2-deoxygalactose, a specific substrate of the Salmonella typhiimurium galactose permease: its use for the isolation of galP mutants.

2-Deoxygalactose is a specific substrate of the galactose permease. The apparent Km is about 500 micron, compared to 45 micron for galactose, whereas the maximal rate of uptake is one-half to one-third of that of galactose. None of the other galactose transport systems, including methyl beta-D-thiogalactosides I and II, the beta-methyl-galactoside permease, and both arabinose systems, is able to catalyze transport of 2-deoxygalactose to a significant extent. 2-Deoxygalactose can also be used to isolate mutants defective in galactose permease, since it is bacteriostatic. Colonies that grow with lactate, malate, or succinate as a carbon source in the presence of 0.5 to 2 mM 2-doexygalactose were found to be mostly galP mutants, lacking galactose permease. Spontaneous 2-deoxygalactose-resistant strains arose with a frequency of about 2 X 10(-6). galP mutants have also been derived from pts deletion mutants that require galactose permease for growth on glucose. Revertants have been obtained that have acquired the parental phenotype.     

D-galactose accumulation in rabbit ileum. Effects of theophylline on serosal permeability.

The effects of theophylline and dibutyryl cyclic AMP, on in vitro unidirectional galactose fluxes across the mucosal and serosal borders of rabbit ileum have been studied. 1. When Ringer [galactose] = 2mM, theophylline and dibutyryl cyclic AMP reduce both mucosal-serosal and serosal-mucosal galactose flux by approx. 50%. The K1 for theophylline inhibition of flux in both directions is 2 mM. 1 mM dibutyryl cyclic AMP elicits a maximal inhibitory response. Concurrent with the inhibition in transmural galactose fluxes, theophylline and dibutyryl cyclic AMP increase the tissue accumulation of [galactose] and the specific-activity ratio R of 3H : 14C-labelled galactose coming from the mucosal and serosal solutions respectively. It is deduced that theophylline and dibutyryl cyclic AMP are without effect on the mucosal unidirectional permeability to galactose but cause a symmetrical reduction in serosal entry and exit permeability. 2. Reduction in the asymmetry of the mucosal border to galactose by reducing Ringer [Na], raising Ringer [galctose] or adding ouabain reduces the theophylline-dependent increase in galactose accumulation. 3. Hypertonicity in the serosal solution increases the permeability of the serosal border to galactose and reduces tissue galactose accumulation. Serosal hypertonicity partially reverses the theophylline-depedent effects on galactose transport. Replacing Ringer chloride by sulphate abolishes the theophylline-dependent effects on galactose transport. 4. It is considered that the theophylline-dependent increase in galactose accumulation results from the reduction in serosal permeability. This is shown to be a quantitatively consistent inference. 5. Further support for the view that the asymmetric transport of galactose in rabbit ileum results from convective-diffusion is presented.     

Galactose catabolism in Caulobacter crescentus.

Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.     

Proton movements coupled to sugar transport via the galactose transport system in Salmonella typhimurium.

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium. The addition of galactose to lightly buffered suspensions of anaerobic, non-metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellularpH as galactose and protons enter the cell together. Other substrates for this transport system, D-fucose, 2-deoxygalactose, glucose and 2-deoxyglucose similarly cause an influx of protons when transported. In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx. Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non-energized conditions. At pH 7.2 the permeant anion SCN- increases both the rate and extent of galactose-induced proton influx; at pH 6 the rate, but not the extent is increased by SCN-.     

Allelism of IMP1 and GAL2 genes of Saccharomyces cerevisiae.

Cloning and characterization of the previously described Saccharomyces cerevisiae IMP1 gene, which was assumed to be a nuclear determinant involved in the nucleomitochondrial control of the utilization of galactose, demonstrate allelism to the GAL2 gene. Galactose metabolism does not necessarily involve the induction of the specific transport system coded by GAL2/IMP1, because a null mutant takes up galactose and grows on it. Data on galactose uptake are presented, and the dependence on ATP for constitutive and inducible galactose transport is discussed. These results can account for the inability of imp1/gal2 mutants to grow on galactose in a respiration-deficient background. Under these conditions, uptake was affected at the functional level but not at the biosynthetic level.     

Effects of ethanol and diabetes on galactose oxidative metabolism and elimination in rats.

Blood galactose clearance after an intravenous galactose load has been widely used for years as an index of liver function. We developed a noninvasive [13C]galactose breath test, which explores galactose oxidative metabolism; this test is well correlated with liver fibrosis in patients with chronic viral hepatitis. The goal of this study was to evaluate the influence of nonhepatic factors such as diabetes and ethanol on whole-body galactose clearance (measured as the serum galactose elimination capacity test) and oxidative metabolism (measured as the [13C]galactose-induced breath 13CO2 production) in rats. Acute ethanol administration induced a significant decrease of galactose clearance and 13CO2 production. There was a significant correlation between the amount of ethanol given and the inhibition of galactose metabolism (R2 = 0.72, p < 0.0001). In streptozotocin-induced diabetic rats, the [13C]galactose-induced breath 13CO2 production was significantly reduced (p < 0.0001) and normalized by insulin treatment. However, diabetes did not decrease whole-body galactose clearance, indicating an isotopic dilution of [13C]glucose produced from [13C]galactose metabolism into the enlarged glucose pool. These results must be taken into account when using the [13C]galactose breath test as a quantitative liver function test.     

Purification and mechanism of action of a plant galactokinase]

The previously described galactokinase from Fenugreek seeds, has been purified by affinity chromatography on a column of galactosamine-CH Sepharose. This material ensures a more specific fixation than does ATP-Sepharose. A 400 fold purification was achieved in a single step, with a 80 per cent yield. Km's for galactose and for Mg/ATP2- complex were respectively 0.54 x 10-3 M and 5, 10-3 M. Galactose-1-phosphate is a competitive inhibitor of galactose while the inhibition for Mg-ATP2- is not a competitive one. The Mg-ADP complex is a non-competitive inhibitor of both galactose and Mg-ATP2-. Moreover, the Km of the enzyme for M-ATP2- complex is modified when 2-deoxy- and 6-deoxy-galactose are used instead of galactose. These results are consistent with an ordered sequential mechanism for this galactokinase: galactose binds to the enzyme before Mg-ATP2-, and galactose-1-phosphate is the last reaction product liberated. The affinity of the kinase for 6-deoxygalactose is lower than for 2-deoxygalactose. This observation reveals the importance of the hydroxyl in C6 position for the binding on the enzyme.     

Galactose transport in Streptococcus thermophilus

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Galactose transport in Salmonella typhimurium.

We have studied the various systems by which galactose can be transported in Salmonella typhimurium, in particular the specific galactose permease (GP). Mutants that contain GP as the sole galactose transport system have been isolated, and starting from these mutants we have been able to select point mutants that lack GP. The galP mutation maps close to another mutation, which results in the constitutive synthesis of GP, but is not linked to galR. Growth of wild-type strains on glaactose induces GP but not the beta-methylgalactoside permease (MGP). Strains lacking GP are able to grow slowly on galactose, and MGP is induced; however, D-fucose is a much better inducer of MGP. Induction of GP or MGP is not prevented by a pts mutation, although this mutation changes the apparent Km of MGP for galactose. pts mutations have no effect on GP. GP has a rather broad specificity: galactose, glucose, mannose, fucose, 2-deoxygalactose, and 2-deoxyglucose are substrates, but only galactose and fucose can induce this transport system.     

Conversion of 14C-galactose into amino acids in tissue culture cells and its inhibition by manganese

The incorporation of 14C-galactose into primary AGMK-cells was studied in the presence and absence of Mn2+. The transport of galactose into the cells is not influenced by Mn2+. 1 mM MnCl2 inhibits the incorporation of galactose into acid-precipitable material up to 50% after 6 hours incubation. In the absence of Mn2+ a substantial amount of galactose is converted to glucose, which is mainly metabolized into aspartic acid and serine. The conversion of galactose into glucose is inhibited by the addition of Mn2+. However, Mn2+ does not influence the activity of the UDP-galactose-4'-epimerase in vitro. Using the SDS-polyacrylamide electrophoresis the labelling of protein bands is similar with 14C-galactose or a 14C-amino acid mixture, respectively. In the presence of Mn2+ the incorporation of both galactose or amino acids is inhibited: With amino acids the inhibition is observed in all protein bands, whereas with galactose some bands remain unaffected. It is concluded that these are galactoproteins.     

A fluorometric method for monitoring the enzymic hydrolysis of terminal galactose from GM1-ganglioside.

A fluorometric method for monitoring the enzymic hydrolysis of the terminal galactose from GM₁-ganglioside has been developed. The released galactose is oxidized with galactose dehydrogenase and NAD and the fluorescence of the product NADH measured. This method can detect as little as 0.1 nmol of galactose. β-Galactosidase from the gastropod Turbo cornutus was employed for the hydrolysis reaction. The rate of GM₁-ganglioside hydrolysis is linearly proportional to incubation time for 30 min under the assay conditions employed. In addition to galactose, the other product of hydrolysis, GM₂-ganglioside, is identified by thin-layer chromatography. This procedure provides a convenient and specific method for measuring the release of galactose from GM₁-ganglioside.     

A coupled enzyme assay for measurement of sialidase activity.

A multi-coupled enzyme assay system for determining sialidase activity is described. Enzymes, substrates and chromogens are reacted in situ and determined spectrophotometrically in ELISA microtiter plates. Sialidase is assayed by the extent of desialylated galactose on an appropriate sialoglycoconjugate (fetuin), which is otherwise unavailable for oxidation by galactose oxidase. The oxidation is monitored by the coupling of H2O2 released to a third enzyme, peroxidase. The rate of change of absorbance at 405 nm, resulting from the oxidized chromogen is a measure of the reaction rate of the coupled enzyme system. A similar system can be used for determining galactose oxidase in solution, or on blots using galactose as substrate. Due to the small-scale single-step measurement, the described assay is a sensitive, convenient, and inexpensive alternative to the classic colorimetric determination.     

Formation and properties of retinylphosphate galactose.

Crude cell membrane fractions from a number of tissues can form acidic glycolipids. The formation of acidic galactose lipid and mannose lipid was greatly reduced in vitamin A deficiency, primarily in tissues known to be mucus-producing. Mouse mastocytoma tissue was active in forming acidic galactose lipids with UDP-galactose as substrate. One of the products was identified as retinylphosphate galactose. The synthetase reaction producing this compound exhibited an apparent pH optimum at 6.3. The presence of detergent and retinol stimulated the synthetase reaction, which exhibited an absolute requirement for Mn2+ or Mg2+. The synthetase reaction was readily reversible. Incubation of particulate enzyme with retinylphosphate galactose and UDP yielded UDP-galactose and a compound tentatively identified as retinylphosphate. The galactose lipid was isolated by column chromatography on DEAE-cellulose and silica gel. The retinylphosphate galactose was homogeneous when examined by thin layer chromatography. Mild acid hydrolysis of labeled retinylphosphate galactose yields [14C]galactose, whereas alkaline hydrolysis and hydrogenolysis produced [14C]galactose 1-phosphate. Retinylphosphate galactose bound to vitamin A-depleted, retinol-binding protein.     

Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP- galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled alpha-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12(+) and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the Delta mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than alpha1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S. pombe alpha1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galalpha1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S. cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S. cerevisiae.      

Enzymes related to galactose utilization inPseudomonas cepacia

Pseudomonas cepacia produced a characteristic green sheen on EMB-galactose plates owing to production of galactonic acid by the constitutive membrane-associated glucose dehydrogenase of this bacterium. Mutants isolated as glucose dehydrogenase deficient (Gcd^–) also were deficient in membrane-associated galactose dehydrogenase. A strain that formed glucose dehydrogenase at 30°C but not at 40°C was also temperature sensitive with respect to formation of galactose dehydrogenase. The Gcd^– strains still utilized galactose. A second, NAD-specific, galactose dehydrogenase (not membrane associated) along with a transport system for galactose were induced during growth on galactose and constituted an alternative pathway of conversion of galactose to galactonate. Enzymes of the De Ley-Doudoroff pathway of conversion of galactonate to pyruvate and glyceraldehyde-3-phosphate were induced during growth on galactose. Unexpectedly, growth on galactose also elicited formation of enzymes of the Entner-Doudoroff (ED) route. Furthermore, mutants blocked in the ED pathway grew poorly on galactose. One interpretation of these findings is that glyceraldehyde-3-phosphate formed from galactose via the De Ley-Doudoroff route (by cleavage of 2-keto-3-deoxy-6-phosphogalaconate) is reconverted to hexose phosphate and metabolized via the ED pathway.     

The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose

The enzyme UDP-galactose-4-epimerase (GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDP-glucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C. albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of beta-d-galactose to alpha-d-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. Absence of C. albicans GAL10 (CaGAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H2O2. While the S. cerevisiae GAL10 (ScGAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C. albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose.