Submicrosomal distribution of dolichol kinase and dolichyl phosphate phosphatase in the microsomes of germinating soybeans

The availability of dolichyl phosphate is a major factor in the rate of formation of N-linked glycoproteins in mammalian cells. Recent studies in our laboratory suggested that glycoproteins required for seed germination and early plant development are formed via the dolichyl phosphate pathway. Soybean microsomes contain dolichol kinase and dolichyl phosphate phosphatase, enzymes that regulate dolichyl phosphate levels by interconversion of dolichyl phosphate and dolichol. In the present study, soybean microsomes were fractionated into rough and smooth endoplasmic reticulum and Golgi, and the activities of dolichol kinase and dolichyl phosphate phosphatase were measured in each. Submicrosomal fractions were obtained using a procedure developed for rat liver, and were characterized by marker enzymes, RNA content and electron microscopy. The site of N-glycosylation, the rough endoplasmic reticulum, contained high levels of both dolichol kinase and dolichyl phosphate phosphatase. This makes possible a mechanism whereby glycoprotein formation during seed germination is regulated by availability of dolichyl phosphate.     

Metabolic interconversion of dolichol and dolichyl phosphate during development of the sea urchin embryo

The in vivo and in vitro synthesis and turnover of dolichol and dolichyl phosphate have been studied over the course of early development in sea urchin embryos. Synthesis of dolichol and dolichyl phosphate was studied in vivo and in vitro using [3H]acetate and [14C] isopentenylpyrophosphate, respectively, as precursors. Both the in vivo and in vitro results indicate that the principal labeled end product of de novo synthesis is the free alcohol, and that this alcohol is subsequently phosphorylated to produce dolichyl phosphate. The presence of 30 microM compactin inhibits the de novo synthesis of dolichol from [3H]acetate by greater than 90%, but has no effect on the incorporation of 32Pi into dolichyl phosphate for more than 6 h, thus suggesting that during this time interval the major source of dolichyl phosphate is preformed dolichol. The rate of turnover of the [3H]acetate-labeled polyisoprenoid backbone of dolichyl phosphate is very slow (t1/2 = 40-70 h). In contrast, the rate of loss of the [32P]phosphate headgroup is more rapid (t1/2 = 5.7-7.7 h) and increases over the course of development. Finally, dolichyl phosphate phosphatase activity has been measured in vitro. The activity of this enzyme, which can be distinguished from phosphatidic acid phosphatase, was found to increase as a function of development, in qualitative agreement with the increased turnover of 32P from dolichyl phosphate observed in vivo. These results suggest that the phosphate moiety of dolichyl phosphate is in a dynamic state, and that dolichol kinase and dolichyl phosphate phosphatase play key roles in regulating the cellular level of dolichyl phosphate.     

The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes

The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P NMR, small-angle x-ray scattering, differential scanning calorimetry, and freeze-fracture electron microscopy. These studies suggest that dolichol and dolichol derivatives destabilize unsaturated phosphatidylethanolamine containing bilayer structures and promote hexagonal II phase formation; high concentrations of dolichol induce lipid structures characterized by "isotropic" 31P NMR and particulate fracture faces; dolichol, contrary to cholesterol, has no effect on the thermotropic behavior of membranes consisting of phosphatidylcholine, while dolichyl-P incorporation abolishes the transition from the gel to liquid crystalline phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine; both dolichol and dolichyl-P increase the fatty acid fluidity in phosphatidylethanolamine mixtures; the effect of dolichol on bilayer structure and fluidity is more pronounced with increasing number of isoprene residues; dolichol esters are only soluble to a limited extent in the bilayer and segregates into domains at low concentrations; the results are consistent with a localization of dolichyl-P in which the phosphate group is oriented to the water interphase. The induction of hexagonal II phase by dolichyl-P may elicit the transmembrane movement of glycosylated lipid intermediate.     

Time course of dolichol and dolichyl phosphate during chemical carcinogenesis in rat liver

Hyperplastic liver nodules were induced in rats by administration of an initiator (diethylnitrosamine or 3'-methyl-4-dimethylaminoazobenzene) and/or a promoter (phenobarbital) by the method reported by Tatematsu et al. (1983, Carcinogenesis 4, 381-386). The dolichol content in the liver and liver microsomes of the rats treated with the initiator were approx. 1.5-times higher than that of the control and rats treated with only the promoter. However, the composition of dolichols was not changed. The time course of the dolichyl phosphate concentration in the rat liver treated with both initiator and promoter showed a pattern different from that in the control liver, the initiator-treated liver or the promoter-treated liver. The main component of dolichyl phosphate in liver treated with both the initiator and promoter changed from that with 18 isoprene units to that with 19. It is suggested that the changes in liver dolichols and dolichyl phosphates may be related to the formation of hyperplastic liver nodules.     

Interaction of dolichol and dolichyl phosphate with phospholipid bilayers

The thermotropic phase transition of dipalmitoylphosphatidylcholine vesicles reconstituted with dolichol or dolichyl phosphate was investigated as a function of the lipid-to-polyisoprenoid ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. At the concentrations studied, dolichol and dolichyl phosphate lowered and broadened the transition temperature of dipalmitoylphosphatidylcholine bilayers. Dolichol was found to increase the motional freedom of the bilayer both below and above the transition temperature as determined by fluorescence depolarization. In contrast, low concentrations of dolichyl phosphate decreased the bilayer motional freedom below the transition temperature while high concentrations increased the motional freedom. Above the transition temperature, dolichyl phosphate decreased bilayer 'fluidity' at all concentrations. The data suggest that these polyisoprenoids perturb the bilayer lattice, with the neutral species dolichol increasing membrane 'fluidity', while dolichyl phosphate acts to 'stiffen' the membrane.     

Hydrolysis of dolichyl esters by oviduct membranes and characterization of endogenous dolichyl esters

The chick oviduct system has been employed to study whether dolichol esters might serve as a storage form of dolichol to be converted to dolichyl phosphate (Dol-P) during periods when Dol-P levels increase. Chicken oviduct membranes catalyze the hydrolysis of dolichyl-[14C]oleate; the reaction is dependent on detergent (0.04% NP-40 is optimal), is unaffected by divalent cations and EDTA, and exhibits a pH optimum of 6.0. Oviduct membranes also hydrolyze cholesteryl-[14C]oleate, which exhibits similar properties except the pH optimum is 5.0-5.5. Neither Dol-[14C]palmitate nor Chol-[14C]palmitate is hydrolyzed by membranes. Chol-ester hydrolysis is more sensitive to heat-denaturation than is Dol-ester hydrolysis. Esterase activity was assayed in membranes prepared from immature chicks, chicks treated with diethylstilbestrol, chicks withdrawn from diethylstilbestrol, and mature hens. The highest esterase specific activity was observed in membranes obtained from chicks withdrawn from hormone. In order to characterize the fatty acid composition of Dol-esters they were purified from mature hen oviducts by chromatography on DEAE-cellulose and Fractogel ORPVA-6000, reverse-phase HPLC, and TLC. About 15-25% of oviduct dolichol is in the esterified form. Fatty acid analysis revealed that approximately 85% of the dolichol was esterified to oleic acid. The fact that the highest esterase activity is found in membranes from chicks withdrawn from hormone and that only 20% of the dolichol is esterified argues against a role for Dol-esters as a reservoir of dolichol for conversion to Dol-P.     

Quantitation of excretion of dolichol and dolichyl phosphate from the rat

In order to gain information on the metabolism of dolichol, the rates of excretion of dolichol and doJichyl phosphate were determined in rats maintained on a dolichol-free diet for l0 days . Analysis of fecal samples collected every 49 h yielded mean output values of 9.0±1,4 and 20.7±3,0 g/rat/day of dolichot and dolichy] phosphate, respectively. Urinary output rates were 0.56±0.12 and 0.50±0.2g g/rat/ day of dolichol and dolichyt phosphate, respectively. Thus, excretion is one fate of endogenously synthesized dolichoI compounds in the rat.     

Changes in hepatic dolichol and dolichyl monophosphate caused by treatment of rats with inducers of the endoplasmic reticulum and peroxisomes and during ontogeny

Rats were treated with various inducers of the endoplasmic reticulum and peroxisomes and the properties and distributions of dolichol and dolichyl phosphate analyzed. The treatment of rats with carcinogenic agents 2-acetylaminofluorene, N-nitrosodiethylamine and 3-methylcholanthrene and with the compounds such as phenobarbital, terpentine, cholestyramine and di(2-ethylhexyl)phthalate have all caused changes in the microsomal or lysosomal contents of dolichol to various extents, but only the latter group influenced dolichyl-P concentration. Shortly after birth, the hepatic content of dolichyl-P reaches the adult level, whereas the level of the free alcohol is low at birth but increases continuously thereafter. Incorporation of [3H]mevalonate into dolichol was also dependent on factors other than de novo synthesis, e.g., the pool size. Rates of glycosylation reactions dependent on dolichyl-P exhibit considerable changes but are independent of the existing levels of lipid intermediate. GDP-mannosyl transferase activity increases greatly with birth, but the enzyme activity returns to the adult level within a day after birth. These results demonstrate that structural and functional modifications induced with drugs can greatly influence the content and distribution of dolichol which are independent of the existing levels of dolichyl-P.     

Metabolic labeling of dolichol and dolichyl phosphate in isolate hepatocytes

Isolated hepatocytes from rat liver were incubated with [3H]mevalonate, and the labeling of polyprenols in the microsomal fraction was followed. After a 1-min incubation the alpha-unsaturated forms of polyprenyl-P2, -P, and polyprenol were mainly labeled and at this time point only 2, 8, and 17%, respectively, of the label was associated with the saturated forms. In the case of the free alcohol 2 h of incubation was required before all the labeling was recovered in the saturated form. After 1 min polyprenols and polyprenyl-P with 20 and 21 isoprene residues demonstrated much higher specific labeling than the shorter compounds, but after 5 min these differences were greatly reduced. In experiments utilizing short incubation times and chasing no evidence has been obtained that the phosphorylated form is a precursor of the free alcohol or vice versa, that the free alcohol is a precursor of the phosphorylated form. In human liver about 1% of the dolichol is present in the alpha-unsaturated form. These experiments suggest that: 1) the alpha-unsaturated form is the precursor of the alpha-saturated free alcohol, 2) dolichol does not necessarily arise directly from dephosphorylation of the phosphorylated form, and 3) the free alcohol is for the most part not phosphorylated under in vivo conditions in rat liver.     

The half-lives of dolichol and dolichyl phosphate in rat liver

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 -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 T_1/2. The half-lives of dolichols in lysosomes were between 115 and 137 h, while microsomal dolichyl-P exhibited a T_1/2 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.     

Spectrophotometric determination of dolichol and dolichyl derivatives using the Chugaev color reaction

A simple colorimetric method for the assay of microamounts of dolichol is described. It is based essentially on the Chugaev color reaction. The procedure allows the rapid (1 h after purification of the sample, less than half a day for the whole procedure) and reliable (% SE ≤ 5%) determination of dolichol and dolichyl derivatives in the microgram range. Color production is linear within the concentration range 2 to 20 μg dolichol. With biological samples, prior to colorimetric assay, dolichol and dolichyl derivatives are isolated by solvent extraction and TLC. Recoveries are monitored by isotope dilution analysis. The method has been used for the analysis of dolichol and dolichyl ester levels in bovine thyroid tissue. The procedure can also be applied for the analysis of other isoprenoids.     

Location and biosynthesis of monoterpenyl fatty acyl esters in rose petals

The upper epidermal layer of cells and the epicuticular wax surface of Lady Seton rose petals are sites of biosynthesis and accumulation, respectively, of a family of terpenyl fatty acyl esters. These esters are based mainly on the acyclic monoterpene alcohol geraniol coupled primarily to fatty acids of chain lengths 16-20 and in mass terms represent from 14% to 64% of the total monoterpenes present in the petals. The lipophilic nature of these non-volatile esters of the monoterpene alcohols contrasts with that of the lipophilic volatile parent alcohols themselves and with the hydrophilic, non-volatile, glucoside derivative of the other principal petal fragrant compounds, the phenylpropanoids, beta-phenyl ethanol and benzyl alcohol. These latter compounds are also synthesised and are resident in the petal. Biosynthetic studies confirmed that the petal upper epidermal cell layer has the capacity to incorporate mevalonic acid into the monoterpene component of the fatty acyl ester. The biosynthesis of the monoterpene component of the fatty acyl ester occurs via the mevalonic acid pathway in Lady Seton as well as in the hybrid tea rose Fragrant Cloud. In the latter flower the biosynthesis of geraniol was biosynthetically trans as was the formation of nerol and citronellol. Both geraniol and nerol were shown to be precursors of citronellol via an NADPH dependent reductase reaction. Oleic acid is assimilated into the acyl moiety of the terpenyl ester in Lady Seton isolated petal discs. It is probable that the lipophilic non-volatile terpenyl fatty acyl esters represent a stable storage form of the corresponding alcohols from their residency within the epicuticular wax layer. These acyl esters may realise, on hydrolysis, additional aroma notes from the living flower and potentially commercially significant quantities of the fragrant terpenols during oil of rose essence production.     

Extraction and quantitation of dolichol and dolichyl phosphate in soybean embryo tissue

A procedure for the quantitative extraction of both dolichol and dolichyl phosphate (Dol-P) in plant tissue (soybean embryos) into diethyl ether from an alkaline saponification mixture is described. A complete and quantitative separation of total dolichol and total Dol-P is then obtained based on their respective solubilities in diethyl ether and water. After separation dolichol and Dol-P can both be analyzed and quantitated directly by reverse-phase HPLC on C18 columns without additional purification. The two major homologs of dolichol and Dol-P are those with 17 and 18 isoprene units. The total dolichol and total Dol-P contents of dry embryos were 96.3 +/- 0.8 and 5.3 +/- 0.1 micrograms/g, respectively. The post-HPLC recoveries for dolichol and Dol-P were 101 +/- 2 and 84 +/- 3% respectively, using [1-14C]dolichol and Dol-P containing 20 isoprene units as recovery standards. Dol-P estimations could be carried out on material equivalent to as little as 65 mg embryo tissue.     

Cellular energy metabolism during fetal development. IV. Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.     

Changes in polyamine contents during development and germination of rice seeds

In rice seed, polyamine concentration on a fresh weight basis increased for 16 days after fertilization, followed by a gradual decline. Arginine decarboxylase activity also followed the same pattern. On germination, the polyamine concentration was greatest after 24 hr and the arginine decarboxylase showed a peak after 48 hr.     

Nature of proteinase inhibitors released from soybeans during imbibition and germination

Proteinase inhibitors are released to a smaller extent from soybeans which have been pre-equilibrated to an atmosphere of high relative humidity (r.h.) compared to those equilibrated to low r.h. Seeds pre-equilibrated to high r.h. also exhibited better germination. Regardless of the states of seed hydration, both Kunitz and Bowman-Birk soybean trypsin inhibitors are released in parallel with respect to each other and to other proteins at germination times up to 100 hr. After 48 hr of germination, new forms of trypsin inhibitor appear in the leachate which react with anti-Bowman-Birk trypsin inhibitor antibodies. No new forms of Kunitz trypsin inhibitor were observed immunochemically during the first 100 hr of germination.     

Changes in the fatty acid composition of Drosophila melanogaster during development and ageing.

In Drosophila melanogaster the saturated fatty acids increase in amount early in pupal development relative to the concentrations in late third instar larvae, then decline to the levels characteristic of one-day-old adults. Conversely, the monounsaturated fatty acids decline in content early in pupal development, then increase late in the pupal period. Lauric acid (12:0), myristic acid (14:0) and palmitoleic acid (16:1) become more prominent and oleic acid (18:1) and palmitic acid (16:0) less prominent as the adult ages. At about 40 days of adult age myristic acid (14:0) begins to decrease and oleic acid (18:1) to increase. Within 20 days of eclosion males and females contain different amounts of myristic acid (14:0), palmitic acid (16:0) and oleic acid (18:1).     

Effects of dolichol and dolichyl phosphate on in vitro differentiation of hematopoietic progenitors

The exogenous addition of dolichyl phosphate (Dol-P), an active form of dolichol (Dol) that carries oligosaccharide chains for protein-N-glycosylation, significantly enhanced colony formation of mouse bone marrow hematopoietic progenitors (CFU-e, BFU-e, and CFU-gm) was stimulated by erythropoietin (Epo) and colony-stimulating factor (CSF), but Dol enhanced colony formation of CFU-e only. The effects of Dol or Dol-P on these hematopoietic progenitors were fully dependent on stimulation by Epo or CSF. Other mevalonate-metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, had no effect on hematopoietic progenitors. These studies suggest that exogenous Dol-P enhances the frequency of differentiation of hematopoietic progenitors stimulated by Epo or CSF, and there may be a diversity in cellular response of these progenitors to Dol.     

Separation and quantitative determination of dolichol and dolichyl phosphate in rat and trout liver

The dolichol concentrations in rat and trout liver were found respectively to be 50-59 and 16-21 micrograms/g using three experimental methods: densitometric scanning of thin-layer plates, colorimetric assay and HPLC analysis. By HPLC of benzoylated dolichols, the distribution of the dolichols according to the number of their isoprene residues, was determined in rat and trout liver. The major component was dolichol -18 in rat and dolichol -19 in trout liver. Dolichyl phosphate concentrations were found to be 6-7 micrograms/g of rat liver and 8-9 micrograms/g of trout liver by densitometric scanning of thin-layer plates.     

Intersubunit transfer of fatty acyl groups during fatty acid reduction

Fatty acid reduction in Photobacterium phosphoreum is catalyzed in a coupled reaction by two enzymes: acyl-protein synthetase, which activates fatty acids (+ATP), and a reductase, which reduces activated fatty acids (+NADPH) to aldehyde. Although the synthetase and reductase can be acylated with fatty acid (+ATP) and acyl-CoA, respectively, evidence for acyl transfer between these proteins has not yet been obtained. Experimental conditions have now been developed to increase significantly (5-30-fold) the level of protein acylation so that 0.4-0.8 mol of fatty acyl groups are incorporated per mole of the synthetase or reductase subunit. The acylated reductase polypeptide migrated faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the unlabeled polypeptide, with a direct 1 to 1 correspondence between the moles of acyl group incorporated and the moles of polypeptide migrating at this new position. The presence of 2-mercaptoethanol or NADPH, but not NADP, substantially decreased labeling of the reductase enzyme, and kinetic studies demonstrated that the rate of covalent incorporation of the acyl group was 3-5 times slower than its subsequent reduction with NADPH to aldehyde. When mixtures of the synthetase and reductase polypeptides were incubated with [3H] tetradecanoic acid (+ATP) or [3H]tetradecanoyl-CoA, both polypeptides were acylated to high levels, with the labeling again being decreased by 2-mercaptoethanol or NADPH. These results have demonstrated that acylation of the reductase represents an intermediate and rate-limiting step in fatty acid reduction. Moreover, the activated acyl groups are transferred in a reversible reaction between the synthetase and reductase proteins in the enzyme mechanism.