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PubMed1976

Transport of 2-deoxy-D-galactose in Saccharomyces fragilis.

2-Deoxy-D-galactose (dGal) transport in Saccharomyces fragilis is characterized by energy requirement and accumulation of the free sugar against a concentration gradient, indicating active transport. Besides free sugar dGal-1-phosphate, UDP-dGal and a trehalose-like derivative were found inside the cells. The accumulation of the phosphorylated derivatives was balanced by a concomitant decrease of ATP, orthophosphate and polyphosphates. With pulse labeling experiments it could be shown that the free sugar is transported into the cells. This conclusion was supported by several other experimental results, e.g. the lack of correlation between the sugar transport parameters and the dGal phosphorylation capacity, and the countertransport of free dGal evoked by galactose in the medium. The typical differences between this active transport mechanism and the transport-associated phosphorylation system, described previously, are discussed.

Adenosine Triphosphate↗
PubMed1989

Construction of an artificial bifunctional enzyme, beta-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling.

The in-frame fusion between two oligomeric enzymes, beta-galactosidase and galactose dehydrogenase, is described. The lacZ gene was fused to the 3' end of the galdh gene with a linker encoding only three amino acids. The purified artificial bifunctional enzyme displayed the enzymic activity of both gene products. The hybrid protein was found in two major forms, consisting of four and six subunits, but other forms could also be identified. The molecular weight of each subunit was determined to be 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bifunctional enzyme shows kinetic advantages over the identical native system in conversion of lactose to galactonolactone. A higher steady-state rate and a reduction of the transient time are observed. This phenomenon is especially pronounced at low initial substrate concentrations and when the pH is adjusted to a level at which the galactose dehydrogenase activity is much higher than that of the beta-galactosidase.

Carbohydrate Dehydrogenases↗
PubMed2004

Refinement of the conformation of UDP-galactose bound to galactosyltransferase using the STD NMR intensity-restrained CORCEMA optimization.

The STD NMR technique has originally been described as a tool for screening large compound libraries to identify the lead compounds that are specific to target proteins of interest. The application of this technique in the qualitative epitope mapping of ligands weakly binding to proteins, virus capsid shells, and nucleic acids has also been described. Here we describe the application of the STD NMR intensity-restrained CORCEMA optimization (SICO) procedure for refining the bound conformation of UDP-galactose in galactosyltransferase complex using STD NMR intensities recorded at 500 MHz as the experimental constraints. A comparison of the SICO structure for the bound UDP-galactose in solution with that in the crystal structure for this complex shows some differences in ligand torsion angles and V253 side-chain orientation in the protein. This work describes the first application of an STD NMR intensity-restrained CORCEMA optimization procedure for refining the torsion angles of a bound ligand structure. This method is likely to be useful in structure-based drug design programs since most initial lead compounds generally exhibit weak affinity (millimolar to micromolar) to target proteins of pharmaceutical interest, and the bound conformation of these lead compounds in the protein binding pocket can be determined by the CORCEMA-ST refinement.

Acetylglucosamine↗
PubMed1988

Galactose at the nonreducing terminus of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein ZP3 is essential for the glycoprotein's sperm receptor activity.

During fertilization in mice, zona pellucida glycoprotein ZP3 mediates initial sperm-egg interactions by serving as receptor for sperm. Purified egg ZP3, as well as ZP3-derived O-linked oligosaccharides, exhibit sperm receptor activity in vitro. We report that treatment of purified egg ZP3 and ZP3-derived O-linked oligosaccharides with either alpha-galactosidase or galactose oxidase results in loss of sperm receptor activity. In the latter case, sperm receptor activity can be restored to the oxidized glycoprotein and O-linked oligosaccharides by treatment with sodium borohydride. We conclude that galactose, located in alpha-linkage at the nonreducing terminus of O-linked oligosaccharides, is at least one of the sugar determinants on ZP3 responsible for binding of sperm to the zona pellucida.

Animals↗
PubMed1996

Molecular cloning and characterization of a novel isoform of the human UDP-galactose transporter, and of related complementary DNAs belonging to the nucleotide-sugar transporter gene family.

We described recently the molecular cloning of human UDP-galactose transporter 1 (hUGT1) [Miura, N. et al. (1996) J. Biochem. 120, 236-241]. Now we have characterized its isoform, hUGT2, that is most likely generated through the alternative splicing of a transcript derived from the UGT genomic gene, that also codes for hUGT1. Introduction of the open reading frame sequence of hUGT2 into a mouse cell line, Had-1, that lacks the UDP-galactose transporter, complemented the genetic defect of the mutant, as judged from the lectin-sensitivity spectra of the transformants and the nucleotide-sugar transporting activity of microsomal vesicles isolated from them. UGT-related genes were found through a BLAST search of dbEST based on their significant similarity with hUGT genes. We report here cDNA clones belonging to two subfamilies of the nucleotide-sugar transporter gene family. One is the human CMP-sialic acid transporter gene, and the other is a group of homologous genes with an undefined function that are distributed in man, mouse, and rat, and show significant similarity to the yeast UDP-N-acetylglucosamine transporter.

Amino Acid Sequence↗
PubMed1975

The action of group Bm or CisAB sera on group O red cells in the presence of UDP-D-galactose.

Group B and AB sera, acting on O red cells in the presence of UDP-galactose, each converted them into B active cells, which were agglutinated by anti-B human serum (1:512) at the titer of 128-fold, while group Bm and A-1 Bm sera, converted O red cells similarly incubated into B active cells, which were agglutinated by anti-B human serum (1:512) at the titer of 8- to 16-fold. This indicates that alpha-galactosyltransferase activity in Bm and A-1 Bm sera may be about 1/8-1/16 that in B and AB sera. Group CisAB sera, even after absorption of cold anti-B agglutinins with packed, washed group B red cells, did not convert O red cells in the presence of UDP-galactose in such a way that they might agglutinate against anti-B human serum.

ABO Blood-Group System↗
PubMed1986

Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes.

The biochemical defect in the mutant Chinese hamster ovary cell lines Clone 13 (Briles, E. B., Li, E., and Kornfeld, S. (1977). J. Biol. Chem. 252, 1107-1116) and Lec8 (Stanley, P. (1980) ACS Symp. Ser. 128, 214-221) was examined. These two mutants, which belong to the same genetic complementation group, were shown in previous studies to exhibit an 80-90% reduction in galactosylation and sialylation of proteins and lipids when compared to wild-type cells. The same studies, however, demonstrated that the mutants were not deficient in the corresponding sugar nucleotides, glycosyltransferases, and endogenous acceptors for these transferases. We now provide evidence strongly suggesting that the primary defect in Lec8 and Clone 13 cells is their inability to translocate UDP-galactose into the lumen of the Golgi apparatus. Golgi vesicles from Lec8 and Clone 13 CHO glycosylation mutants translocate in vitro UDP-galactose at only 3-5% the rate of vesicles from wild-type CHO cells. The deficiency is specific because vesicles from the mutant cells can translocate adenosine 3'-phosphate 5'-phosphosulfate, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine at rates comparable to those of vesicles from wild-type cells. These studies also suggest that sugar nucleotides sharing a common uridine nucleotide utilize different translocators present in the Golgi membrane in vivo. The consequence of the above-described mutations and the resulting block in galactosylation of macromolecules in vivo on the translocation of CMP-sialic acid into the Golgi lumen was also examined. As expected, Golgi apparatus vesicles from Lec8 cells were unable to incorporate sialic acid into (endogenous) macromolecules. However, the vesicles were able to transport CMP-sialic acid into their lumen, although the rate of translocation was only 17% of that of wild-type-derived Golgi vesicles.

Animals↗
PubMed1984

Galactose neuropathy: impact of chronic endoneurial edema on nerve blood flow.

Peripheral neuropathy induced by galactose feeding results in endoneurial edema with increased tissue pressure and ultimate demyelination of nerve fibers. To assess the role of ischemia in the pathogenesis of this neuropathy, nerve blood flow was measured 6 months after the start of galactose ingestion, between the onset of edema and nerve fiber changes. Measurement was carried out by a noninvasive technique involving the use of [14C]iodoantipyrine as a radioactive tracer of tissue perfusion. A significant decline in nerve blood flow was found, which correlated positively with increased nerve water content as quantified by a microgravimetric technique. We sought to establish the pathogenic role of nerve edema in the development of schwannopathy and demyelination by morphological examination. It was found that Schwann cells appeared normal in areas of the peripheral nervous system in which edema does not occur, such as spinal ganglia and roots, whereas in regions in which there was edema, massive glycogen accumulation in Schwann cells and demyelination occurred. These findings suggest that edema, rather than hyperactivity in the sorbitol pathway, is responsible for the pathological changes in galactosemic neuropathy and that ischemia resulting from edema and increased endoneurial fluid pressure is the mechanism responsible for fiber injury.

Animals↗
PubMed2003

MM3 potential energy surfaces of trisaccharides. II. Carrageenan models containing 3,6-anhydro-D-galactose.

The adiabatic potential energy surfaces (PES) of six trisaccharides-namely 3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp, beta-D-Galp-(1-->4)-3,6-An-alpha-D-Galp-(1-->3)-beta-D-Galp, and their derivatives sulfated on positions 2 and 4 of the beta-galactose unit-were obtained using the MM3 force field. Each PES was described by a single contour map for which the energy is plotted against the two psi glycosidic angles, given the small variations of the phi glycosidic torsional angle in the low-energy regions of disaccharide maps. In five of the six examples, the surfaces are those expected from the maps of the disaccharidic repeating units of carrageenans, with less important factors altering the additive effect of both linkages. However, when a sulfate group is present on C2 of a beta-galactose reducing end, a new low-energy minimum in a different region is produced, originated in a hydrogen bond between the first and third monosaccharidic moieties of the trisaccharide. The flexibility of the beta-linkages is nearly identical to that in their disaccharide counterparts, while that of the alpha-linkages is slightly reduced, independent of their presence closer or further away from the reducing end. A fair agreement is observed between the x-ray fiber diffraction analysis for a kappa-carrageenan double helix and the surfaces obtained for the trisaccharide analogs of that polymer.

Carbohydrate Conformation↗
PubMed1976

Galactose oxidase: applications of the covalently immobilized enzyme in a packed bed configuration.

Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.

Alcohol Oxidoreductases↗
PubMed2004

Prospective evaluation of a novel approach for the use of a quantitative galactose oxidase-Schiff reaction in ductal fluid samples from women with breast carcinoma.

BACKGROUND: The galactose oxidase-Schiff reaction (GOS) yields positive findings in a number of malignant solid tumors. The goals of the current study were to develop a novel technique for quantifying GOS reactivity in nipple aspirate fluid (NAF) samples from women with invasive breast carcinoma and to assess the clinical utility of the technique in this setting. METHODS: Patients with biopsy-proven unilateral invasive breast carcinoma were eligible for study entry. Before definitive surgery, NAF samples were obtained from healthy breast tissue and malignant breast tissue from 23 women with breast carcinoma. Under blind conditions with respect to clinical data, 10 microL NAF samples were applied to a glass fiber membrane and incubated with 100 microL galactose oxidase and 1 mL Schiff reagent. The stain was developed and the color reaction quantitated by measuring hue (shade) and chroma (intensity) using a spectrophotometer. RESULTS: GOS reactivity was quantitated using two color parameters, hue and chroma. Because chroma varies with concentration, this measurement was adjusted for the concentration of NAF in each sample. After adjustment for NAF concentration, chroma was found to be statistically significantly different in the affected breast tissue sample and the healthy contralateral internal control sample (P = 0.001). CONCLUSIONS: A quantitative measure of GOS reactivity based on spectrophotometric measurement of intensity of color has been developed and was found to be significantly different in the affected breast compared with the unaffected breast in the current population of patients with breast carcinoma. The preliminary results support further exploration of this novel quantitative test in patients with breast carcinoma.

Adult↗
PubMed1983

Characterization of two mutations in the Escherichia coli galE gene inactivating the second galactose operator and comparative studies of repressor binding.

A series of plasmids has been constructed which contain either a single one of the two operators O1 and O2 of the Escherichia coli galactose operon or different combinations thereof. This permits comparison of the two operators with respect to their repressor binding ability. A plasmid containing only the second operator, O2, located within the structural gene galE, was found to titrate repressor in vivo and in vitro with essentially the same efficiency as a plasmid containing only the 'classical' galactose operator, O1, located upstream of the start of transcription. Whereas some cooperativity between the two sites seems possible in vivo, they bind repressor independently under the conditions of the in vitro assay. After hydroxylamine treatment of plasmid DNA in vitro, two different mutations have been isolated each of which inactivates the second operator, O2. Both are GC to AT transitions located at equivalent positions relative to the axis of rotational symmetry within the second gal operator. Subdividing O1 and O2 according to their 2-fold symmetry yields four half-sites with the consensus sequence (5')gTGnaAnC(3'). All known single point mutations of O1 and O2 affect the frame of invariant residues. The two half-sites of the lac operator also coincide with this frame.

Alleles↗
PubMed1992

Human serum amyloid P is a multispecific adhesive protein whose ligands include 6-phosphorylated mannose and the 3-sulphated saccharides galactose, N-acetylgalactosamine and glucuronic acid.

Carbohydrate recognition by amyloid P component from human serum has been investigated by binding experiments using several glycosaminoglycans, polysaccharides and a series of structurally defined neoglycolipids and natural glycolipids. Two novel classes of carbohydrate ligands have been identified. The first is 6-phosphorylated mannose as found on lysosomal hydrolases, and the second is the 3-sulphated saccharides galactose, N-acetyl-galactosamine and glucuronic acid as found on sulphatide and other acidic glycolipids that occur in neural or kidney tissues or on subpopulations of lymphocytes. Binding to mannose-6-phosphate containing molecules and inhibition of binding by free mannose-6-phosphate and fructose-1-phosphate are features shared with mannose-6-phosphate receptors involved in trafficking of lysosomal enzymes. However, only amyloid P binding is inhibited by galactose-6-phosphate, mannose-1-phosphate and glucose-6-phosphate. These findings strengthen the possibility that amyloid P protein has a central role in amyloidogenic processes: first in formation of focal concentrations of lysosomal enzymes including proteases that generate fibril-forming peptides from amyloidogenic proteins, and second in formation of multicomponent complexes that include sulphoglycolipids as well as glycosaminoglycans. The evidence that binding to all of the acidic ligands involves the same polypeptide domain on amyloid P protein, and inhibition data using diffusible, phosphorylated monosaccharides, is potentially important leads to novel drug designs aimed at preventing or even reversing amyloid deposition processes without interference with essential lysosomal trafficking pathways.

Acetylgalactosamine↗
PubMed2003

Evaluation of a galactose-carrying gelatin sponge for hepatocytes culture and transplantation.

This study proposes a new three-dimensional culture of mouse hepatocytes in a porous galactose-carrying modified gelatin sponge matrix. The modification of gelatin using galactose residues significantly increased the attachment of hepatocytes on the substrate. A modified gelatin sponge with lactobionic acid (MGLA) was prepared to increase the specific interaction between the hepatocytes and the matrix. Hepatocytes cultured in a three-dimensional MGLA sponge released much less lactate dehydrogenase than those cultured on a collagen Type I-coated monolayer. Moreover, the survival rate of hepatocytes cultured on an MGLA sponge was longer than the survival rate of hepatocytes cultured on a collagen Type I-coated monolayer. Hepatic specific metabolic functions, namely, the secretion of serum albumin and the synthesis of urea, were well maintained and promoted by spheroidal hepatocytes formed in the MGLA sponge.

Animals↗
PubMed1984

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells.

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.

Animals↗
PubMed1987

L-fucose, D-mannose, L-galactose, and their BSA conjugates stimulate macrophage migration.

The effect of selected monosaccharides on the random migration of normal adult rabbit alveolar macrophages (AM) was investigated. It was observed that 10 mM of L-fucose, L-galactose, or D-mannose stimulated AM migration 1.5-2.0 times. In addition, derivatives of L-fucose and D-mannose occupying the carbon-6 position such as L-fucosyl-lactose, D-mannose-6-phosphate, D-mannitol, and mannan enhanced the migration of AM, whereas derivatives of L-fucose and D-mannose in the carbon-1 position produced no migration enhancement. Macrophage migration enhancement activity that was produced spontaneously by spleen cell cultures from normal young rabbits was destroyed by treatment with L-fucosidase. Accordingly, the migration enhancement factor (MEF) found in spleen cell culture supernatants appeared to depend on L-fucose conjugated to some protein carrier because MEF was non-dialyzable. When normal adult AM were treated with L-fucosidase, they lost their responsiveness to migration inhibitory factor (MIF) but retained their responsiveness to MEF. We have interpreted this to mean that the MIF and MEF receptors are distinct. Synthetic MEFs were prepared by conjugating L-fucose, D-mannose, of L-galactose to bovine serum albumin (BSA). It was noted that these sugar-BSA conjugates were about 200 times more effective than the corresponding free sugars in producing migration enhancement. In addition, these sugar-BSA conjugates neutralized MIF activity in a migration inhibition test.

Animals↗
PubMed2003

Identification of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone in galactose oxidase-producing isolates of Fusarium graminearum.

Fusarium graminearum strains are well known for their role as plant pathogens and for their production of mycotoxins, and less known for their secretion of galactose oxidase, a well-studied and useful enzyme. Three galactose oxidase-producing isolates of F. graminearum were grown on rice to identify the production of zearalenone and trichothecenes through the use of thin layer chromatography and gas chromatography coupled to mass fragmentation. Detection and identification of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone were accomplished.

Fusarium↗
PubMed1990

Spontaneous mutations in the galactose operons of Streptomyces coelicolor A3 (2) and Streptomyces lividans 66.

Mutations can be divided into two classes: point mutations (base changes, frame shifts) and DNA rearrangements (deletions, insertions, inversions etc.). In Escherichia coli K 12, DNA insertions account for up to 40% of spontaneous mutations that inactivate genes (for review, see CULLUM 1985) and the Insertion Sequences involved can also mediate deletions and inversions. We started to study spontaneous mutations in Streptomyces in the expectation of isolating transposable elements and chose the galactose genes as convenient system. When selection is made for resistance to the galactose analogue 2-deoxygalactose (2dgalR), gal K mutants are obtained that are defective in the enzyme galactokinase (Kendall et al. 1987). We isolated spontaneous 2dgalR mutations in the closely related strain S. coelicolor A 3 (2) and S. lividans 66 and cloned the gal K gene of the former strain. Adams et al. (1988) have cloned and sequenced the gal genes of S. lividans 66 and shown that they form an operon, the restriction map appears to be identical to that of S. coelicolor A 3 (2). In this paper we describe the characterisation of spontaneous 2 dgalR mutants in S. coelicolor A 3 (2) and S. lividans 66.

Chromosome Deletion↗