A Synthesis of Dolichyl Phosphorofluoridate

An improved method for the synthesis of dolichyl H-phosphonate was developed using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (salicyl chlorophosphite) as a reagent. Dolichyl phosphorofluoridate was for the first time synthesized from dolichyl H-phosphonate by its treatment with chlorotrimethylsilane,followed by oxidation with iodine in the presence of fluoride in pyridine.     

On the feasibility of electron transfer to singlet oxygen from mitochondrial components

The incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into dolichyl mannosyl phosphate in rat liver microsomes showed a biphasic time-course; an initial rapid incorporation of mannose which ceased within 2 min and a much slower incorporation which continued for 30 min. In the presence of 0.18 mM (250 μg/ml) bacitracin, the rapid incorporation proceeded normally whereas the slow incorporation was inhibited by about 70%. Upon addition of dolichyl pyrophosphate, the microsomes catalyzed the dephosphorylation of the added compound which was also inhibited by bacitracin. The results, coupled with several other observations, suggest that the rapid reaction represents the transfer of mannose to endogenous dolichyl phosphate whereas the bacitracin-sensitive, slow reaction represents a more complex process in which the enzymatic dephosphorylation of dolichyl pyrophosphate is involved as a rate-limiting step.     

The Synthesis of Moraprenyl and Dolichyl Hydrogen Succinates and Maleates

The reactions of moraprenol and dolichol with succinic and maleic anhydrides in the presence of pyridine or triethylamine were studied, and the conditions were found for the efficient synthesis of moraprenyl and dolichyl hydrogen succinates and maleates. These may be of interest as analogues of moraprenyl and dolichyl hydrogen phosphates with modified anionic groups.     

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.     

Identification and quantification of dolichol and dolichoic acid in neuromelanin from substantia nigra of the human brain

Neuromelanin (NM) isolated from the substantia nigra of the human brain is found to contain a series of dolichoic acids (dol-CA) containing 14-20 isoprene units. This is the first observation of dol-CA in a natural system. Using internally spiked nor-dolichol and nor-dolichoic acid standards, the concentrations of dolichol (dol) and dol-CA present in NM were determined. Remarkably, dol was only four times as abundant as dol-CA in NM. The distribution of dol-CA chains lengths in NM also differed from that of dol, suggesting that the enzyme(s) responsible for the conversion of dol to dol-CA prefer a dolichol substrate containing 19 isoprene units.     

Solubilization of an alpha-(1 leads to 3)-D-mannosyltransferase from pancreas which utilizes synthetic dolichyl pyrophosphate trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc as substrate

Calf pancreas microsomes were treated with 0.5-1% Triton X-100 and the resulting soluble enzyme preparation was incubated with GDP-D-[14C]mannose. The addition of synthetic Dol-PP derivative of the trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc stimulated the synthesis of labeled lipid-bound tetrasaccharide 50-100-fold. The labeled tetrasaccharide thus formed was identified as alpha-Man-(1 leads to 3)-beta-Man-(1 leads to 4)-beta-GlcNAc- (1 leads to 4)GlcNAc by its chromatographic properties and by its sensitivity to alpha-mannosidase and to endo-beta-N-acetylglucosaminidase D. The solubilized alpha-(1 leads to 3)mannosyltransferase did not require divalent cation and was active in the presence of 10 mM EDTA.     

Novel Functional Aspects of the Membrane-Bound exo-Pyrophosphatase of the Hyperthermoacidophilic Archaeon Sulfolobus Are Provided by Analysis of Its Gene and the Adjacent Gene Cluster

The gene of the previously described plasma-membrane-bound acidic pyrophosphatase (exo-PPase) and adjacent genes of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639) were cloned and sequenced. The 4-kb gene cluster comprises four open reading frames (sepp, simp, sabc, and satr) encoding the pyrophosphatase, a small hydrophobic protein of unknown cellular function, a hydrophilic ABC transport ATPase, and an amino transferase. The four proteins have deduced molecular masses of 21, 16, 34, and 48 kDa, respectively. Sepp, simp, and sabc are transcribed as monocistronic mRNAs from which sepp and sabc have been heterologously expressed by in vitro translation using reticulocyte lysates. The Sulfolobus acidocaldarius acidic exo-pyrophosphatase is a membrane-residing protein anchored with five transmembrane alpha-helices. Alignments with protein sequences from databases together with predictions of membrane topology reveal a novel group of proteins with the conserved phosphatase motif KxxxxxRP-(x12-54)-PSGH-(x31-54)-SRxxxxxHxxxD. For none of them a phosphatase or pyrophosphatase activity has yet been described except for the authentic Sulfolobus acidocaldarius protein. On the basis of these investigations a direct role of the exo-PPase in dolichyl phosphate or pyrophosphate hydrolysis and in resistance to the peptide antibiotic bacitracin is discussed.     

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.     

Biosynthesis and characterization of large dolichyl diphosphate-linked oligosaccharides in Sacchromyces cerevisiae

Yeast membranes incorporate radioactivity from GDP[¹⁴C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components.The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditios. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol.When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharide is formed besides the previously-described Dol-PP-(GlcNAc₂ mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [¹⁴C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.     

Generation of reactive oxygen species is an early event in dolichyl phosphate-induced apoptosis

The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.     

Synthesis of Dolichyl and Polyprenyl Sulfates

Dolichyl and polyprenyl sulfates were synthesized as analogues of dolichyl and polyprenyl phosphates by the interaction of dolichols and polyprenols with the pyridine–sulfuric anhydride complex.