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)

Transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) from Streptoverticillium mobaraense has been used to stabilize immobilisates produced with beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae and acid-processed gelatins of different qualities as support. The isopeptide level of N epsilon-(gamma-L-glutamyl)-L-lysine bonds formed by transglutaminase was determined to estimate their influence on the kinetic properties of the enclosed beta-galactosidase. An HPLC procedure using precolumn derivatization of the gelatin hydrolysates with FMOC-chloride was chosen which permits the analysis of cross-linked lysine with satisfactory precision. Depending on the gelatin quality, the degree of cross-links necessary for the transformation of gelatin into an insoluble protein was in the range 0.3-32.3% of the available lysine residues. beta-Galactosidase was entrapped in the gelatin matrices with a yield of 8-46% of the initial activity. Long reaction times for cross-linking were due to low yields rather than to the number of isopeptide bonds. Repeated use of the immobilisates did not lead to an appreciable loss of activity. The Vmax of beta-galactosidase were diminished by immobilization caused by a tighter package of the protein chains rather than by the extent of cross-links, while the obtained Km values of the free enzyme and the immobilisates were quite similar. Also, the pH and temperature of optima of the free enzyme and the gelatin immobilisates differ only slightly. The data suggest that the immobilization procedure only moderately affects the activity of enzymes catalysing the reaction of a small compound if gelatin with high jelly strength is cross-linked in a 10% solution with transglutaminase.
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PMID:Influence of gelatin matrices cross-linked with transglutaminase on the properties of an enclosed bioactive material using beta-galactosidase as model system. 885 18

Human lactase-phlorizin hydrolase (EC 3.2.1.23/62) is a major disaccharidase in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a single-chain precursor protein and undergoes proteolytic processing during maturation. We studied proteolytic processing of human lactase-phlorizin hydrolase in transfected COS-1, Caco-2, and MDCK cells using metabolic labeling, surface immunoprecipitation, protease sensitivity assays, and microsequencing. Furthermore, we generated mutated forms of the enzyme to alter potential proteolytic cleavage sites and expressed these in Caco-2 and COS-1 cells. Since the N-terminal amino acid of microvillus lactase-phlorizin hydrolase corresponds to Ala869 in the precursor protein, it has been speculated that processing occurs at position Arg868-Ala869. Substitution of Arg868 with isoleucine, lysine, or glutamic acid had no effect on the proteolytic processing of pro-LPH in Caco-2 cells. As in wild-type enzyme a processed 160-kDa form was generated. These data are not consistent with a primary proteolytic processing at position Arg868-Ala869. Using amino-terminal amino acid sequencing of this processed form isolated from stable transfected MDCK cells we identified the cleavage site at Arg734-Leu735. Treatment of pro-lactase-phlorizin hydrolase expressed in COS-1 and MDCK cells by trypsin yielded a 145-kDa form with an identical amino terminal as the mature microvillus enzyme isolated from intestinal mucosa (Ala869). These data provide unambiguous evidence of a two-step processing of human lactase-phlorizin hydrolase. The first cleavage occurs intracellularly after a dibasic site (Arg734-Leu735) and yields the 160-kDa intermediate form. In a second step the intermediate form inserted into the microvillus membrane is trimmed to the mature enzyme by luminal trypsin.
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PMID:Proteolytic processing of human lactase-phlorizin hydrolase is a two-step event: identification of the cleavage sites. 895 Oct 31

Hydrophobically modified dextrans, benzoyl dextran and valeryl dextran, have been used to study the interactions between tryptophan residues and benzoyl or valeryl groups by partitioning of tryptophan, tryptophan-tryptophan, (tryptophan)3, poly(lysine, tryptophan), beta-galactosidase and lysozyme in polymer aqueous two-phase systems. The two-phase systems used were polyethylene glycol (PEG)-benzoyl dextran, PEG-valeryl dextran, dextran-benzoyl dextran and dextran-valeryl dextran. Interaction between tryptophan residues and benzoyl or valeryl groups was observed by partitioning of tryptophan containing compounds to the phase containing hydrophobically modified dextran. At a certain phase composition the interactions were increased with increasing number of tryptophan per molecule. In a PEG-dextran system the partitioning of tryptophan peptides to the PEG phase was increased with increased number of tryptophan. In a PEG-benzoyl dextran system the opposite effect was obtained. At similar conditions benzoyl groups showed stronger interactions with tryptophans compared to valeryl groups. The partition coefficient of salts (sodium phosphate, NaCl, Nal and NaClO4) was determined in PEG-benzoyl dextran and PEG-valeryl dextran aqueous two-phase systems. The effect of addition of these salts on partitioning of poly(lysine, tryptophan), beta-galactosidase and lysozyme was studied. Salt effects on partitioning could be explained by the relative affinities of the ions for the polymers in the system. Charged molecules containing tryptophan were to an increasing degree partitioned to the phase for which the counterions had highest affinity. Strong effects on the partitioning of positively charged poly(lysine, tryptophan) and lysozyme were obtained with the ions I- and ClO4-.
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PMID:Interaction between tryptophan residues and hydrophobically modified dextran. Effect on partitioning of peptides and proteins in aqueous two-phase systems. 913 30

An adenovirus/DNA complex was constructed by chemically linking poly-L-lysine to the capsid of the replication-defective adenovirus dl312, allowing for coupling with plasmid DNA by an ionic interaction. We have previously demonstrated that this adenovirus/DNA complex can efficiently transduce malignant cells with a plasmid expressing the beta-galactosidase gene both in vitro and in vivo. In this report, we show that this system can deliver a therapeutic gene that encodes for the tumor suppressor protein p53 to lung cancer cells, both in vitro and in vivo, leading to significant biological effects. Transfection of the p53-negative human lung cancer cell line H1299 with the adenovirus/DNA complex carrying a plasmid expressing the p53 gene resulted in high levels of p53 protein and induction of apoptosis. Injection of the complex carrying the p53 gene to subcutaneous tumor sites 5 days after tumor cell implantation resulted in a significant inhibition of tumorigenicity as measured by the number and size of tumors that developed 21 days after treatment. Three and six injections of the complex carrying the p53 gene into H1299 subcutaneous tumor nodules led to significant dose-related tumor growth suppression 18 days after the first injection compared with control-treated tumors. This adenovirus/DNA complex, therefore, is capable of efficiently delivering the p53 gene into malignant cells in vitro and in vivo and now provides a general gene delivery vector that is simple to construct and capable of testing therapeutic genes in malignant cells.
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PMID:Delivery of the p53 tumor suppressor gene into lung cancer cells by an adenovirus/DNA complex. 917 38

The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.
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PMID:Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor. 927 36

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the sigma D form of RNA polymerase. The level of beta-galactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No beta-galactosidase could be detected in a sigD genetic background.
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PMID:Functional and genetic characterization of mcpC, which encodes a third methyl-accepting chemotaxis protein in Bacillus subtilis. 935 24

Transglutaminase 1 (TGase 1) is a tissue-specific enzyme which is expressed in the keratinized stratified squamous epithelia and which catalyzes straightepsilon-(gamma-glutamyl) lysine cross-links of proteins to form the cell envelope at the periphery of cornified cells. A transient expression assay using a luciferase reporter gene linked to the 2.5 kb 5' upstream region of the human TGase 1 gene (TGM1) showed phorbol ester-responsive promoter activity in cultured normal human keratinocytes. To assess its promoter activity in vivo, we generated transgenic mice expressing the Escherichia coli beta-galactosidase gene (lacZ) directed by the 5' upstream region. beta-Galactosidase histochemistry revealed that the TGM1-lacZ transgene was expressed in terminally differentiating keratinocytes in upper layers of stratified squamous epithelia in embryonic, neonatal and adult transgenic mice. The expression pattern was similar to that of endogenous TGase 1 mRNA detected by in situ hybridization. Furthermore, topical application of a phorbol ester to adult tail skin enhanced expression of the transgene as well as TGase 1 mRNA in the epidermis. Thus, the 2.5 kb 5' upstream sequence of TGM1 includes elements regulating tissue- and terminal differentiation-specific gene expression in stratified squamous epithelia.
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PMID:Activation of the human transglutaminase 1 promoter in transgenic mice: terminal differentiation-specific expression of the TGM1-lacZ transgene in keratinized stratified squamous epithelia. 936 Oct 26

A sandwich transfer enzyme immunoassay for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The assay procedure consisted of the reaction of SCT with 2,4-dinitrophenyl biotinyl anti-SCT IgG and anti-SCT Fab'-beta-galactosidase conjugate, trapping onto (anti-2,4-dinitrophenyl bovine serum albumin) IgG-coated polystyrene balls, eluting with epsilon N-2,4-dinitrophenyl-L-lysine and transferring to streptavidin-coated polystyrene balls and fluorometric detection of beta-D-galactosidase activity. The practical detection limit of SCT was 0.05 pg (15 amol)/50 microliters of sample and 1 pg/ml as the concentration. The application of this method has enabled us to directly estimate the bioavailability of SCT dosed intranasally at the therapeutic level (160 IU, 31 micrograms) for its anti-osteoporotic effect as compared to an intramuscular dose (10 IU, 1.9 micrograms). The pharmacokinetic parameters of the intranasal SCT (n = 6) thus estimated were as follows: the area under the blood concentration-time curve (AUC) 9400 +/- 5400 (SD) pg.h/ml, and the mean residence time (MRT) = 42 +/- 14 (SD) min, when the AUC for the intramuscular SCT (n = 3) = 5600 +/- 2000 (SD) pg.h/ml and the MRT = 39 +/- 19 (SD) min.
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PMID:A sandwich transfer enzyme immunoassay for salmon calcitonin: determination of the bioavailability of intranasal salmon calcitonin in human. 940 61

DNA topoisomerase II alpha is the intracellular target for several important chemotherapeutic agents, and drug-resistant human tumor cell lines have been described in which deletions in the C-proximal region of this enzyme are associated with its cytoplasmic localization. We have identified multiple potential bipartite nuclear localization signal (NLS) sequences in this region using a modified definition of the motif, and in the present study, we have expressed five of these as fusion proteins with beta-galactosidase. Only one sequence (spanning amino acids 1454 to 1497) was sufficient to cause strong nuclear localization. Subsequent mutation analyses indicated that this NLS sequence was bipartite and that both domains contain more than two basic amino acids. Substitution of the lysine residue at position 1492 in the second basic domain with glutamine resulted in a fusion protein that localized inefficiently to the nucleus, indicating that all three basic residues in this domain are necessary. Our results confirm that a broader definition is required to detect all potential bipartite NLS motifs in a polypeptide sequence, although functional tests are still essential for identification of those sequences actually capable of directing nuclear localization.
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PMID:Bipartite nuclear localization signals in the C terminus of human topoisomerase II alpha. 943 41

The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex. Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-28). To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed. From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains. Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs. The domain responsible for these two activities was mapped to a conserved region near the N terminus. Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs. This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast. Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo.
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PMID:Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1. 944 33


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