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)

Antisera with specificity for the product of a yeast cell-division-cycle (CDC) gene were prepared by immunizing rabbits to a novel hybrid polypeptide. A segment of the yeast gene CDC28 was fused to the Escherichia coli lacZ gene, which encodes beta-galactosidase, by insertion of yeast sequences into the plasmid pBGF1. pBGF1 contains the lac promoter-operator and most of the lacZ gene. An EcoRI site, 16 codons upstream from the carboxyterminus of the beta-galactosidase coding region, served as a convenient splicing site for the heterologous sequences. To insure that an open reading frame be maintained between the two gene segments for some portion of the fusions, the CDC28-encoding segments were first subjected to limited digestion with nuclease BAL31 to produce random junction points. A hybrid polypeptide encoded by such a continuous open reading frame was purified from E. coli by preparative SDS-polyacrylamide gel electrophoresis and used to immunize rabbits. The resulting antisera were shown to have specificity for CDC28 gene product synthesized by cell-free translation of yeast mRNA.
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PMID:Preparation of product-specific antisera by gene fusion: antibodies specific for the product of the yeast cell-division-cycle gene CDC28. 629 92

The Tn10 tetR gene encodes the repressor that regulates transcription of the Tn10 tetracycline resistance determinant. We have determined the DNA sequence of the tetR gene and a 905 base pair region immediately 3' to tetR. The tetR gene is located on a 701 base pair HincII restriction fragment. Deletions at either end of this region eliminate synthesis of the wild-type TetR protein in E. coli minicells, and eliminate TetR activity as measured by repression of beta-galactosidase synthesis in tetA-lacZ operon fusion strains. Taken together, the DNA sequence and the genetic data indicate that tetR encodes a 207 amino acid protein with a calculated molecular weight of 23,328. This value is in good agreement with estimates of 23,000-25,000 based on electrophoretic mobility in SDS-polyacrylamide gels. There is 47% amino acid sequence homology between the deduced sequences of the Tn10 and RP1/Tn1721 TetR proteins. There is, in addition, significant amino acid sequence homology between an NH2-terminal region of the Tn10 TetR repressor and the DNA recognition regions of other DNA-binding proteins.
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PMID:Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant. 633 Jun 87

Two important features of infection of mice with larvae of Taenia taeniaeformis are the ready demonstration of host protective antibodies and the ability to immunize susceptible strains of mice against first infection using crude parasite preparations. Candidate immunogens in established larvae and the invasive oncosphere have been identified by immunoprecipitation of radiolabeled parasite proteins with host-protective antibodies. To overcome the difficulties associated with purification of these antigens from parasite material, the alternative strategy of expressing parasite proteins in Escherichia coli has been adopted. Double stranded DNA complementary to mRNA from 28 day old liver larvae was inserted into the beta-galactosidase gene of the bacteriophage lambda Amp 3. Some recombinants express a fusion protein with additional parasite-encoded epitopes located at the C-terminal end of the beta-galactosidase protein. Four clones that reacted with antibodies in an E. coli colony immunoassay were selected for detailed characterization. Analysis of lysates of the selected clones by SDS-PAGE and Western blotting revealed that each clone produced an abundant fusion protein that reacted specifically with a hyperimmune anti-oncosphere serum. Sibling analysis revealed that the four antiserum-positive clones encoded three immunologically-distinct parasite antigens. The identity of the native protein of larvae encoded by one clone (designated TA10) was an abundant antigen of Mr 70,000. This approach allows the assessment of antigens expressed in E. coli as vaccines in susceptible strains of mice by direct immunization and challenge and thus the development of a model defined-antigen vaccine against a larval cestode parasite.
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PMID:Expression of Taenia taeniaeformis antigens in Escherichia coli. 639 85

In search of a beta-galactosidase which specifically hydrolyses beta 1----3 bound galactose residues in galacto-glycoconjugates, an acid beta-galactosidase from chicken liver was investigated. The isolation procedure involved ammonium sulphate precipitation followed by lectin chromatography on Con A-Sepharose 4B, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sepharose 6B and affinity chromatography on p-aminophenyl-thio-beta-D-galactoside-agarose. The beta-galactosidase was purified 3000-fold with 11% recovery of enzyme activity. The purified protein showed an apparent molecular mass of above 200000 in SDS-polyacrylamide gel electrophoresis. A few minor bands were also present. The reduced and denatured beta-galactosidase migrated as a single major band with an apparent molecular mass of 67000. The enzyme released galactose from lactose and from the synthetic substrates Gal beta 1----3Gal, Gal beta 1----6Gal and Gal beta 1----3Ara. However, the enzyme did not release galactose from the snail gland galactans and the high molecular weight galacto-glycoconjugates and it did not hydrolyse the peanut agglutinin receptor of the red blood cell membrane.
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PMID:Isolation of a beta-galactosidase from chicken liver. 644 30

Plasma membrane glycoconjugates, enzymatically labelled with [3H]galactose, were used as an autoradiographic membrane marker for a morphometric analysis of membrane traffic during fluid phase pinocytosis in the amoeba, Dictyostelium discoideum. The fraction of grains associated with the plasma membrane decreased exponentially from 99% for cells fixed directly after labelling on the cell surface, to a steady-state value of 45% after about 1 h of pinocytotic activity. The complementary fraction of grains was observed on vacuolar membranes. Only after a lag of about 20 min, a small but significant fraction (3%) of the total grains, was found in the region of the Golgi membranes. During two subsequent doublings of cell number, over a period of 24 h, label was lost into the medium at a constant rate of 1% per h. The cell bound label remained fully membrane bound over the entire period. The beta(1-4) linkage was not noticeably modified, as judged by its susceptibility to hydrolytic release by beta-galactosidase. An analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed an identical labelling pattern for total membrane fractions when prepared directly after labelling or after 24 h of membrane flow.
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PMID:Membrane traffic in Dictyostelium discoideum: plasma membrane glycoconjugates internalized and recycled during fluid phase pinocytosis enter the Golgi complex. 664 36

beta-Galactosidase from Alternaria tenius was purified to homogeneity from the cultural fluid using acetone precipitation, ion-exchange chromatography on DEAE-cellulose, adsorption on hydroxylapatite and affinity chromatography on N-(beta-D-galactopyranosyl-thiocarbamoyl)-beta-aminocaproyl-AN-Sepharose 4B. The enzyme homogeneity was demonstrated by ultracentrifugation and polyacrylamide gel electrophoresis with SDS or without it. The specific activity of the homogeneous enzyme is 160 u. per mg of protein; mol. weight as determined by various methods is 142 000-176 000, pI = 4.6, temperature optimum is 60-65 degrees, pH optima for o-nitrophenyl-beta-D-galactopyranoside (o-NPG) and lactose are 3.8--4.4 and 3.6--4.8, respectively. The Km values for o-NPG and lactose are 0.21 . 10(-3) and 6.57 . 10-3 M, respectively. The enzyme is a glycoprotein and contains up to 30% of carbohydrates. EDTA and pCMB have no effect on the beta-galactosidase activity. Galactose acts as a competitive inhibitor, while glucose has no inhibiting effect on the enzyme activity.
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PMID:[Purification and properties of beta-galactosidase from Alternaria tenius]. 679 53

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase.
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PMID:Further characterization of intestinal lactase/phlorizin hydrolase. 681 89

An analysis was made of the pinocytosis-derived internalization and recycling of membrane in the macrophage cell line, P388D1. Plasma membrane glycoconjugates, reversibly labelled with [3H]galactose, were used as a membrane marker. Label internalized with the plasma membrane was no longer accessible to release by externally added beta-galactosidase and could therefore be distinguished quantitatively from label remaining on the cell surface. Direct experimental evidence for membrane recycling was obtained by demonstrating that previously internalized label reappeared at the cell surface. The composition of labelled membrane glycoconjugates, as analysed by SDS-polyacrylamide gel electrophoresis, remained unaltered before and after internalization. The label remained membrane-bound in an unmodified way during the entire period of 8 h investigated, corresponding to about twenty-four cycles of membrane flow. Membrane flow led to a steady-state distribution of label between the plasma membrane and intracellular membranes. The redistribution of label occurred with biphasic kinetics, which could be described as the sum of two exponential functions. This behavior is explained by presenting a model of membrane flow between the plasma membrane and two consecutive intracellular membrane compartments, which we assume to consist of pinosomal membranes and of pinosome-derived membrane of secondary lysosomes. The relative membrane surface areas turn out to be in the ratio of 100:12.5:7.3, respectively. At the observed rate of pinocytosis, the equivalent of the plasma membrane is internalized once every 21 min, in the form of primary pinosomes of the size 0.24 micrometer. The residence time of membranes in the pinosome compartment is about 3 min. The rate at which membranes enter the lysosomal compartment is 31 times lower than the rate of membrane internalization. We conclude that only 3% of the amount of membrane internalized at any one time subsequently enters the secondary lysosome compartment. After a residence time of 49 min this membrane fraction is finally recycled to the cell surface. The results are discussed in terms of mixing and sorting-out of pinosomal and lysosomal membranes.
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PMID:Internalization and recycling of plasma membrane glycoconjugates during pinocytosis in the macrophage cell line, P388D1. Kinetic evidence for compartmentation of internalized membranes. 684 Jan 99

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [125I] insulin binding to soluble receptors from mouse liver was inhibited by digestion with beta-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[3H] hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10-7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified with two galactoglycoprotein of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with beta-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10-7 M proinsulin slightly hindered labeling, while pretreatment with 10-7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate that glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.
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PMID:Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding. 703 Mar 99

Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled [Fe4S4]-siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia. An expression system which produced enzymatically active spinach nitrite reductase (NiR) in Escherichia coli was developed in order to study the structure-function relationships of the coupled active site using site-directed mutagenesis. The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a beta-galactosidase fusion containing five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive. After optimizing growth conditions, active NiR represented 0.5-1.0% of the total protein. E. coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis. The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E. coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A278/A386 = 1.9. The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum. The specific activity for the methyl viologen-dependent reduction of nitrite of E. coli-expressed NiR was 100 U/mg and the Km determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that the E. coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of spinach nitrite reductase in Escherichia coli: site-directed mutagenesis of predicted active site amino acids. 748 61


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