Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Urea, at concentrations which do not interfere with bacterial growth, specifically inhibits the expression of catabolite sensitive operons. To search for the target and the mechanism of urea action we measured lactose (lac) and tryptophanase (tna) specific mRNA synthesis in vivo and in vitro. We show that urea acts by two different mechanisms at these two catabolite sensitive operons, resembling the manner in which catabolite repression regulates lac and tna. At the lac promoter, urea abolishes transcription initiation or blocks an early step in mRNA elongation without interfering with the binding of RNA polymerase and catabolite gene activator protein (CAP). At the tna promoter, urea does not abolish transcription initiation but could interfere with tnaC translation.
Mol Gen Genet 1990 Feb
PMID:Two different mechanisms for urea action at the LAC and TNA operons in Escherichia coli. 216 52

The catabolite gene activator protein (CAP) and the lac repressor regulate the transcriptional activity of the lactose operon. An early step in the regulatory functions of these proteins is their binding to specific DNA sequences within the lac promoter-operator region. Using the gel electrophoresis mobility-shift technique, we have found that the ternary complex with CAP and repressor bound to their respective highest affinity sites is 4 to 11-fold more stable than is predicted from the affinities of the independently bound proteins. This favorable binding interaction is unexpected, because CAP and lac repressor exert opposing effects on lac operon transcription. Deoxyribonuclease I footprinting analyses show that interacting proteins remain bound to the sites occupied when the proteins bind singly. These sites have a center-to-center separation of 72 base-pairs (corresponding to 6.9 turns of a B-form DNA helix), and thus occupy the same "face" of the DNA cylinder. Such an orientation is compatible with models of the ternary complex in which DNA curvature facilitates the interaction of CAP and lac repressor.
J Mol Biol 1990 Jul 20
PMID:Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter. 216 65

Cell interactions during mouse development have been shown to involve carbohydrate-containing macromolecules (glycoconjugates). We have therefore used a series of fluorescein-labelled synthetic glycoproteins to determine if mouse oocytes and zygotes also express sugar binding molecules (endogenous lectins) which might participate in such interactions. Unfertilized secondary oocytes did not express endogenous lectins at 4 degrees C but a low level of expression of fucose, mannose, and galactose-binding activity could be detected at 37 degrees C. In contrast, the zygote clearly expressed three classes of endogenous lectins, with preferential binding for i) fucose or mannose, ii) glucose or galactose, and iii) lactose. The expression of these lectins was much reduced at 4 degrees C and maximal binding at 37 degrees C was achieved only after 2 h incubation. We therefore conclude that a low level of endogenous lectin expression in the mouse oocyte is greatly enhanced after fertilisation and that, at both stages, expression, or the detection of expression, is markedly temperature dependent.
Mol Reprod Dev 1990 Aug
PMID:Mouse zygotes express endogenous lectins. 222 79

The crystal structures of complexes of isolectins 1 and 2 of wheat germ agglutinin (WGA1 and WGA2) with N-acetylneuraminyl-lactose (NeuNAc-alpha(2-3)-Gal-beta(1-4)-Glc) have been refined on the basis of data in the 8 to 2.2 A resolution range to final crystallographic R-factors of 17.2% and 15.3% (Fo greater than 1 sigma), respectively. Specific binding interactions and water association, as well as changes in conformation and mobility of the structure upon ligand binding, were compared in the two complexes. The temperature factors (B = 16.3 A2 and 18.4 A2) were found to be much lower compared with those of their respective native structures (19 to 22 A2). Residues involved in sugar binding, dimerization and in lattice contacts exhibit the largest decreases in B-value, suggesting that sugar binding reduces the overall mobility of the protein molecules in the crystal lattice. The binding mode of this sialyl-trisaccharide, an important cell receptor analogue, has been compared in the two isolectins. Only one of the two unique binding sites (4 per dimer), located in the subunit/subunit interface, is occupied in the crystals. This site, termed the "primary" binding site, contains one of the five amino acid substitutions that differentiate WGA1 and WGA2. Superposition of the refined models in each of the independent crystallographic environments indicates a close match only of the terminal non-reducing NeuNAc residue (root-mean-square delta r of 0.5 to 0.6 A). The Gal-Glc portion was found to superimpose poorly, lack electron density, and possess high atomic thermal factors. In both complexes NeuNAc is stabilized through contact with six amino acid side-chains (Ser114 and Glu115 of subunit 1 and Ser62, Tyr64, Tyr(His)66 and Tyr73 of subunit 2), involving all NeuNAc ring substituents. Refinement has allowed accurate assessment of the contact distances for four hydrogen bonds, a strong buried non-polar contact with the acetamido CH3 group and a large number of van der Waals' interactions with the three aromatic side-chains. The higher affinity of N-acetylneuraminyl-lactose observed by nuclear magnetic resonance studies for WGA1 can be explained by the more favorable binding interactions that occur when residue 66 is a Tyr. The tyrosyl side-chain provides a larger surface for van der Waals' stacking against the NeuNAc pyranose ring than His66 and a hydrogen bond contact with Gal (C2-OH), not possible in WGA2.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1990 Oct 20
PMID:2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose--wheat germ agglutinin isolectin complexes. 223 24

A series of deletion mutants of the wild-type Escherichia coli lactose promoter, with endpoints at +25, +19, +14, +1 and -6 (relative to the start of transcription at +1), was constructed and the deleted DNA replaced with non-lac DNA. These mutants were used to show that no specific DNA sequences downstream from -6 are required for efficient promoter utilization in vitro. In all cases transcription is dependent on the presence of the catabolite activator protein (CAP) and cAMP, and begins at +1 at a level indistinguishable from that at the wild-type promoter. A set of lac DNA fragments deleted to -6 was constructed, having an A, C, G or T residue at +1 and heterologous DNA downstream. These synthetic promoters allow systematic testing of the effect of the initiating nucleotide on the transcription process. Again, transcription occurs mainly from +1, at a level similar to the normal wild-type level. No substantial differences between these promoters are observed in the rates of formation of stable complexes, in the degree of complex formation, in the rate at which polymerase "escapes" from the complex or in abortive transcription products. Equivalent results are seen with a related set of constructs based on the CAP-insensitive lac UV5 promoter. Thus, lac promoter sequences including consensus hexamers at -10 and -35, plus the spacer region between them, provide specificity and efficiency both in initiation of transcription by RNA polymerase and in CAP-polymerase interactions. A question as to whether there is a third RNA polymerase binding site at lac, in addition to the known overlapping P1 and P2 regions, was not unambiguously answered. However, if a "P3" site does exist, it must lie between P1 and P2. Alternatively, the variety of polymerase interactions at wild-type lac may reflect different structural states of the enzyme. The results presented here indicate that DNA downstream from -6 plays little part in determining the conformation of the enzyme at the lactose promoter.
J Mol Biol 1990 Nov 20
PMID:Specific sequences downstream from -6 are not essential for proper and efficient in vitro utilization of the Escherichia coli lactose promoter. 225 29

We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three alpha helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.
Mol Gen Genet 1990 Aug
PMID:Genetic analysis of the LexA repressor: isolation and characterization of LexA(Def) mutant proteins. 225 42

Recent progress in the analysis of mutants of the Escherichia coli lactose carrier function is reviewed, with special emphasis on the structural basis for energy barriers which prevent 'forbidden' conformational changes. Mutations which break down the barriers to forbidden isomerizations involving the binary carrier:sugar (CS) and carrier:proton (CH) complexes have been obtained in several laboratories. These mutants allow uncoupled transport of H+ or galactoside in the lactose carrier which normally couples cation and sugar movement in a 1:1 stoichiometry. These uncoupled mutants appear to be associated with changes in both sugar and cation recognition, suggesting that the physical interactions forming the basis for co-substrate recognition and uncoupling are not independently variable. By postulating that translocation involves transformation of the stable intermediate of the co-transport cycle to unstable transition state conformations of the carrier, it is possible to consider the consequences of mutagenesis in terms of transition state theory. Consistent with several experimental observations, the analysis predicts in each mutant the occurrence of more than one abnormality in the transport cycle (such as changes in sugar recognition, cation recognition or the coupling reaction). We have called the general phenomenon a 'mutational double-effect' because any mutation which alters the Gibbs free energy change of one reaction in the transport cycle must affect the free energy change of at least one other reaction in this cycle.
Mol Microbiol 1990 Sep
PMID:Towards an understanding of the structural basis of 'forbidden' transport pathways in the Escherichia coli lactose carrier: mutations probing the energy barriers to uncoupled transport. 228 70

Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.
Mol Reprod Dev 1990 Jun
PMID:Effect of equilibration period on the viability of frozen-thawed mouse morulae after rapid freezing. 237 93

To elucidate the details of pH-induced conformational transformation of ricin [I] in the region surrounding tryptophan residues, we studied parameters of fluorescence of the native toxin and its isolated A- and B-subunits at pH 4.0, 5.0 and 7.4. The studies were carried out using resolution of fluorescence spectra according to different degree of tryptophan accessibility to ionic (iodide) and non-ionic organic (acrylamide) quenchers. Application of the new method allowed to reveal three classes of tryptophan residues differing in their accessibility to quenchers alpha-residues are accessible neither to ions nor to organic molecules; beta-residues are accessible only to organic molecules; while surface gamma-residues are accessible to both types of quenchers. The fluorescence spectra were assessed for each class of tryptophan residues. The major part of them was shown to be localized in apolar rigid microenvironment. Fluorescence of ricin and especially of its isolated subunits proved to be strongly dependent on the pH value. At pH less than 5 the structure of B-chain loosens, this process being reflected by an increase in accessibility of tryptophan residues to quenchers. In acidic solution at least one out of seven tryptophan residues in the ricin molecule undergoes conformational transformation. Positive charge prevails in the regions surrounding quencher-accessible tryptophan residues. Binding of lactose leads to a slight compactization of the toxin structure that causes, in its turn, short-wave shifts of the fluorescence spectra and reduction of Stern-Volmer constants for intraglobular tryptophan residues.
Mol Biol (Mosk)
PMID:[Ricin structure: the study by the fluorescence quenching method]. 240 31

Sialo- and asialoglycoconjugates were isolated from Trypanosoma cruzi epimastigotes and their composition determined. Sialoglycoconjugates bound to wheat germ agglutinin (WGA)-Sepharose and were precipitated by concanavalin A, Wistaria floribunda hemagglutinin and WGA. Asialoglycoconjugate bound to concanavalin A-Sepharose and precipitated with concanavalin-A and W. floribunda hemagglutinin but not with WGA. Cells grown in the presence of fetal calf serum were agglutinated by WGA but not by peanut agglutinin. The reverse was true for cells grown without fetal calf serum. Neuraminidase-treated cells incorporated sialic acid or its 7-carbon analog, 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid (AcNeu7) from sialylated compounds such as fetuin or sialyl-lactose but did not incorporate free sialic acid. Restoration of the WGA sialylreceptors in neuraminidase-treated cells, as determined by cell agglutination with WGA, was also obtained by incubation with fetuin or sialyl-lactose but not with free sialic acid. Moreover, restoration of agglutinability by WGA in neuraminidase-treated cells or cells grown in medium without fetal calf serum occurred equally well in energy-rich or energy-depleted cells. A transglycosilase reaction for sialic acid incorporation in T. cruzi epimastigotes is suggested.
Mol Biochem Parasitol 1985 Jun
PMID:Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route. 241 16


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