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)

The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.
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PMID:In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin. 848 Sep 97

The Escherichia coli codBA operon encodes cytosine permease (CodB) and cytosine deaminase (CodA). CodB mediates uptake of exogenously supplied cytosine, and CodA catalyses the hydrolytic deamination of cytosine to uracil and ammonia. The hydropathic profile of CodB indicates that it is an integral cytoplasmic membrane protein possessing several transmembrane-spanning domains. The membrane topology of CodB was investigated by using gene fusions containing varying lengths of the amino-terminus of CodB fused to either alkaline phosphatase (AP) or beta-galactosidase (BG). The AP activities expressed by the CodB-AP fusions are consistent with a topological model in which the amino- and the carboxy-termini of CodB are located in the cytoplasm, and in which CodB possesses 12 membrane-spanning segments. The enzyme activities of most of the CodB-BG fusions support the model. However, the results obtained with some of the CodB-BG fusions illustrate the limitations of using BG as a reporter protein in studies of membrane protein topology.
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PMID:Membrane topology analysis of the Escherichia coli cytosine permease. 853 18

Light-induced carotenogenesis in Myxococcus xanthus is under the control of the carQRS operon. CarQ, a proposed extracytoplasmic (ECF) RNA polymerase sigma factor, is required for expression of the operon and the carC gene that encodes phytoene dehydrogenase. CarR, an inner membrane protein in Escherichia coli, is essential for carQRS promoter inactivation in the dark. CarS is required for the light-dependent expression of the promoter of the carB gene cluster that encodes the rest of the structural genes for carotenogenesis. Regulation of carQRS is dependent on the stoichiometry of CarQ and CarR. Increasing the copy number of carQ over carR led to constitutive carotenogenesis, as did loss of translational coupling between carQ and carR. The severity of the constitutive phenotype depended on the distance between the uncoupled genes. When expressed in M. xanthus, a CarR:beta-galactosidase fusion protein disappeared in the light. We propose that anti-sigma factor CarR sequesters CarQ to the membrane in the dark, but, in the light, loss of CarR leads to release of the sigma factor.
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PMID:Light-induced carotenogenesis in Myxococcus xanthus: light-dependent membrane sequestration of ECF sigma factor CarQ by anti-sigma factor CarR. 882 46

The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid. VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and 'phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2). Conversely, VirB4::beta-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low beta-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high beta-galactosidase activities. Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K treatment of A. tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of approximately 35 and approximately 45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains.
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PMID:The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface. 899 Feb 98

Proteins can be remarkably tolerant of major mutational changes. Sites that accomodate large insertions without loss of function ("permissive" sites) appear generally to correspond to surface regions at which the added sequences do not disrupt overall folding. The identification of such sites can aid in the engineering of functional derivatives of a protein with novel properties. To screen for permissive sites, we developed a simple two-step procedure for generating 31-codon insertions in cloned genes. In a first step, a beta-galactosidase or alkaline phosphatase gene fusion is generated by insertion of a transposon derivative into the target gene. Requiring beta-galactosidase or alkaline phosphatase activity fixes the translational reading frame of the transposon relative to the target gene. In a second step, most of the transposon sequences are excised in vitro, leaving the in-frame insertion. Insertions may be targeted either to cytoplasmic or exported protein sequences, and the inserted sequence acts as an epitope in a variety of proteins. As a test case, a set of 31-codon insertions in the Escherichia coli lac permease gene was generated. The lactose transport activities of the mutant proteins followed a simple pattern: most of the proteins (10/12) with insertions in sequences thought to face the cytoplasm or periplasm were at least partially active, whereas all proteins (9/9) with insertions in membrane-spanning sequences were inactive. The only exceptions were two inactive proteins with insertions in the third cytoplasmic region. Most of the inactive proteins were detected at reduced levels in cells, presumably due to proteolytic breakdown. These studies thus illustrate the use of the new method to identify permissive sites and help document the remarkable sequence flexibility of many of the hydrophilic loops in lac permease. In addition to screening for permissive sites, 31-codon insertion mutagenesis may be useful in epitope-tagging proteins at multiple internal positions, in analyzing membrane protein topology, and in dissecting structure-function relationships in proteins.
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PMID:A simple screen for permissive sites in proteins: analysis of Escherichia coli lac permease. 909 23

An antigen in the outer membrane protein (OMP) fraction of Campylobacter jejuni was identified and characterized. Western blot analysis demonstrated antigenic differences in this protein between two congenic C. jejuni strains. Strain A74/C, which colonizes chickens, expressed the antigen at 34 kDa, while strain A74/O, which poorly colonizes chickens, expressed the antigen at 32 and 34 kDa. A genomic library was constructed in lambdagt11 with DNA from A74/O and screened with antibody raised against C. jejuni OMPs. A clone that possessed a 1.3-kb insert and expressed an immunoreactive protein fused to beta-galactosidase was isolated and purified. DNA sequence analysis revealed the insert contained one open reading frame 864 bases long. The deduced amino acid sequence demonstrated 56.3% similarity with Bacillus steorothermophilus glnH, a glutamine-binding protein, and 54.0% similarity with C. jejuni PEB1, a putative colonization adhesin. Southern hybridization, Northern hybridization, and DNA sequence analyses of the congenic colonizing and noncolonizing strains of C. jejuni failed to distinguish the two strains and revealed only one copy of the gene. Post-translational modification may be an alternate explanation for the antigenic differences seen between the two strains.
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PMID:Cloning of an outer membrane protein gene from Campylobacter jejuni. 914 43

Tgl protein is required for the production of the type IV pili found at a pole of the Myxococcus xanthus cell. These pili are essential for social motility. Evidence is presented that Tgl is a membrane protein, based on experiments with polyclonal antibody specific for Tgl that was raised against the fusion proteins beta-galactosidase-Tgl and TrpE-Tgl. Immunoaffiity-purified antibody reacted with a protein in M. xanthus having an apparent molecular mass of 27.5 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sequence of the tgl gene translates into a polypeptide of 27 kDa. Although these numbers are close, it is likely that the primary tgl translation product is processed and modified in M. xanthus. The N terminus has a signal peptidase II recognition sequence, cleavage of which is expected to remove 19 amino acid residues. When the tgl gene is expressed in Escherichia coli, the protein product consistently migrates faster in the gel than mature Tgl expressed in M. xanthus, suggesting a second modification by addition which slows migration of the protein from M. xanthus. Tgl, as detected by its specific antibody, sediments with the membrane fraction of cells. It can be extracted with detergents but not with salt or by the addition of chelators for divalent cations. In an equilibrium gradient, Tgl bands at the buoyant density of membranes and with the NADH-oxidase activity. Intact cells failed to bind anti-Tgl antibody, and less than 2% of the total Tgl is released in soluble form from the periplasm. Yet, cells that had been osmotically shocked and treated with paraformaldehyde were able to react with the specific antibody--a reaction absent from cells with a deletion of the tgl transcription unit. Assuming that osmotic shock disrupts the outer membrane, the fractionation and localization data imply that Tgl is attached to the inner or outer membranes, from which it is exposed to the intermembranous space. Tgl is necessary for synthesis of pili in M. xanthus and is the only pilus protein that can be donated by other cells (stimulation). Tgl contains six tandem copies of the tetratrico peptide repeat structural motif. Its membrane localization, capacity for stimulation, and content of tetratrico structural repeats together suggest that Tgl may be necessary for the assembly of pilin subunits into filaments.
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PMID:Identification and localization of the Tgl protein, which is required for Myxococcus xanthus social motility. 920 56

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

The drrAB operon of Streptomyces peucetius encodes for resistance to the antibiotics doxorubicin and daunorubicin. Subcloning of the drrAB genes in Escherichia coli has previously been shown to result in expression of DrrA and DrrB proteins and resistance to doxorubicin in a sensitive strain of E. coli. DrrA, a peripheral membrane protein, binds ATP in a UV-catalyzed reaction in a doxorubicin-dependent manner; DrrB, a hydrophobic protein, is localized to the inner membrane of E. coli (Kaur, P. (1997) J. Bacteriol. 179, 569-575). The present study provides evidence that DrrB, the membrane component of the complex, is stably maintained in the cell only if DrrA is present. Furthermore, it was found that the catalytic component DrrA is in an active conformation only when it is in a complex with DrrB. In a subclone containing the drrB gene by itself, no DrrB protein could be detected, although a translational fusion of the first 15 amino acids of DrrB to beta-galactosidase indicated that DrrB is translated in the absence of DrrA. Upon co-transformation with a plasmid containing the drrA gene in trans, DrrB could again be detected in these cells. UV cross-linking studies with [alpha-32P]ATP showed that only the membrane-bound form of DrrA in cells containing both DrrA and DrrB was in a conformation competent to bind ATP. Chemical cross-linking studies also provided direct evidence for interaction between the two proteins. Based on these analyses, a model for interaction between DrrA and DrrB proteins is presented.
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PMID:Biochemical coupling between the DrrA and DrrB proteins of the doxorubicin efflux pump of Streptomyces peucetius. 965

The Escherichia coli gamma-aminobutyric acid permease (GabP) is a plasma membrane protein from the amine-polyamine-choline (APC) superfamily. On the basis of hydropathy analysis, transporters from this family are thought to contain 12, 13 or 14 transmembrane domains. We have experimentally analysed the topography of GabP by using the cytoplasmically active LacZ (beta-galactosidase) and the periplasmically active PhoA (alkaline phosphatase) as complementary topological sensors. The enzymic activities of 32 GabP-LacZ hybrids and 43 GabP-PhoA hybrids provide mutually reinforcing lines of evidence that the E. coli GabP contains 12 transmembrane segments that traverse the membrane in a zig-zag fashion with both N- and C-termini facing the cytoplasm. Interestingly, the resulting model predicts that the functionally important 'consensus amphipathic region' (CAR) [Hu and King (1998) Biochem. J. 330, 771-776] is at least partly membrane-embedded in many amino acid transporters from bacteria and fungi, in contrast with the apparent situation in mouse cationic amino acid transporters (MCATs), in which this kinetically significant region is thought to be fully cytoplasmic [Sophianopoulou and Diallinas (1995) FEMS Microbiol. Rev. 16, 53-75]. To the extent that conserved domains serve similar functions, the resolution of this topological disparity stands to have family-wide implications on the mechanistic role of the CAR. The consensus transmembrane structure derived from this analysis of GabP provides a foundation for predicting the topological disposition of the CAR and other functionally important domains that are conserved throughout the APC transporter superfamily.
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PMID:Membrane topology of the Escherichia coli gamma-aminobutyrate transporter: implications on the topography and mechanism of prokaryotic and eukaryotic transporters from the APC superfamily. 980 86


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