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PubMed2001

Alpha-retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors.

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of alpha,alpha-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme-substrate complexes formed from binary enzyme-phosphate and enzyme-alpha-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and alpha-d-glucopyranosyl phosphate, and binds 3 x 10(4)-fold tighter (K(i) approximately 1 microM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope=1.14; r(2)=0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (K(m)/k(cat))] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log K(i)). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite -1 in the enzyme-phosphate complex with a dissociation constant of 56 microM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of alpha-retaining glucosyltransferase mechanisms that occur with and without a beta-glucosyl enzyme intermediate.

Binding Sites↗

PubMed1972

Interaction of soluble glucosyl- and mannosyl-transferase enzyme activities in the synthesis of a glucomannan.

A neutral-detergent-solubilized-enzyme preparation derived from Phaseolus aureus hypocotyls contains two types of glycosyltransferase activity. One, mannosyltransferase enzyme activity, utilizes GDP-alpha-d-mannose as the sugar nucleotide substrate. The other, glucosyltransferase enzyme activity, utilizes GDP-alpha-d-glucose as the sugar nucleotide substrate. The soluble enzyme preparation catalyses the formation of what appears to be a homopolysaccharide when either sugar nucleotide is the only substrate present. A beta-(1-->4)-linked mannan is the only polymeric product when only GDP-alpha-d-mannose is added. A beta-(1-->4)-linked glucan is the only polymeric product when only GDP-alpha-d-glucose is added. In the presence of both sugar nucleotides, however, a beta-(1-->4)-linked glucomannan is formed. There are indications that endogenous sugar donors may be present in the enzyme preparation. There appear to be only two glycosyltransferases in the enzyme preparation, each catalysing the transfer of a different sugar to the same type of acceptor molecule. The glucosyltransferase requires the continual production of mannose-containing acceptor molecules for maintenance of enzyme activity, and is thereby dependent upon the activity of the mannosyltransferase. The mannosyltransferase, on the other hand, does not require the continual production of glucose-containing acceptors for maintenance of enzyme activity, but is severely inhibited by GDP-alpha-P-glucose. These properties promote the synthesis of beta-(1-->4)-linked glucomannan rather than beta-(1-->4)-linked glucan plus beta-(1-->4)-linked mannan when both sugar nucleotide substrates are present.

Carbon Isotopes↗

PubMed1974

A comparison of glucoside formation by liver preparations from the rabbit and the mouse.

Liver homogenates from mice and from rabbits transfer glucose from UDP-[6-(3)H]glucose, at pH7.0, to oestradiol-17alpha, oestradiol-17beta, oestradiol-17alpha 3-glucuronide, p-nitrophenol and diethylstilboestrol. In the rabbit the phenolic steroids were better substrates than p-nitrophenol for the glucosyltransferase, whereas the reverse was true in the mouse. At pH8.0, rabbit liver, but not mouse liver, transferred glucose to oestradiol-17alpha 3-glucuronide in better yield than that at pH7.0. Evidence is presented for the presence of two glucosyltransferases in rabbit liver. One of these has a pH optimum at about 8.0, and is highly specific for oestradiol-17alpha 3-glucuronide, whereas the other, which has a pH optimum at about 7.0, is similar in this respect to the transferase in mouse liver.

Animals↗

PubMed2000

Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization.

Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the alpha-amylase family (glycoside hydrolase family 13), which all have a (beta/alpha)8 barrel structure. In the glucansucrases, however, the alpha-helix and beta-strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In alpha-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis-Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (beta/alpha)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which alpha1,3-linked glucosyl units are joined sequentially to maltose.

Enzyme Stability↗

PubMed2000

Congenital disorder of glycosylation-Ic: case report and genetic defect.

The clinical phenotype and the molecular defect of a patient with a new subtype of congenital disorders of glycosylation (CDG-Ic, formerly designated as CDGS type V) characterized by a deficiency of Dol-P-Glc: Man9GlcNAc2-PP-Dol glucosyltransferase is described. The clinical picture presents with several features similar to CDG-Ia (phosphomannomutase 2 deficiency) such as hypotonia and atactic-dystonic movements. In contrast to CDG-Ia, the course of the disease appears milder. The head growth, the functioning of the peripheral nerves and the initial cerebellar development were normal. Sequencing of the patient's Dol-P-Glc: Man9GlcNAc2-PP-Dol glucosyltransferase cDNA revealed an in-frame deletion of three nucleotides leading to the loss of isoleucine 299.

Brain↗

PubMed1974

Initiation characteristics for the synthesis of five T4 phage-specific messenger RNAs in vitro.

The involvement of the nucleoside triphosphates in the initiation of the synthesis of the messenger ribonucleic acid of five T4 specific enzymes has been studied. Only one of these, the messenger RNA for deoxynucleosidemonophosphate kinase, can be initiated in the presence of one nucleoside triphosphate, namely ATP. All of the remaining four require the presence of at least two nucleoside triphosphates during the initiation period. The combination of ATP and UTP was best for the initiation of messenger RNA for dihydrofolate reductase, ATP and CTP for deoxycytidylate hydroxymethyltransferase and beta-glucosyltransferase, and ATP and GTP for alpha-glucosyltransferase. We have concluded that there is a great variation in the nucleotide composition and sequence of the initiation sites in T4 DNA. No correlation in the requirements of nucleoside triphosphates during the initiation period could be observed among the five systems studied according to their classification as one type or another of "early" T4 messenger RNA.

Adenosine Triphosphate↗

PubMed1996

Complete removal of sphingolipids from the plasma membrane disrupts cell to substratum adhesion of mouse melanoma cells.

GM-95, a mutant cell line derived from mouse melanoma MEB-4 cells, is deficient in glycosphingolipids (GSLs) due to the lack of ceramide glucosyltransferase-1 activity (Ichikawa, S., Nakajo, N., Sakiyama, H., and Hirabayashi, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2703-2707). In this study, we examined the involvement of the complex sphingolipids in cell to substratum adhesion. Immunofluorescent and chemical analyses revealed that the complex sphingolipids were significantly concentrated in the detergent-insoluble substrate attachment matrix of both GM-95 and MEB-4 cells. In spite of the absence of GSLs, GM-95 cells retained the ability to adhere to extracellular matrix (ECM) proteins such as fibronectin, collagen, and laminin. When both GM-95 and MEB-4 cells were treated with neutral sphingomyelinase, GM-95 cells were rounded up and detached from all ECM proteins examined. In contrast, neither the morphology nor the adherence of MEB-4 cells was altered. Under this treatment, sphingomyelin (SM) became undetectable in both cells. A similar inhibition was observed upon pretreatment of cells with fumonisin B1 or ISP-1, both of which block the synthesis of ceramide, a common precursor of both GSLs and SM. Stable transfectants expressing GSLs, which were established by transfection of glucosyltransferase-1 cDNA into GM-95 cells, became resistant to neutral sphingomyelinase-mediated rounding up and detachment from ECM proteins. In conclusion, the complex sphingolipids play critical roles in cell to substratum adhesion, and the presence of either GSLs or SM is sufficient for the adhesion.

Animals↗

PubMed1997

Heat stress induces a glycosylation of membrane sterol in myxoamoebae of a true slime mold, Physarum polycephalum.

To know the very early events occurring after heat shock, the changes of membrane lipids were examined. Heat stress induced the production of a certain glycolipid in the myxoamoebae of Physarum polycephalum in a few minutes. The purified glycolipid was determined to be a poriferasterol monoglucoside by structural studies that was previously reported to be expressed during the differentiation of Physarum cells from haploid myxoamoebae to diploid plasmodia (Murakami-Murofushi, K., Nakamura, K., Ohta, J., Suzuki, M., Suzuki, A., Murofushi, H., and Yokota, T. (1987) J. Biol. Chem. 262, 16719-16723). The activity of UDP-glucose:poriferasterol glucosyltransferase (Murakami-Murofushi, K., and Ohta, J. (1989) Biochim. Biophys. Acta 992, 412-415) was also expressed rapidly after heat shock. Thus, the activation of sterol glucosyltransferase and the production of sterol-glucoside were considered to be important events that were involved in the signal transduction system to induce some succeeding heat-shock responses, such as the synthesis of heat-shock proteins.

Animals↗

PubMed1998

A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins.

A fragment of the N-terminal 546 amino acid residues of Clostridium sordellii lethal toxin possesses full enzyme activity and glucosylates Rho and Ras GTPases in vitro. Here we identified several amino acid residues in C. sordellii lethal toxin that are essential for the enzyme activity of the active toxin fragment. Exchange of aspartic acid at position 286 or 288 with alanine or asparagine decreased glucosyltransferase activity by about 5000-fold and completely blocked glucohydrolase activity. No enzyme activity was detected with the double mutant D286A/D288A. Whereas the wild-type fragment of C. sordellii lethal toxin was labeled by azido-UDP-glucose after UV irradiation, mutation of the DXD motif prevented radiolabeling. At high concentrations (10 mM) of manganese ions, the transferase activities of the D286A and D288A mutants but not that of wild-type fragment were increased by about 20-fold. The exchange of Asp270 and Arg273 reduced glucosyltransferase activity by about 200-fold and blocked glucohydrolase activity. The data indicate that the DXD motif, which is highly conserved in all large clostridial cytotoxins and also in a large number of glycosyltransferases, is functionally essential for the enzyme activity of the toxins and may participate in coordination of the divalent cation and/or in the binding of UDP-glucose.

Amino Acid Sequence↗

PubMed2003

The folate precursor p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol.

Plants synthesize p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. It has generally been supposed that pABA exists as the free acid in plant cells and that it moves between organelles in this form. Here we show that fruits and leaves of tomato and leaves of a diverse range of other plants have a high capacity to convert exogenously supplied pABA to its beta-D-glucopyranosyl ester (pABA-Glc), whereas yeast and Escherichia coli do not. High performance liquid chromatography analysis indicated that much of the endogenous pABA in fruit and leaf tissues is esterified and that the total pool of pABA (free plus esterified) varies greatly between tissues (from 0.2 to 11 nmol g-1 of fresh weight). UDP-glucose:pABA glucosyltransferase activity was readily detected in fruit and leaf extracts, and the reaction was found to be freely reversible. p-Aminobenzoic acid beta-D-glucopyranosyl ester esterase activity was also detected in extracts. Subcellular fractionation indicated that the glucosyltransferase and esterase activities are predominantly if not solely cytosolic. Taken together, these results show that reversible formation of pABA-Glc in the cytosol is interposed between pABA production in chloroplasts and pABA consumption in mitochondria. As pABA is a hydrophobic weak acid, its uncharged form is membrane-permeant, and its anion is consequently prone to distribute itself spontaneously among subcellular compartments according to their pH. Esterification of pABA may eliminate such errant behavior and provide a readily reclaimable storage form of pABA as well as a substrate for membrane transporters.

4-Aminobenzoic Acid↗

PubMed2005

Golgi and endoplasmic reticulum functions take place in different subcellular compartments of Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite that causes dysentery in developing countries of Africa, Asia, and Latin America. The lack of a defined Golgi apparatus in E. histolytica as well as in other protists led to the hypothesis that they had evolved prior to the acquisition of such organelle even though glycoproteins, glycolipids, and antigens have been detected, the latter of which react with antibodies against Golgi apparatus proteins of higher eukaryotes. We here provide direct evidence for Golgi apparatus-like functions in E. histolytica as well as for components of glycoprotein folding quality control. Using a combination of bioinformatic, cell biological, and biochemical approaches we have (a) cloned and expressed the E. histolytica UDP-galactose transporter in Saccharomyces cerevisiae; its K(m) for UDP-galactose is 2.9 microm; (b) characterized vesicles in an extract of the above protist, which transport UDP-galactose into their lumen with a K(m) of 2.7 microm;(c) detected galactosyltransferase activity(ies) in the lumen of the above vesicles with the K(m) for UDP-galactose, using endogenous acceptors, being 93 microm;(d) measured latent apyrase activities in the above vesicles, suggesting they are in the lumen; (e) characterized UDP-glucose transport activities in Golgi apparatus and endoplasmic reticulum-like vesicles with K(m)s for UDP-glucose of approximately 2-4 microm. Although the endoplasmic reticulum-like fraction showed UDP-glucose: glycoprotein glucosyltransferase activity, the Golgi apparatus-like fraction did not. This fraction contained other glucosyltransferases. Together, these studies demonstrate that E. histolytica has different vesicles that play a role in protein glycosylation and folding quality control, analogous to the above organellar functions of higher eukaryotes.

Amino Acid Sequence↗

PubMed2002

Organizational diversity among distinct glycoprotein endoplasmic reticulum-associated degradation programs.

Protein folding and quality control in the early secretory pathway function as posttranslational checkpoints in eukaryote gene expression. Herein, an aberrant form of the hepatic secretory protein alpha1-antitrypsin was stably expressed in a human embryonic kidney cell line to elucidate the mechanisms by which glycoprotein endoplasmic reticulum-associated degradation (GERAD) is administered in cells from higher eukaryotes. After biosynthesis, genetic variant PI Z underwent alternative phases of secretion and degradation, the latter of which was mediated by the proteasome. Degradation required release from calnexin- and asparagine-linked oligosaccharide modification by endoplasmic reticulum mannosidase I, the latter of which occurred as PI Z was bound to the molecular chaperone grp78/BiP. That a distinct GERAD program operates in human embryonic kidney cells was supported by the extent of PI Z secretion, apparent lack of polymerization, inability of calnexin to participate in the degradation process, and sequestration of the glycoprotein folding sensor UDP-glucose:glycoprotein glucosyltransferase in the Golgi complex. Because UDP-glucose:glycoprotein glucosyltransferase sustains calnexin binding, its altered distribution is consistent with a GERAD program that hinders the reentry of substrates into the calnexin cycle, allowing grp78/BiP to partner with a lectin, other than calnexin, in the recognition of a two-component GERAD signal to facilitate substrate recruitment. How the processing of a mutant protein, rather than the mutation itself, can contribute to disease pathogenesis, is discussed.

Alkaloids↗

PubMed1998

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain.

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans -stereoconfiguration in the beta-position are described. The water-soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc-P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water-soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.

Animals↗

PubMed1998

Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo.

In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and is composed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase. It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway. To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins. These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains as compared to wild-type and G1 strains. Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae. Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S. cerevisiae.

Animals↗

PubMed1999

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells.

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide-lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.

Animals↗

PubMed2005

Processive lipid galactosyl/glucosyltransferases from Agrobacterium tumefaciens and Mesorhizobium loti display multiple specificities.

The glycosyltransferase family 21 (GT21) includes both enzymes of eukaryotic and prokaryotic organisms. Many of the eukaryotic enzymes from animal, plant, and fungal origin have been characterized as uridine diphosphoglucose (UDP-Glc):ceramide glucosyltransferases (glucosylceramide synthases [Gcs], EC 2.4.1.80). As the acceptor molecule ceramide is not present in most bacteria, the enzymatic specificities and functions of the corresponding bacterial glycosyltransferases remain elusive. In this study, we investigated the homologous and heterologous expression of GT21 enzymes from Agrobacterium tumefaciens and Mesorhizobium loti in A. tumefaciens, Escherichia coli, and the yeast Pichia pastoris. Glycolipid analyses of the transgenic organisms revealed that the bacterial glycosyltransferases are involved in the synthesis of mono-, di- and even tri-glycosylated glycolipids. As products resulting from their activity, we identified 1,2-diacyl-3-(O-beta-D-galacto-pyranosyl)-sn-glycerol, 1,2-diacyl-3-(O-beta-D-gluco-pyranosyl)-sn-glycerol as well as higher glycosylated lipids such as 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol, 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->6)-O-beta-D-gluco-pyranosyl]-sn-glycerol, and the deviatingly linked diglycosyldiacylglycerol 1,2-diacyl-3-[O-beta-D-gluco-pyranosyl-(1-->3)-O-beta-D-galacto-pyranosyl]-sn-glycerol. From a mixture of triglycosyldiacylglycerols, 1,2-diacyl-3-[O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl-(1-->6)-O-beta-D-galacto-pyranosyl]-sn-glycerol could be separated in a pure form. In vitro enzyme assays showed that the glycosyltransferase from A. tumefaciens favours uridine diphosphogalactose (UDP-Gal) over UDP-Glc. In conclusion, the bacterial GT21 enzymes differ from the eukaryotic ceramide glucosyltransferases by the successive transfer of up to three galactosyl and glucosyl moieties to diacylglycerol.

Agrobacterium tumefaciens↗

PubMed1990

Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts.

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.

Animals↗

PubMed1996

Molecular and biochemical characterization of three anthocyanin synthetic enzymes from Gentiana triflora.

Full length cDNA clones of flavonoid 3',5'-hydroxylase, dihydroflavonol 4-reductase and flavonoid 3-glucosyltransferase were cloned from petals of Gentiana triflora. Their sequences were homologous to counterparts from other plants. Flavonoid 3',5'-hydroxylase and flavonoid 3-glucosyltransferase were enzymatically characterized by expressing cDNAs in heterologous expression systems.

Alcohol Oxidoreductases↗