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PubMed1980

[Alpha-methylglucoside transmembrane phosphorylation and regulation of the beta-galactoside permease activity in E. coli K12].

The interaction between alpha-methylglucoside (MeGlc) and beta-galactoside transport systems in E. coli K12 was studied. It was shown that an addition of MeGlc to bacterial cells leads to repression of [14C] lactose accumulation and o-nitrophenyl-beta-D-galactopyranoside (NO2PheGal) hydrolysis. The mutational damage of one of the components of the MeGlc transport system is accompanied by elimination of the glucoside inhibitory action. Intracellular MeGlcP and GlcP lower the efficiency of the transmembraneous transfer of beta-galactosides. The data obtained suggest that repression of the beta-galactoside permease activity during transport of the MeGlc is a result of two processes: i.e. phosphorylation-coupled translocation of MeGlc and intracellular accumulation of MeGlcP. An assumption on the intramembraneous interaction of enzyme IIGlc with beta-galactoside permease is made.

Bacterial Proteins↗
PubMed1981

Glycosyltransferases of the human cervical epithelium. I. Characterization of a beta-galactoside alpha-2-L-fucosyltransferase and the identification of a beta-N-acetylglucosaminide alpha-3-L-fucosyltransferase.

Using phenyl beta-D-galactoside as an acceptor, alpha-2-L-fucosyltransferase activity was identified in human cervical epithelium with pH optima at 6.0 and 7.2. The different response to p-chloromercuribenzoate, and ability to utilise asialofetuin as an acceptor, suggests the presence of two fucosyltransferases. The acid form is probably involved in glycoprotein synthesis in vivo. At pH 6.0, fucosyltransferase has a temperature optimum of 25 degrees C, requires the presence of Triton X-100 and either manganese or magnesium for maximal activity, and has Km values for GDP-L-[14-C]fucose and phenyl beta-D-galactoside of 32.1 . 10(-6) M and 8.2 . 10(-3) M, respectively. Guanosine nucleotides are potent inhibitors of the fucosyltransferase reaction; GDP is a competitive inhibitor while, depending on its concentration, GTP can either inhibit or activate the reaction. The alpha-L-fucosidase present in cervical tissue has negligible activity towards the enzyme product, phenyl-alpha-2-L-[14C]fucosyl-beta-D-galactoside. The use of high and low molecular weight acceptors indicates the presence of a beta-N-acetylglucosaminide alpha-3-L-fucosyltransferase and an N-acetylgalactosaminide fucosyltransferase.

Asialoglycoproteins↗
PubMed1981

Inhibition of growth of Escherichia coli by lactose and other galactosides.

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a beta-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in delta pH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-beta-D-thiogalactoside.

Escherichia coli↗
PubMed1987

The role of protons in the mechanism of galactoside transport via the lactose permease of Escherichia coli.

The kinetic mechanism of lactose transport across the cytoplasmic membrane has been investigated and the results related to standard models for the lactose-H+ symport reaction using computer simulation. It is shown that the biphasic kinetics reported for lactose uptake (Kaczorowski, G.J. and Kaback, H.R. (1979) Biochemistry 18, 3691-3697) are consistent with random binding of lactose and protons and rapid subsequent translocation of the ternary lactose-H+-permease complex. Such a model is also shown to explain the observed dependence of the kinetic parameters on the magnitude of the protonmotive force. Both sugar and protons are shown to cause product inhibition of lactose flux and the ability of standard models to account for the pattern of inhibition is discussed. Three apparent dissociation constants have been determined for the protonation reactions in the external medium: two (pKa 6.3 and 9.6) control the activity of the permease, whilst the third (pKa 8.3) controls the affinity of the permease for galactosides. A similar set of dissociation constants has been determined for the internal reactions. Again two (pKa 6 and 9.8) control activity and a third (pKa 8.8) controls the affinity for galactosides. The dissociation reactions characterised by pKa 8.3, 8.8, 9.6 and 9.8 are attributed to the dissociation of the substrate (symported) proton from the binary proton-permease complexes (pKa 8.3 and 8.8) and the ternary proton-galactoside-permease complexes (pKa 9.6 and 9.8). The third pair (pKa 6.3 and 6.0) must be interpreted as describing a separate protonation reaction which may have a regulatory or auxiliary role in transport.

Bacterial Proteins↗
PubMed1994

Expression of galactoside-specific endogenous lectins and their ligands in human oral squamous cell carcinoma.

Human endogenous lectins have a wide spectrum of biological functions. The present study analyses the expression of beta-galactoside specific and N-acetyl-D-galactosamine specific endogenous lectins in oral squamous cell carcinomas using biotinylated neoglycoproteins. The expression pattern of beta-galactosyl-containing glycoconjugates or ligands of beta-galactoside specific lectins in these tissues was also studied using an endogenous biotinylated lectin, the human 14-kDa lectin. For comparison a galactoside specific plant lectin from mistletoe, Viscum album was also employed. The results demonstrate that oral squamous cell carcinomas mainly express accessible binding sites for lactosylated neoglycoprotein (90%) while few carcinomas expressed mild amount of N-acetyl-D-galactosamine specific binding sites (40%). There was no difference in the binding patterns of these probes between well and less differentiated carcinomas. Expression of these neoglycoprotein binding sites were mostly concentrated in immature basaloid cells, indicating a possible association with cell proliferation. The binding pattern of D-galactosyl specific lectins (human 14-kDa and mistletoe lectins) showed conspicuous differences. This feature emphasizes the caution that needs to be exercised in interpreting the biological significance of results attained using plant lectins on human tissue.

Acetylgalactosamine↗
PubMed1987

Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain.

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.

Amino Acids↗
PubMed2006

Synthesis and biological evaluation of the suberoylanilide hydroxamic acid (SAHA) beta-glucuronide and beta-galactoside for application in selective prodrug chemotherapy.

The beta-O-glucuronide and beta-O-galactoside of SAHA have been prepared and evaluated as prodrugs for selective cancer chemotherapy (ADEPT, PMT). These new compounds are stable under physiological conditions and do not exhibit any antiproliferative activity compared to the parent drug after a 48-h treatment of H661 cells. The glucuronide derivative did not lead to the release of the drug in the presence of either Escherichia coli or bovine liver beta-glucuronidase. On the other hand, under enzymatic cleavage of galactoside prodrug by the corresponding enzyme, a rapid release of SAHA was observed demonstrating that the beta-O-galactoside of SAHA is a promising candidate for in vivo investigations.

Animals↗
PubMed2004

The cleavage of vitamin E galactoside in the rat tissue homogenates.

The stability of alpha-tocopheryl beta-galactoside in the presence of endogenous galactosidases in selected tissue homogenates (liver, kidney, ileum and brain) was estimated. High degree release of alpha-tocopherol from alpha-tocopheryl beta-galactoside in tissues of ileum, kidney and brain was observed (82%, 75% and 72%, increase above endogenous alpha-tocopherol, respectively). A possible enzymatic mechanism of the galactoside decomposition was proposed.

Animals↗
PubMed1992

Dichotomy in the laminin-binding properties of soluble and membrane-bound human galactoside-binding protein.

Recent studies indicate that galactoside-binding proteins may bind the poly-N-acetyllactosamine sequences of laminin. We questioned whether human galactoside-binding protein (hL-31) binds to laminin and whether cells that express hL-31 on their surface use it as a laminin receptor to promote cellular attachment. The data show that both lectin and cells bind to immobilized laminin. The binding of soluble lectin to laminin is inhibited by lactose, while cell adhesion to it is not. The results indicate that laminin may be a ligand for soluble galactoside-binding proteins.

Binding Sites↗
PubMed1998

Properties of an alpha-galactosidase, and structure of its gene galA, within an alpha-and beta-galactoside utilization gene cluster of the hyperthermophilic bacterium Thermotoga maritima.

Thermotoga maritima represents one of the few hyperthermophilic bacteria currently known. The chromosomal alpha-galactosidase gene of T. maritima strain MSB8 has been cloned and its nucleotide sequence was determined. The gene, designated galA, has coding capacity for a 552 residue polypeptide with a calculated molecular mass of 63,653 Da. GalA was found to be flanked by other genes probably involved in galactoside breakdown and utilization. The previously sequenced beta-galactosidase gene, lacZ, is localized immediately upstream of galA while two open reading frames that putatively encode enzymes of galactose catabolism, i.e. galactose-1-phosphate uridylytransferase (galT) and galactokinase (galK), were found downstream of galA. The identified genes are extremely close together or even overlap and have the same orientation, so they could all be part of one galactoside utilization operon of T. maritima MSB8. GalA displayed low-level amino acid sequence similarity with alpha-galactoside of glycosyl hydrolase family 36. However, GalA is smaller than the other members of this enzyme family. The galA gene was expressed in Escherichia coli and the recombinant alpha-galactosidase was purified and characterized. The molecular mass of the recombinant enzyme was estimated at about 62 kDa by denaturting gel electrophoresis. Maximal hydrolysis of the chromogenic substrate p-nitrophenyl-alpha-D-galactopyranoside was measured at pH 5.0-5.5 and 90-95 degrees C (5 min assay). Divalent cations were not required for activity. The enzyme released galactose from raffinose, melibiose and the synthetic substrates p-nitrophenyl-and omicron-nitrophenyl-alpha-D-galactopyranoside. The T. maritima alpha-galactosidase thus was highly specific for the galactose moiety and the alpha-anomeric configuration of the glycosidic linkage. Its extreme thermal stability (t 1/2 = 6.5 h at 85 degrees C) makes this enzyme an interesting candidate for biotechnological applications.

Amino Acid Sequence↗
PubMed1993

Two step single primer mediated polymerase chain reaction. Application to cloning of putative mouse, beta-galactoside alpha 2,6-sialyltransferase cDNA.

Using the 2 step single primer mediated polymerase chain reaction(PCR), mouse beta-galactoside alpha 2,6-sialyltransferase cDNA was cloned. Single primer mediated PCR is a method to amplify a particular DNA fragment beyond its known sequence region. It employs only one primer for the reaction. Compared to other PCR methods to amplify an adjacent sequence of known DNA fragment, this method requires no enzymatic manipulation on template DNA and is applicable to a template on long DNA fragment. First, a short DNA fragment of the enzyme was obtained from mouse cDNA by the usual PCR method using degenerate primers synthesized according to a relatively conserved region in rat and human beta-galactoside alpha 2,6-sialyltransferase. Four primers were synthesized based on this sequence, then 2 step single primer mediated PCR were performed to obtain 5' and 3' flanking sequences of this short fragment resulting in 1.0 kb and 1.3 kb fragments being amplified respectively. The integrity of the two fragments was confirmed by an additional PCR using primers synthesized according to the joined sequence, which contained 1.2kb complete putative mouse beta-galactoside alpha 2,6-sialyltransferase coding region. The result showed that the specificity and consequently applicability of the single primer mediated PCR for amplifying a particular DNA fragment beyond known sequence region was remarkably improved by the successive 2nd reaction.

Amino Acid Sequence↗
PubMed1991

Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes.

Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of Mr 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2004

Functionalization of OEP-based benzochlorins to develop carbohydrate-conjugated photosensitizers. Attempt to target beta-galactoside-recognized proteins.

meso-(2-Formylvinyl)octaethylporphyrin on reaction with cyanotrimethylsilane in the presence of various catalysts [copper triflate [Cu(OTf)(2)], indium triflate [In(OTf)(3)], or magnesium bromide diethyl etherate (MgBr(2).Et(2)O)] produced a mixture of the intermediate 3-hydroxy-3-cyanopropenoporphyrin, the corresponding trimethylsilyl ether derivative, and the unexpected propenochlorins. The yields of the reaction products were found to depend on the reaction conditions and the catalysts used. The intermediate porphyrins on treatment with concentrated sulfuric acid yielded the free-base cyanobenzochlorins in major quantity along with several other novel benzochlorins as minor products. Reduction of ethyl-3-hydroxy-1-pentenoate-porphyrin with DIBAL-H/NaBH(4) and subsequent acid treatment provided the corresponding free-base 10(3)-(2-hydroxyethyl)benzochlorin, which upon a sequence of reactions gave a free-base benzochlorin bearing a carboxylic acid functionality in good yield. It was then condensed with a variety of carbohydrates (glucosamine, galactosamine, and lactosamine), and the related conjugates were screened using the galectin-binding-ability assay. Among the carbohydrate conjugates investigated, the lactose and galactose analogues displayed the galectin-binding ability with an enhancement of about 300-400-fold compared to lactose. In preliminary studies, all photosensitizers (with or without carbohydrate moieties) were found to be active in vitro [radiation-induced fibrosarcoma (RIF) tumor cells]. However, the cells incubated with lactose (known to bind to beta-galactoside-recognized proteins) prior to the addition of the photosensitizers containing the beta-galactose moiety (e.g., galactose and lactose) produced a 100% decrease in their photosensitizing efficacy. Under similar experimental conditions, benzochlorin without a beta-galactoside moiety or the related glucose conjugate did not show any inhibition in its photosensitizing efficacy. These results in combination with the galectin-binding data indicate a possible beta-galactoside-recognized protein specificity of the galactose- and lactose-benzochlorin conjugates.

Carbohydrates↗
PubMed1984

The transient kinetics of uptake of galactosides into Escherichia coli.

The uptake of galactosides into Escherichia coli via the lactose permease was studied in the time range 0.01-10s by rapid mixing and quenched flow. An initial transient was observed under two conditions. Firstly, a lag in the approach to the steady state was observed at low galactoside concentrations (less than Km). Secondly, a burst of uptake was observed when anaerobic cell suspensions were mixed with aerobic substrate solutions. However, the cause of the burst of uptake appears to be a burst in the rate of respiration. The rate of galactoside uptake during this phase is 10-fold greater than during the steady state.

Antimetabolites↗
PubMed1999

Nutrient content and protein and energy digestibilities of ethanol-extracted, low alpha-galactoside soybean meal as compared to intact soybean meal.

A study was conducted with adult Leghorn roosters to determine TMEn, dry matter digestibility, amino acid content and digestibility, and cellulose and hemicellulose content and digestibility of ethanol-extracted, low alpha-galactoside soybean meal (SBM). Results were compared to that of commercially available 47% CP SBM. Twenty-one roosters were utilized; seven roosters were precision-fed each of the two soybean meal treatments, and seven served as feed-deprived controls. Excreta were collected for 48 h after feeding. A second experiment compared the TMEn, as determined with roosters, to AMEn, as determined with male broiler chicks, of 44% and 47% CP SBM and three different ethanol extraction procedures to remove alpha-galactosides. The ethanol extraction procedure concentrated CP, gross energy, and TMEn. Dry matter digestibility of SBM increased from 52.1% (control SBM) to 63.3% (ethanol-extracted SBM) because of the ethanol extraction procedure. The average amino acid digestibility of ethanol-extracted SBM was 91.6%, compared to 88.0% for control SBM. The digestibility of methionine, alanine, valine, and lysine was improved by the ethanol extraction procedure. Total cellulose and hemicellulose digestion, measured as digestion of specific monosaccharide components, was increased by the ethanol extraction. The TMEn and AMEn values for the different control and extracted SBM were similar. These data indicate that the ethanol extraction of SBM to remove alpha-galactosides resulted in a product that is more protein and energy dense than commercial SBM. Advantages in digestion of amino acids and fiber may contribute to the increase in TMEn observed in both young and adult birds.

Animal Feed↗
PubMed1977

beta-D-Galactoside transport in Escherichia coli: substrate recognition.

1. A number of galactosides and other sugar compounds were examined as inhibitors of facilitated or active transport by the lactose permease system of Escherichia coli. Efficient inhibition required an alpha- or beta-anomeric galactopyranosyl ring of D-configuration, a free 6-hydroxyl group, and a certain aglycone size which was reached, for example, by monosaccharide or nitrophenyl substituents. 2. Aromatic alpha-D-galactopyranosides acted as high-affinity inhibitors (Ki, below 50 micrometer). At least two of them were not transported, in contrast to alpha-galactoside disaccharides and to aromatic beta-D-galactopyranosides. 3. beta-D-Galactoside transport was not significantly inhibited by specific inhibitors and transitionstate analogues of beta-galactosidase (D-galactal, D-galactonolascone). 4. The beta-D-galactopyranoside, lactitol, and alpha-D-galactopyranoside, galactinol, were not efficiently bound by the lactose permease system, although the maximal rate of uptake of lacitol was similar to that of lactose. By comparison with several structurally related D-galactopyranosides, the decreased affinity was attributed to an effect of the membrane/water interface. A model for substrate recognition by the lactose permease system is presented.

Biological Transport↗
PubMed1982

Lactose carrier protein of Escherichia coli. Reconstitution of galactoside binding and countertransport.

A procedure for the reconstitution of the lactose carrier protein, a galactoside:proton symporter in Escherichia coli, is described. Starting from cytoplasmic membranes derived from carrier-overproducing strains, essentially all proteins including 89% of the carrier are solubilized by a mixture of dodecyl/tetradecyl polyoxyethylene (n = 9.5) ether and dodecyl O-beta-D-maltoside. In the micellar state the carrier binds substrates with reduced affinity. Addition of E. coli phospholipids and removal of detergents by a hydrophobic column yields small vesicles (50-60-nm diameter). In these vesicles, about 70% of the carrier is recovered and reconstituted carrier is identical to native carrier in terms of substrate binding. After fusion of the small vesicles into larger vesicles (1-5 micrometers), rapid countertransport of galactosides is demonstrated. Attempts to show active galactoside transport by the imposition of artificial electrical potential or pH gradients were unsuccessful, most likely because the reconstituted vesicles are in fact highly permeable to protons.

Bacterial Proteins↗
PubMed1983

Chemical modification of the lactose carrier of Escherichia coli by plumbagin, phenylarsinoxide or diethylpyrocarbonate affects the binding of galactoside.

The effects of chemical modification of the lactose carrier of Escherichia coli on galactoside binding (in overproducing strains) and on transport was examined. Both the modifying reagents diethylpyrocarbonate and rose bengal and the thiol reagents phenylarsinoxide and plumbagin can completely inhibit the binding of the substrate p-nitrophenyl alpha-D-galactopyranoside to the carrier. If care is taken to inhibit galactoside binding only partially, the loss of transport is found to parallel the loss of binding sites. The modified carrier molecules are completely inactive, while the remaining active carrier molecules evince normal transport and binding parameters. The binding of galactoside protects the carrier partially against these forms of chemical modification. In view of these observations, the results of previous chemical modification studies [Padan, E., Patel, L. and Kaback, H.R. (1979) Proc. Natl Acad. Sci. USA, 76, 6221-6225; Konings, W.N. and Robillard, G.T. (1982) Proc. Natl Acad. Sci. USA, 79, 5480-5484] must be re-interpreted. Our results stress the utility of studying substrate binding, the first step in the transport cycle.

Arsenicals↗