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)

A peptide containing residues 1-50 of the Aalpha-chain of fibrinogen, expressed as a fusion peptide with beta-galactosidase, is rapidly cleaved by thrombin at Arg-16, similarly to whole fibrinogen. When Phe-8, which is highly conserved, is replaced with tyrosine (F8Y), the cleavage is slowed drastically [Lord, Byrd, Hede, Wei and Colby (1990) J. Biol. Chem. 265, 838-843]. To examine the structural basis for this result, we have determined the crystal structure of bovine thrombin complexed with a synthetic peptide containing residues 1-23 of fibrinogen Aalpha and the F8Y mutation. The crystals are in space group P43212, with unit-cell dimensions of a = 88.3 A (1 A = 0.1 nm), c = 195.5 A and two complexes in the asymmetric unit. The final R factor is 0.183 for 2sigma data from 7.0 to 2.5 A resolution. There is continuous density for the five residues in the P3, P2, P1, P1' and P2' positions of the peptide (Gly-14f to Pro-18f) at the active site of thrombin, and isolated but well-defined density for Tyr-8f at position P9 in the hydrophobic pocket of thrombin. The tyrosine residue is shifted relative to phenylalanine in the native peptide because the phenol side chain is larger and makes a novel, intrapeptide hydrogen bond with Gly-14f. Adjacent peptide residues cannot form the hydrogen bonds that stabilize the secondary structure of the native peptide. Consequently, the 'reaction'geometry at the scissile bond, eight residues from the mutation, is perturbed and the peptide is mostly uncleaved in the crystal structure.
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PMID:Crystal structure of fibrinogen-Aalpha peptide 1-23 (F8Y) bound to bovine thrombin explains why the mutation of Phe-8 to tyrosine strongly inhibits normal cleavage at Arg-16. 930 32

A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation. The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated phi 31p trigger promoter fused to the lethal LlaIR+ restriction endonuclease cassette. Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the presence of phi 31 selected for phage phi 31 derivatives which were markedly less sensitive to phi 31p-LlaIR(+)-encoded restriction than the parental phage, phi 31. The efficiency of plaquing (EOP) on L. lactis NCK690(pTRK414H) was 10(-4) for phi 31 versus 0.4 for the derived phages. The mutant phages remained fully sensitive to LlaIR+ restriction, suggesting an alteration in the recognition or firing of the phi 31p promoter. Sequencing over the promoter region in four mutant phages revealed the identical C-to-A transversion, generating a Phe-to-Leu substitution, in a transcriptional activator of the phi 31p promoter, designated ORF2. The mutant phages were analyzed for their ability to induce the native phi 31p promoter element fused to a lacZst reporter gene. Compared to the parental phage, phi 31, lower levels of beta-galactosidase activity were induced throughout the lytic cycle, indicating that the strength at which the mutant phages activated the phi 31p promoter was altered. Based on these observations, improvements were made in promoter strength and restriction activity in an attempt to elevate the effectiveness of the phage-triggered suicide system. When the phi 31p-LlaIR+ cassette was paired with other abortive defense systems, Per31 and AbiA, the EOP of phi 31 was reduced to < 10(-10) and the level of phage in the culture was lowered below the detection limits of the assay.
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PMID:Bacteriophage-triggered defense systems: phage adaptation and design improvements. 936 24

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a beta-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectable signal (Leu-14 --> Arg and Glu-46 --> Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.
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PMID:Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system. 937

The His at position 357 of beta-galactosidase (Escherichia coli) was substituted by an Asp, an Asn, a Leu, and a Phe, and studies done with the substituted enzymes showed that the main role of His-357 is to stabilize the transition state by interacting with the C3 hydroxyl. The substituted enzymes were less stable to heat than was wild-type enzyme (40-90% of the activity was lost in 10 min at 52 degreesC compared to wild-type beta-galactosidase which lost no activity), but the gross physical properties of the substituted enzymes at normal temperatures were not changed. There were also no differences in the ability to bind or to be activated by Mg2+. The substitutions (except Asp) did not affect the pKa for binding substrate in the ground state, but the pKa of the kcat was altered as would be expected for a residue important for binding the transition state. Substitution by Asp may cause a conformational change at high pH values. Activation energy differences (Delta DeltaGS), as determined by differences in kcat/Km values, indicated that substitutions for His-357 caused significant destabilizations of the first transition state (for the step in which the galactoside bond is cleaved and the covalent reaction intermediate is formed). This resulted in decreases of up to 900-fold in k2 for the mononitrophenyl substrates. In contrast, the k3 values (which depend on the energy level of the second transition state) were not decreased as much (<90-fold). In some cases, the k3 values even increased (when Asn was substituted for His-357). The importance of His-357 for stabilization of the transition state was confirmed by studies with transition state analogue inhibitors that showed that His-357 forms strong specific interactions with the C3 hydroxyl of the galactose moiety of the transition state. Studies with substrate analogue inhibitors indicated that His-357 is probably not important for the binding of the substrates themselves.
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PMID:His-357 of beta-galactosidase (Escherichia coli) interacts with the C3 hydroxyl in the transition state and helps to mediate catalysis. 966 15

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC50 8.02 x 10(-11) M (+/- 0.30 x 10(-11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K(m) and Vmax values were 7.2 x 10(-5) (+/- 0.7 x 10(-5) M-1 and 1.13 x 10(-3) (+/- 0.05 x 10(-3) OD unit-1 min-1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21-24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.
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PMID:Hydrolytic enzymes of Psoroptes cuniculi (Delafond). 1007 Jul 42

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.
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PMID:Bivalent inhibitor of the N-end rule pathway. 1036 69

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, n-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were active in extracts of scab mites (Psoroptes spp.) raised on sheep or rabbits. Trypsin and chymotrypsin activities were not detected. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts in a pH-dependent fashion with an optimum of pH 3-5. Acid proteinase activity was greater in mites raised on rabbits than in those raised on sheep. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)Arg-Leu-OH while pepstatin A, a specific inhibitor of aspartic proteinases, totally inhibited its hydrolysis at a concentration of 1 nM.
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PMID:Immunological control of scab mites: digestive enzymes as candidate compounds. 1042 5

Extracts of ectoparasitic mites of birds (Dermanyssus gallinae), sheep (Psoroptes ovis) and plants (Tetranychus urticae) and of free-living mites (Acarus siro) contained acid and alkaline phosphatase, C4 and C8 esterases, lipase, leucine and valine aminopeptidases and a range of glycosidase activities. Dermanyssus gallinae and P. ovis, species highly adapted to an animal parasitic lifestyle, had very similar profiles and contained low activities of glycosidases. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only beta-galactosidase and beta-glucuronidase. All extracts hydrolysed haemoglobin with optima below pH6, and this hydrolysis was associated with an aspartic proteinase and variable cysteine proteinase activity dependent on species. Inhibitor-labelling with biotinyl-Phe-Ala-FMK revealed the presence of cysteine proteinases with molecular masses of 25-33.5kDa. Each mite species contains the enzymes necessary to complete digestion of the diet in the intracellular lysosomal compartment. The absolute and relative activities of each enzyme varied, and are discussed according to phylogeny and dietary habit.
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PMID:A comparative survey of the hydrolytic enzymes of ectoparasitic and free-living mites. 1067 40

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.
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PMID:Cystic fibrosis transmembrane conductance regulator does not affect neutrophil migration across cystic fibrosis airway epithelial monolayers. 1075 64

The genus Proteus belongs to the tribe of Proteae in the family of Enterobacteriaceae, and consists of five species: P. mirabilis, P. vulgaris, P. morganii, P. penneri and P. myxofaciens. They are distinguished from the rest of Enterobacteriaceae by their ability to deaminate phenylalanine and tryptophane. They hydrolyze urea and gelatin and fail to ferment lactose, mannose, dulcitol and malonate; and do not form lysine and arginine decarboxylase or beta-galactosidase [1]. Colonies produce distinct "burned chocolate" odor and frequently show the characteristics of swarming motility on solid media. P. mirabilis, P. vulgaris and P. morganii are widely recognized human pathogens. They have been isolated from urinary tract infections, wounds, ear, and nosocomial bacteremic infections, often in immuncompromised patients [2-6]. P. myxofaciens has no clinical interest to this time. P. penneri as species nova was nominated by the recommendation of Hickman and co-workers [7]. Formerly it was recognized as P. vulgaris biogroup 1 or indole negative P. vulgaris [8, 9]. Although it has been less commonly isolated from clinical samples than the other three human pathogenic Proteus species, it has nevertheless been connected with infections of the urinary tract, wounds and has been isolated from the feces of both healthy and diarrheic individuals [10-12]. Potential virulence factors responsible for virulence of Proteae are: IgA protease, urease, type3 fimbriae associated with MR/K haemagglutinins of at least two antigenic types, endotoxin, swarming motility and HlyA and/or HpmA type hemolysins [for review see ref. 13]. In the followings we give a survey of accumulated concepts about the position and characteristics of HlyA type alpha-hemolysins both in general and with emphasis on virulence functions in the tribe of Proteae.
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PMID:Proteus virulence: involvement of the pore forming alpha-hemolysin (a short review). 1105 65


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