Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During short incubations of a Golgi apparatus-enriched subcellular fraction from rat liver with UDP-[3H]GlcNAc, label is efficiently transferred to endogenous acceptors. Most of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that it is mainly associated with N-linked oligosaccharides. The glycoprotein acceptors are resistant to proteases unless detergent is added in amounts greater than the critical micellar concentration. This shows that the acceptors are within the lumen of intact compartments, which have the correct topological orientation expected for the Golgi apparatus in intact cells. Structural characterization of the radiolabeled N-linked oligosaccharides shows a variety of distinct neutral and anionic species. The neutral chains include bi-, tri-, and tetra-antennary molecules with terminal beta-[3H] GlcNAc residues. In vitro sialylation shows that some of the tetra-antennary chains have beta 1,3-linked Gal residues on their unlabeled antennae. An unknown modification appears to block the action of beta-galactosidase on these galactosylated oligosaccharides. Chasing the labeling reaction with a mixtures of UDP-Gal, CMP-Neu5Ac, and adenosine 3'-phosphate,5'-phosphosulfate causes an increase in the percent of radiolabeled anionic oligosaccharides. Most of the negative charge is due to sialic acid (Sia), and some appears to be in phosphodiester-linked [3H]GlcNAc. The sialylated oligosaccharides are a mixture of bi-, tri-, and tetra-antennary species with 1-3-Sia residues, and some of the [3H]GlcNAc residues are directly covered with unlabeled Gal and Sia residues. This in vitro approach should recapitulate reactions that occur in the biosynthesis of N-linked oligosaccharides in the Golgi apparatus of the intact cell. Since the conditions during labeling do not permit inter-compartmental transport, the oligosaccharides produced should represent the biosynthetic capabilities of individual Golgi compartments. Evidence is presented for a functional association of GlcNAc transferases I, II, and alpha-mannosidase II, with separation from GlcNAc transferase IV and/or V. The structures also indicate co-compartmentalization of several GlcNAc transferase(s) with beta-galactosyltransferase(s) and sialyltransferase(s). The compartmental organization of the Golgi apparatus is discussed in light of these findings.
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PMID:Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]N-acetylglucosamine. 834 99

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.
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PMID:Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon. 903 99

Metabolic labelling of Plasmodium falciparum parasites with [3H]GlcN, [3H]Man, [3H]Gal and [3H]ethanolamine, and subsequent purification by SDS-PAGE of the labelled material provided effective labelling of the MSP-1, 195 kDa, and MSP-2, 42-53 kDa, glycoproteins. Reductive beta-elimination of the MSP-2 released from the gel consisted of glycopeptides containing labelled sugars. Processing of the eliminated components and identification of the sugar residues demonstrated the presence of N-acetylglucosaminitol and N-acetylgalactosaminitol amongst other labelled sugars. Reductive beta-elimination with sodium hydroxide-sodium borotritide-borohydride showed the presence of glucosaminitol and alanine in the hydrolysis products. The MSP-2 was retained on solid phase wheat-germ agglutinin and was released from the lectin by treatment with GlcNAc. Upon treatment with O-glycanase the MSP-2 glycoprotein released labelled amino sugar, and derived oligosaccharides on treatment with exoglycosidases released labelled components corresponding to the metabolically incorporated sugars. Labelled Gal was incorporated into the MSP-2 glycoprotein using [3H]UDP-Gal and galactosyltransferase. The galactosylated glycoprotein released labelled Gal upon treatment with beta-galactosidase. The results of the present study suggest that the carbohydrate chains of the MSP-2 glycoprotein are attached to the protein backbone via GlcNAc- and GalNAc-serine/threonine in O-glycosyl linkage and the glycoprotein has terminal GlcNAc and Gal residues. The carbohydrate moieties of MSP-2, glycoprotein consist mainly of short chains linked to the protein core.
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PMID:Carbohydrate moiety of Plasmodium falciparum glycoproteins: the nature of the carbohydrate-peptide linkage in the MSP-2 glycoprotein. 935 84

Non-acid glycosphingolipids were isolated from small intestinal epithelial cells of a single blood group A pig. One very predominant blood group compound was obtained chemically pure upon HPLC fractionation. It was characterized by mass spectrometry and 1H NMR spectroscopy to be the type 1 chain blood group A hexaglycosylceramide. Support for the presence of minute amounts of additional A glycolipids was obtained by mass spectrometry and immunostaining of TLC plates with anti-A antibodies specific for A type 2 chain, A type 3 and 4 chain, and the ALe(b) determinant. Among precursor chains, globoside (type 4) and lactotetraosylceramide (type 1) were immunologically identified, whereas no neolactotetraosylceramide (type 2) and gangliotetraosylceramide reactivities were detected. We addressed the question whether the predominant expression of type 1 chain based A glycolipids reflects a restricted glycolipid precursor chain specificity of the alpha 1-2 fucosyl- and/or the alpha 1-3 N-acetylgalactosaminyltransferases, or if the biosynthesis of the precursor chains themselves is regulated. All precursor core saccharides, lacto- (type 1), neolacto-(type 2), and gangliotetraosylceramide as well as globopentaosylceramide (type 4), could serve as acceptors for fucose in vitro when a crude microsomal fraction obtained from mechanically released, porcine intestinal epithelial cells was used as an enzyme source. Under the same conditions an N-acetylgalactosamine residue could be transferred to the blood group H structures based on these core saccharide chains. Lactotriaosylceramide, but not gangliotriaosylceramide, could serve as an acceptor for UDP-galactose. When the product was digested with beta-galactosidase (EC 3.2.1.23) from S.pneumoniae, under conditions where it specifically cleaves Gal beta 1-4 residues, approximately 40% of the radioactivity was cleaved off, indicating that a substantial amount of neolactotetraosylceramide was made in vitro, as opposed to the predominance of lactotetraosylceramide-based structures found in vivo.
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PMID:Biochemical and enzymatic characterization of blood group ABH and related histo-blood group glycosphingolipids in the epithelial cells of porcine small intestine. 936 37

We have found that spontaneous galactosylation of GlcNAc residues occurs in bovine colostrum, but not in dialyzed colostrum, without adding UDP-Gal as a donor substrate. UDP-Gal was shown to be present in bovine colostrum at a level ranging from 200 to 600 microM. When a tracer UDP-[(14)C]Gal was added to the dialyzed colostrum together with a Gal beta1,4-specific beta-galactosidase, remarkable incorporation of radioactivity into 24-28 kDa and 33 kDa RCA1-positive glycoproteins was demonstrated by SDS-PAGE/autoradiography. Some 100-140 kDa agalactoglycoproteins of a CHO mutant cell line were also galactosylated on a blotted membrane by the incubation in the colostrum.
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PMID:Spontaneous galactosylation of agalactoglycoproteins in colostrum. 1081 67

The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored. The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase from Bacillus circulans using lactose as donor substrate. The UDP-disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4)Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively. Their chemical structures were characterized by 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 35 mg with 96% purity) for Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained. UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP). The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta-galactosidase from Bacillus circulans.
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PMID:Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans. 1130 28

Galactosyltransferases (GalTs), capable of transferring a galactosyl residue from UDP-galactose (UDP-Gal) to polysaccharide acceptor, were solubilized from flax (Linum usitatissimum L.) membranes using 0.5% CHAPS. The observed requirement for a rhamnogalacturonan I (RG-I) exogenous substrate to stimulate the solubilized GalT activity provided the first evidence for the presence of RG-I GalT activities in flax cells. An assay to measure specifically the products of this RG-I GalT activity was designed, based on size-exclusion chromatography. Labelled products were characterized as an RG-I polymer by using purified RG-I hydrolase or lyase. At pH 8 and in the presence of 5 mM CaCl2, beta-D-galactosyl residues were specifically transferred onto RG-I branches of short beta-(1 --> 4)-D-galactan side chains. These side chains were liable to hydrolysis by beta-galactosidase and endo-beta-(1 --> 4)-D-galactanase. The RG-I GalT had a temperature optimum of 30 degrees C. an apparent Km for UDP-Gal and exogenous RG-I substrate of 460 +/- 40 microM and 1.1 +/- 0.1 mg ml(-1) respectively, and a Vmax of 3.0 +/- 0.5 pkat mg(-1) protein.
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PMID:Solubilization of rhamnogalacturonan I galactosyltransfrases from membranes of a flax cell suspension. 1150 67

The nucleotide sequences of the Lactobacillus helveticus lactose utilization genes were determined, and these genes were located and oriented relative to one another. The lacLM genes (encoding the beta-galactosidase protein) were in a divergent orientation compared to lacR (regulatory gene) and lacS (lactose transporter). Downstream from lacM was an open reading frame (galE) encoding a UDP-galactose 4 epimerase, and the open reading frame had the same orientation as lacM. The lacR gene was separated from the downstream lacS gene by 2.0 kb of DNA containing several open reading frames that were derived from fragmentation of another permease gene (lacS'). Northern blot analysis revealed that lacL, lacM, and galE made up an operon that was transcribed in the presence of lactose from an upstream lacL promoter. The inducible genes lacL and lacM were regulated at the transcriptional level by the LacR repressor. In the presence of glucose and galactose galE was transcribed from its promoter, suggesting that the corresponding enzyme can be expressed constitutively. Lactose transport was inducible by addition of lactose to the growth medium.
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PMID:Unusual organization for lactose and galactose gene clusters in Lactobacillus helveticus. 1278 21

Galactosyltransferase (GalT) activity that results in the transfer of galactose (Gal) from UDP-Gal to exogenous (1-->4)-beta-galactooligosaccharides labeled with 2-aminobenzamide (2AB) at their reducing ends was identified in a particulate preparation obtained from 2-day-old mung bean (Vigna radiata L. Wilezek) hypocotyls. The enzymes responsible were shown, by high-performance anion-exchange chromatography and normal-phase liquid chromatography-electrospray ionization mass spectrometry, to transfer up to eight Gals to the non-reducing end of 2AB-labeled galactooligosaccharide. Using 1H nuclear magnetic resonance spectroscopy, and beta-galactosidase and endo-beta-(1-->4)-galactanase treatments of the enzymatically formed 2AB-labeled galactooligosaccharides, the newly incorporated Gal residues were shown to be beta-(1-->4) linked. Time-course studies indicated that at least two different types of GalT isoform are involved in the elongation of the acceptor substrates. 2AB-labeled galactoheptaose was the most effective acceptor substrate analyzed, although galactooligosaccharides with a degree of polymerization between 4 and 6 were also acceptor substrates. 2AB-labeled penta- and heptasaccharides (RG5 and RG7) generated from rhamnogalacturonan I (RG-I) were not acceptor substrates, suggesting that the GalTs were not capable of adding Gal residues directly to the RG-I backbone. Maximum GalT activity was obtained at pH 6.5 and 20 degrees C in the presence of 25 mM Mn2+ and 0.75% (w/v) Triton X-100. The enzyme had an apparent Km of 20 microM for 2AB-labeled galactoheptaose and 32 microM for UDP-Gal. The characteristics of the enzyme in mung bean microsomal membranes and the usefulness of fluorogenic 2AB-labeled galactooligosaccharides for the assay of GalT are discussed.
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PMID:Identification of elongating beta-1,4-galactosyltransferase activity in mung bean (Vigna radiata) hypocotyls using 2-aminobenzaminated 1,4-linked beta- D-galactooligosaccharides as acceptor substrates. 1498 44

A number of selection systems have been developed for direct selection of recombinant plasmids in cloning experiments (positive selection). In this study, the Commonly used LacZ-based alpha-complementation plasmid vectors have been used for designing a positive selection system for the selection of recombinants. The basis for the Strategy is the phenomenon of galactose sensitivity exhibited by galactose epimerase (galE) mutants of Escherichia coli. It is known that lacZ+ galE, but not lacZ- galE cells are killed upon addition of lactose due to the accumulation of a toxic intermediate, UDP-galactose, by hydrolysis of lactose. Using a galE mutant strain of E. coli that carries the lacZAM15 allele, various alpha-complementation plasmids that vary in their copy number were examined for their ability to be killed following addition of lactose. The results show that some plasmids that exhibit relatively high beta-galactosidase enzyme activity can be used effectively for positive selection. This selection would be extremely useful during primary cloning experiments such as construction of genomic or cDNA libraries and also in instances involving selection for rare recombinants.
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PMID:Positive selection system for identification of recombinants using alpha-complementation plasmids. 1559 44


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