The beta-glucosidase from Botryodiplodia theobromae Pat. Kinetics of enzyme-catalysed hydrolysis of o-nitrophenyl beta-D-glucopyranoside in dioxan/water.
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1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Botryodiplodia theobromae Pat. has been studied in the presence of added dioxan. 2. At donor saturation, the maximum rate of hydrolysis in the presence of up to 50%(v/v) dioxan was pH4.3-4.5 (pH of the buffer system in water) in McIlvaine's buffer. 3. Increasing dioxan concentrations progressively decreased the maximum rate of hydrolysis. 4. The rate of enzyme-catalysed reaction was enhanced at high donor concentrations, but inhibited at low donor concentrations in the presence of glycerol, methanol, fructose of sucrose. 5. The hydrolytic reaction was found to proceed with retention of configuration at the anomeric carbon atom. 6. The kinetics of the enzyme-catalysed process in the presence of added acceptors indicated that water was necessary for the maintenance of the active enzyme conformation apart from its acceptor function. (+info)
Oriented channels reveal asymmetric energy barriers for sugar translocation through maltoporin of Escherichia coli.
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Sugar transport through maltoporin of Escherichia coli was investigated. This protein facilitates maltooligosaccharide translocation via a binding site in the channel. Because incorporation of the protein into the bilayer results in randomly orientated channels, we re-examined the postulated symmetric translocation model by reconstitution of maltoporin under an externally applied field. Upon binding of bacteriophage lambda, which exploit surface-exposed loops of maltoporin as the receptor, sugar permeation, but not the ion current, was blocked. Thus using the phage-to-probe orientation we were able to show that the channels were approximately 80% directionally inserted into the bilayer. Moreover, asymmetry of the channel was revealed because sugar entrance through the 'open' periplasmic side of maltoporin was similarly reduced. Here a new asymmetrical two-barrier model is presented. Based on liposome-swelling assays and current-fluctuation analysis we conclude that the periplasmic side of the porin shows a two- to threefold higher energy barrier than the extracellular loop-side of the channels. (+info)
Characterization of specific immune responses of mice inoculated with recombinant vaccinia virus expressing an 18-kilodalton outer membrane protein of Brucella abortus.
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Using the shuttle vector pMCO2 and the vaccinia virus wild-type WR strain, we constructed a recombinant virus expressing an 18-kDa outer membrane protein of Brucella abortus. BALB/c mice inoculated with this virus produced 18-kDa protein-specific antibodies, mostly of immunoglobulin G2a isotype, and in vitro stimulation of splenocytes from these mice with purified maltose binding protein-18-kDa protein fusion resulted in lymphocyte proliferation and gamma interferon production. However, these mice were not protected against a challenge with the virulent strain B. abortus 2308. Disruption of the 18-kDa protein's gene in vaccine strain B. abortus RB51 did not affect either the strain's protective capabilities or its in vivo attenuation characteristics. These observations suggest that the 18-kDa protein plays no role in protective immunity. (+info)
Strategies to determine the extent of control exerted by glucose transport on glycolytic flux in the yeast Saccharomyces bayanus.
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The extent to which the transport of glucose across the plasma membrane of the yeast Saccharomyces bayanus controls the glycolytic flux was determined. The magnitude of control was quantified by measuring the effect of small changes in the activity of the glucose transport system on the rate of glucose consumption. Two effectors were used to modulate the activity of glucose transport: (i) maltose, a competitive inhibitor of the glucose transport system in S. bayanus (as well as in Saccharomyces cerevisiae) and (ii) extracellular glucose, the substrate of the glucose transport system. Two approaches were followed to derive from the experimental data the flux control coefficient of glucose transport on the glycolytic flux: (i) direct comparison of the steady-state glycolytic flux with the zero trans-influx of glucose and (ii) comparison of the change in glycolytic flux with the concomitant change in calculated glucose transport activity on variation of the extracellular glucose concentration. Both these approaches demonstrated that in cells of S. bayanus grown on glucose and harvested at the point of glucose exhaustion, a high proportion of the control of the glycolytic flux resides in the transport of glucose across the plasma membrane. (+info)
Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces.
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Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction. (+info)
The C-terminal portion of the tail fiber protein of bacteriophage lambda is responsible for binding to LamB, its receptor at the surface of Escherichia coli K-12.
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Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, its cell-surface receptor. We fused C-terminal portions of J, the tail fiber protein of lambda, to maltose-binding protein. Solid-phase binding assays demonstrated that a purified fusion protein comprising only the last 249 residues of J could bind to LamB trimers and inhibited recognition by anti-LamB antibodies. Electron microscopy further demonstrated that the fusion protein could also bind to LamB at the surface of intact cells. This interaction prevented lambda adsorption but affected only partially maltose uptake. (+info)
The detergent-soluble maltose transporter is activated by maltose binding protein and verapamil.
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The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V(max) of the ATPase 2.7-fold but had no effect on the K(m) of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN(3), as well as a mercurial, p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation. (+info)
Three pathways for trehalose biosynthesis in mycobacteria.
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Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection. To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria. Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA-OtsB pathway) from glycogen-like alpha(1-->4)-linked glucose polymers (the TreY-TreZ pathway) and from maltose (the TreS pathway). Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG. The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism. The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology. (+info)