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)

Although it is energetically extremely unfavorable to have charged amino acid residues of a polypeptide in the hydrophobic environment of the membrane phospholipid bilayer, a few such charged residues are found in membrane-spanning regions of membrane proteins. Ion pairs (salt bridges) would be much more stable in low dielectric media than single ionized residues. This paper provides indirect evidence for a salt bridge between Asp-240 and Lys-319 in the lactose carrier of Escherichia coli. When Asp-240 was changed to alanine by site-directed mutagenesis, there was a loss of the ability to accumulate methyl-beta-D-thiogalactopyranoside (TMG), melibiose, or lactose. Fast-growing revertants were isolated on melibiose minimal agar plates. Two second-site revertants were isolated: Asp-240-->Ala plus Gly-268-->Val and Asp-240-->Ala plus Lys-319-->Gln. These revertants showed extremely poor accumulation of TMG, melibiose, and lactose, but showed significant "downhill" lactose entry into beta-galactosidase-containing cells with sugar concentrations of 2 and 5 mM. It is concluded that there is some important interaction between Asp-240 and Lys-319, possibly a salt bridge.
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PMID:Possible salt bridges between transmembrane alpha-helices of the lactose carrier of Escherichia coli. 140 Mar 92

Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro-environment of the bacteria-containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin-Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of beta-galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein-di-beta-D-galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post-infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA activity was detected only at early post-infection times (4 h), suggesting that the vacuole may have a mild-acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive beta-galactosidase substrate can provide information about the micro-environment within which an intracellular pathogen, such as S. typhimurium, resides.
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PMID:Characterization of the micro-environment of Salmonella typhimurium-containing vacuoles within MDCK epithelial cells. 148 85

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.
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PMID:The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity. 150 17

For the differentiation of Shigella from Escherichia coli, Indole (tryptophanase), PGUA (beta-glucuronidase) and ONPG (beta-galactosidase) tests were used. A total of 377 Shigella and 124 E. coli strains was examined for each sero- and biosero-type by using these tests. The results were as follows. 1) There were no Shigella strains showing positive reactions for both Indole and ONPG tests. 2) No E. coli strains with Shigella-like characteristics (negative for lysine-decarboxylase, motility and lactose-fermentation tests) showed negative results for both Indole and PGUA tests. 3) The abovementioned strains were classified into twelve types according to the results of these tests. Shigella strains, thus, were differentiated from antigenically Shigella-like E. coli strains. Additional use of these tests together with the conventional methods may valuable for the identification of Shigella strains.
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PMID:[Rapid differentiation method for Shigella and Escherichia coli--application of Indole (tryptophanase), PGUA (beta-glucuronidase) and ONPG (beta-galactosidase) tests]. 162 38

K1 is a monoclonal antibody that reacts with a cell surface antigen (CAK1) found in human mesothelia and nonmucinous ovarian tumors. In this article, the characteristics of the CAK1 antigen have been examined in detail. Using immunofluorescence microscopy, we have found that the CAK1 signal is removed from the cell surface by treatment with proteases or by phosphatidylinositol-phospholipase C, but not by neuraminidase and beta-galactosidase. The phosphatidylinositol-phospholipase C-released material was found to contain the CAK1 antigen which was detected by a competition radioimmunoassay. The phosphatidylinositol-phospholipase C-released CAK1 antigen was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and found to be approximately 40 kDa protein. The CAK1-K1 antibody complex remains on the cell surface and is poorly internalized, as shown by an acid wash immunofluorescence internalization assay. An immunotoxin composed of K1 and Lys-PE40, a mutant form of Pseudomonas exotoxin lacking the cell binding domain, was not cytotoxic, supporting the conclusion that the CAK1-K1 antibody complex is not internalized. However, an immunotoxin composed of K1 and native Pseudomonas exotoxin was selectively cytotoxic to cells expressing the CAK1 antigen. This cytotoxicity is due to the fact that domain I of Pseudomonas exotoxin promotes internalization of antigens which are not internalized or bound to antibody alone. Our results suggest that CAK1 is a polypeptide that is expressed on mesothelial cells and many ovarian cancers, and that K1 may be useful as a targeting agent for the immunotherapy of human ovarian cancer.
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PMID:Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium. 172 78

We have used a microinjection approach to identify a domain of the simian virus 40 (SV40) structural proteins Vp2 and Vp3(Vp2/3) responsible for their nuclear transport. By using both synthetic peptides, containing small regions of Vp2/3 conjugated to bovine serum albumin (BSA), and beta-galactosidase-Vp3 fusion proteins, we have narrowed this nuclear transport signal (NTS) to 9 amino acids (198 to 206 of Vp3 or 316 to 324 of Vp2), Gly-Pro-Asn-Lys-Lys-Lys-Arg-Lys-Leu. The porter proteins carrying the NTS or mutant NTS were microinjected into the cytoplasm of TC7 cells and their subcellular localization following the subsequent incubation period was determined immunologically using anti-BSA IgG or anti-beta-galactosidase. The 9-residue NTS peptide localized BSA into the nucleus of injected cells, changing lysine-202 to threonine or valine abolished this accumulation while changing arginine-204 to lysine did not grossly affect transport. A peptide containing the carboxyl-terminal 13 residues of Vp3 failed to localize BSA to the nucleus. Several single or double point mutations at Vp3 residues 202 and 204 have been introduced by site-directed mutagenesis. Vp3 residues 194-234, containing either a wild-type or mutated sequence at 202 and/or 204, were expressed in Escherichia coli as Vp3-beta-galactosidase fusion proteins. Addition of the carboxyl-terminal 40 residues, but not an internal 150 residues, to otherwise cytoplasmic beta-galactosidase promoted entry of the fusion protein into the nucleus. Changing lysine-202 into threonine, valine, or methionine abolished this nuclear accumulation as did changing arginine-204 into lysine. A double mutant at both positions was also blocked. We have also observed that the lectin wheat germ agglutinin inhibits the nuclear accumulation of BSA carrying the Vp2/3 NTS while the lectin concanavalin A had no effect. These data indicate that even small nuclear proteins can contain NTS's which most likely utilize a mechanism for nuclear import similar to that described for other larger proteins.
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PMID:Simian virus 40 Vp2/3 small structural proteins harbor their own nuclear transport signal. 184 70

The major capsid protein of polyomavirus, VP1, has been expression cloned in Escherichia coli, and the recombinant VP1 protein has been purified to near homogeneity (A. D. Leavitt, T. M. Roberts, and R. L. Garcea, J. Biol. Chem. 260:12803-12809, 1985). With this recombinant protein, a nitrocellulose filter transfer assay was developed for detecting DNA binding to VP1 (Southwestern assay). In optimizing conditions for this assay, dithiothreitol was found to inhibit DNA binding significantly. With recombinant VP1 proteins deleted at the carboxy and amino termini, a region of the protein affecting DNA binding was identified within the first 7 amino acids (MAPKRKS) of the VP1 amino terminus. Southwestern analysis of virion proteins separated by two-dimensional gel electrophoresis demonstrated equivalent DNA binding among the different VP1 isoelectric focusing subspecies, suggesting that VP1 phosphorylation does not modulate this function. By means of partial proteolysis of purified recombinant VP1 capsomeres for assessing structural features of the protein domain affecting DNA binding, a trypsin-sensitive site at lysine 28 was found to eliminate VP1 binding to DNA. The binding constant of recombinant VP1 to polyomavirus DNA was determined by an immunoprecipitation assay (R. D. G. McKay, J. Mol. Biol. 145:471-488, 1981) to be 1 x 10(-11) to 2 x 10(-11) M, which was not significantly different from its affinity for plasmid DNA. McKay analysis of deleted VP1 proteins and VP1-beta-galactosidase fusion proteins indicated that the amino terminus was both necessary and sufficient for DNA binding. As shown by electron microscopy, DNA inhibited in vitro capsomere self-assembly into capsidlike structures (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea, Cell 46:895-904, 1986). Thus, VP1 is a high-affinity, non-sequence-specific DNA-binding protein with the binding function localized near its trypsin-accessible amino terminus. The inhibitory effects of disulfide reagents on DNA binding and of DNA on capsid assembly suggest possible intermediate steps in virion assembly.
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PMID:Characterization of the DNA-binding properties of the polyomavirus capsid protein VP1. 184 46

Using an overlay technique, we previously showed that the Gram-negative periodontal pathogen Fusobacterium nucleatum binds to a glycoprotein of Mr 89,000 (Prakobphol, A., Murray, P., and Fischer, S.J. (1987) Anal. Biochem. 164, 5-11) in the parotid saliva of some individuals. We now show that deglycosylation of the purified glycoprotein results in loss of receptor activity. Amino acid analysis of the protein core showed predominantly proline, glycine, and glutamic acid/glutamine, a characteristic of proline-rich glycoproteins (PRG). The amino terminus contained repeating sequences of Ser-Gln-Gly-Pro-Pro-Pro-Arg-Pro-Gly-Lys-Pro-Glu-Gly-Pro-Pro-Pro- Gln-Gly that had significant compositional and sequence homology to that encoded by exon 3 of the PRB3 gene. We analyzed the PRG oligosaccharides by a combination of mass spectrometry techniques and nuclear magnetic resonance spectroscopy. Twenty-seven highly fucosylated structures were identified. The most abundant was as follows (where Fuc is fucose). (formula; see text) To understand the structural basis of F. nucleatum binding, we screened glycolipids and neoglycolipids carrying carbohydrate structures related to those of the PRG for receptor activity; components with unsubstituted terminal lactosamine residues best supported adherence. Neoglycolipids constructed from PRG oligosaccharides were also receptors. Treatment with beta-galactosidase, but not alpha-fucosidase, abolished binding, suggesting that unsubstituted lactosamine units, including the 6-antenna of the major oligosaccharide, mediate F. nucleatum adherence.
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PMID:Structure and bacterial receptor activity of a human salivary proline-rich glycoprotein. 189 23

The human asialoglycoprotein receptor subunit H2a is cotranslationally inserted into the ER membrane. When expressed together with subunit H1 in mouse fibroblasts part forms a hetero-oligomer that is transported to the cell surface, but when expressed alone it is all rapidly degraded. Degradation is insensitive to lysosomotropic agents and the undegraded precursor is last detected in the ER region of the cell. Small amounts of an intermediate 35-kD degradation product can be detected (Amara, J. F., G. Lederkremer, and H. F. Lodish. 1989. J. Cell Biol. 109:3315). We show here that the oligosaccharides on both precursor H2a and the 35-kD fragment are Man6-9GlcNAc2, structures typically found in pre-Golgi compartments. Subcellular fractionation shows that the intermediate degradation product does not cofractionate with the lysosomal enzyme beta-galactosidase, but is found in a part of the ER that contains ribosomes. Thus the intermediate degradation product is localized in the ER, indicating that the initial degradation event does take place in the ER. All degradation of H2a, including the initial endoproteolytic cleavage generating the 35-kD intermediate, is blocked by the protease inhibitors N-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylalanine chloromethyl ketone. These drugs do not inhibit ER-to-Golgi transport of H1. Depleting the cells of ATP or inhibiting protein synthesis allows the initial endoproteolytic cleavage to occur, but blocks further degradation of the 35-kD intermediate; thus we can convert all cellular H2 into the 35-kD intermediate. Approximately 50% of H2b, a splicing variant differing from H2a by a five amino acid deletion, can be transported to the cell surface, and the rest appears to be degraded by the same pathway as H2a, both when expressed alone in fibroblasts and together with H1 in HepG2 cells. Addition of N-tosyl-L-lysine chloromethyl ketone or N-tosyl-L-phenylalanine chloromethyl ketone blocks degradation of the approximately 50% that is not transported, but does not affect the fraction of H2b that moves to the Golgi region. Thus, a protein destined for degradation will not be transported to the Golgi region if degradation is inhibited.
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PMID:Nonlysosomal, pre-Golgi degradation of unassembled asialoglycoprotein receptor subunits: a TLCK- and TPCK-sensitive cleavage within the ER. 190 64

An open reading frame, rbcR, was identified 226 bp upstream of rbcAB, i.e., the ribulose 1,5-bisphosphate carboxylase genes expressed in the phototrophic purple bacterium Chromatium vinosum. Several features reveal that rbcR encodes a member of the LysR family of transcriptional regulators, in which an anomalous content of lysine and arginine residues (Lys/Arg anomaly) was found. The expression of rbcR in Escherichia coli as a protein fused to the N-terminal region of beta-galactosidase led to reduced expression of rbcAB. Thus, rbcR is likely to encode a trans-acting transcriptional regulator of rbcAB expression in C. vinosum.
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PMID:rbcR [correction of rcbR], a gene coding for a member of the LysR family of transcriptional regulators, is located upstream of the expressed set of ribulose 1,5-bisphosphate carboxylase/oxygenase genes in the photosynthetic bacterium Chromatium vinosum. 190 67


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