental simple AR-HYL-2018

In two experiments with pigs, administration of [2-14C] over 14 hours led to a level of incorporation into the poly cis-isoprenoid alcohol dolichol that was low but significant. The figures for total incorporation of radioactivity into dolichol were 15 and 47% of those for ubiquinone, and 0.1 and 0.16% of those for the squalene-cholesterol system. A similar experiment with rabbits yielded corresponding figures of 3.5 and 0.2%. Rigorous purification failed to change significantly the specific activity of the dolichol. It is concluded that mammalian tissues are capable of synthesizing poly cis-isoprenoids, but that in the case of dolichol in rabbit and pig liver the rate of synthesis is distinctly lower than that of poly-trans-isoprenoids.

Further studies on the structure of the microsomal endogenous substance which accepts labeled glucose from dolichol monophosphate glucose are reported. In order to obtain sufficient amounts of substance for measuring bound dolichol monophosphate, a concentrate was prepared by treating liver with mixtures of chloroform-methanol-water which lead to purification of the radioactive compound. Estimation of dolichol monophosphate, by its accelerating action on dolichol monophosphate glucose formation, showed that it is liberated by mild acid treatment of the concentrate. The dolichol monophosphate-yielding substance behaved like the labeled glucosylated acceptor when tested by its solubility properties in some solvent mixtures, thin-layer and DEAE-cellulose chromatographies and acid or alkaline treatments. Therefore the results provide further evidence that the lipid moiety of the endogenous acceptor of microsomes is dolichol phosphate.

The ability of fourteen marine invertebrates to utilize [14C]mevalonate for the biosynthesis of isoprenoid compounds was investigated. Several of the animals, in particular crustaceans, bivalve molluscs, a coelenterate and a sponge, were unable to synthesize squalene and sterols, whereas gastropod molluscs, echinoderms, an annelid and a sponge could. Regardless of sterol-synthesizing ability the animals (with the exception of a sponge) always made dolichol and ubiquinone, and thus a specific block in squalene and sterol synthesis was indicated in some animals. Radioactivity accumulated in relatively large amounts in farnesol and geranylgeraniol in those animals incapable of making sterols.

— Long-chain polyisoprenols were isolated from brain in yields of 10-15 mg/kg tissue wet weight. On thin-layer chromatography, migrations were identical for brain polyisoprenols and pig liver dolichol which had been isolated as reference material. The IR, 1H NMR and 13C (FT) NMR spectra of calf brain polyisoprenols were consistent with a molecular structure which had a saturated isoprenol unit followed by approximately 19 unsaturated isoprene units. About 3-4 isoprene units possessed trans double bonds. Using reversed phase thin-layer chromatography and pig liver dolichol as a reference, it was estimated that calf brain polyisoprenols had major structures of C95, C100 and C105 while rat brain polyisoprenols contained C90 and C95 as the major components. All data indicated that the brain polyisoprenols were very similar, chemically, to pig liver dolichols except for minor differences in molecular weight.

In some glycoproteins, a glycosylamine bond between 2-acetamido-2-deoxy-~-glucose and the amide group of asparagine serves to link oligosaccharide with peptide. A common oligosaccharide unit containing 2-acetamido-2-deoxy-~-glucose and D-mannose residues has been suggested for the linkage region of these glycoproteins (Lee & Scocca, 1972). Although mannosyltransferase and N-acetylglucosaminyltransferase activities have been demonstrated in several tissues, little is known about the assembly of this unit. Further, the occurrence in some tissues of polyisoprenoid lipids capable of acting as D-mannose and 2-acetamido-2-deoxy-~-g~ucose acceptors has raised the possibility that lipid intermediates are involved in the transfer of these sugars to glycoproteins. Mannosyltransferase activity was investigated in calf pancreas. I t was shown that one of the products formed when a rough-microsomal fraction (microsomes) is incubated with GDP-[14C]mannose in the presence of Mn2+ is an alkali-stable acid-labile [14C]mannolipid (Herscovics et al., 1973). The acidic nature of this lipid was established by its chromatographic behaviour on silicic acid and DEAE-cellulose columns. When a silicic acid column on which the mannolipid was adsorbed was sequentially eluted with chloroform, acetone and chloroform-methanol (1 : 1, v/v), the lipid was recovered in the chloroform-methanol fraction. The radioactive lipid was not eluted from a DEAEcellulose column by chloroform or chloroform-methanol (4: 1, v/v), but could be eluted with chloroform-methanol (4: 1, v/v) containing ammonium acetate. Short-term acid hydrolysis (0.01 M-HCI, 100°C, 1 min) of the pancreatic lipid liberated [14C]mannose but no detectable ['4C]mannosyl phosphate. These properties are consistent with the view that the labelled pancreatic lipid is a polyisoprenyl mannosyl monoor pyro-phosphate (Richards & Hemming, 1972). Production of the mannolipid proceeded linearly with time for approx. lOmin under the incubation conditions employed. The addition of excess of GDP to the microsomes Smin after the beginning of the incubation resulted in a subsequent disappearance of label from the mannolipid. GMP at the same concentration was ineffectual. This evidence suggests that the mannose is linked to the lipid via a phosphodiester rather than a pyrophosphate linkage. Authentic dolichyl a-D-mannopyranosyl phosphate was prepared chemically for comparison with the pancreatic mannolipid. The procedure of MacDonald, as previously modified for the synthesis of 2,3,4,6-tetra-O-acety~-cc-~-galactopyranosyl phosphate (Warren & Jeanloz, 1972), was employed for the preparation of the corresponding a-Dmannopyranosyl derivative. This compound was coupled with pig liver dolichol in the presence of dicyclohexylcarbodi-imide or tri-isopropylbenzenesulphonyl chloride to give the acetylated phosphodiester, which was purified by either silica-gel column chromatography or t.1.c. After deacetylation with sodium methoxide in methanol, the product was purified by preparative t.1.c. (Warren & Jeanloz, 1973). The labelled mannolipid formed in pancreas was compared with the synthetic dolichyl