| 1. |
- Balaz, Martina, et al.
(författare)
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Effects of surface adsorption on catalytic activity of heavy meromyosin studied using a fluorescent ATP analogue
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:24, s. 7233-7251
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Tidskriftsartikel (refereegranskat)abstract
- Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.
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| 2. |
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| 3. |
- Gonzalez Palmén, Lorena, et al.
(författare)
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A Double Role for a Strictly Conserved Serine : Further Insights into the dUTPase Catalytic Mechanism
- 2008
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Ingår i: Biochemistry. - : American Chemical Society. - 0006-2960 .- 1520-4995. ; 47:30, s. 7863-7874
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Tidskriftsartikel (refereegranskat)abstract
- Ser72 at the active site of the Escherichia coli dUTPase has been mutated to an alanine, and the properties of the mutant have been investigated. The serine is absolutely conserved among the monomeric and trimeric dUTPases (including the bifunctional dCTP deaminase:dUTPases), and it has been proposed to promote catalysis by balancing negative charge at the oxygen that bridges the α- and β-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue α,β-imido-dUTP·Mg, the serine β-OH is indeed hydrogen bonded to the α,β-bridging nitrogen of the analogue. However, in the complex of the Asp90→Asn mutant dUTPase with the true substrate dUTP·Mg, the serine β-OH points in the opposite direction and may form a hydrogen bond to Asn84 at the bottom of the pyrimidine pocket. Here we show that the replacement of the β-OH by hydrogen reduces kcat from 5.8 to 0.008 s-1 but also k-1, the rate of substrate dissociation, from 6.2 to 0.1 s-1 (KM = 6 × 10-9 M). We conclude that the serine β-OH exercises both ground state (GS) destabilization and transition state (TS) stabilization, effects not usually linked to a single residue. With experimental support, we argue that the β-OH destabilizes the GS by imposing conformational constraints on the enzyme and that formation ofthe TS depends on a rotation of the serine side chain that not only relieves the constraints but brings the β-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the β-OH to promote both deamination and hydrolysis in the bifunctional deaminases. We find that the E. coli dUTPase does not catalyze the hydrolysis of the α,β-imido-dUTP·Mg, suggesting that the analogue provides the hydrogen in the bond to the serine β-OH.
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| 4. |
- Gonzalez Palmén, Lorena, 1970-
(författare)
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Homotrimeric dUTPases : Principles of Catalysis and Inhibitor Design
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ubiquitous enzyme dUTPase hydrolyzes dUTP into dUMP and pyrophosphate, preventing DNA fragmentation and cell death due to accumulation of dUTP. Inhibitors of dUTPase could serve as drugs in the treatment of cancers and infectious diseases. This thesis presents five studies. A mutational study on the Escherichia coli dUTPase (S72A) provides new insights about the catalytic principles of the homotrimeric dUTPases. A model is presented in which transition state formation is associated with a rotation of the conserved Ser72 side chain. The model can explain the strict order of deamination and hydrolysis catalyzed by the bifunctional dCTP deaminase:dUTPases. The S72A/D90N double mutant is currently investigated. Preliminary data indicate that this form preserves the binding properties of the S72A mutant but is completely inactive, making it attractive for structural studies. In the remaining studies we compare the binding of substrate analogues to the human, the E. coli and the equine infectious anemia virus (EIAV) homotrimeric dUTPases. One study concerns 2´,3´-dideoxy-UTP (ddUTP) and shows that removal of the 3´-hydroxyl group increases KM, ten times with the cellular dUTPases and fifty times with the viral dUTPase, but does not affect kcat with any of these enzymes. Another study concerns the inhibitory effects of 3´-azido-2´,3´-dideoxy-UTP. This derivative binds to the bacterial dUTPase but not to the other forms making it a potential lead for the development of antibacterial dUTPase inhibitors. Yet another study investigates two uracil derivatives. Both compounds are found to inhibit the human, the bacterial but not the viral dUTPase. The inhibition is shown to be competitive.
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| 5. |
- Horsefield, Rob, 1977, et al.
(författare)
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High-resolution x-ray structure of human aquaporin 5
- 2008
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:36, s. 13327-13332
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin 5 (HsAQP5)facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-angstrom resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.
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| 6. |
- Kvassman, Jan, et al.
(författare)
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Mechanism of glyceraldehyde‐3‐phosphate transfer from aldolase to glyceraldehyde‐3‐phosphate dehydrogenase
- 1988
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 172:2, s. 427-431
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic interaction of glyceraldehyde‐3‐phosphate dehydrogenase with glyceraldehydes‐3‐phosphate has been examined by transient‐state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehydes‐3‐phosphate decreases at least 50‐fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6‐bisphosphate, but lend no support to the proposal that glyceraldehydes 3‐phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two‐enzyme system is compatible in all respects tested with a free‐diffusion mechanism for the transfer of glyceraldehydes 3‐phosphate from the producing enzyme to the consuming one.
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| 7. |
- Larsson, Gunilla, et al.
(författare)
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Kinetic Characterization of dUTPase from Escherichia coli
- 1996
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 271:39, s. 24010-24016
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits. In the present work, the catalytic properties of the E. coli dUTPase were investigated in the pH range 5-11. The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed). The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 105 times less efficiently than dUTP, a decline largely accounted for by a higher Km for dCTP. With dUTP·Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s1. Km passed through a broad minimum in the neutral pH range with values approaching 107 M. It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pKa values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP·Mg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step. The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10. Only one enantiomer of the substrate analog 2-deoxyuridine-5-(-thio)-triphosphate was hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the -phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products. The results are discussed with reference to recent data on the structure of the E. coli dUTPase·dUDP complex.
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| 8. |
- Nord, Johan, et al.
(författare)
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dUTPase from the retrovirus equine infectious anemia virus: specificity, turnover and inhibition
- 1997
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Ingår i: FEBS Letters. - 1873-3468. ; 414:2, s. 271-274
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Tidskriftsartikel (refereegranskat)abstract
- The kinetic properties of dUTPase from equine infectious anemia virus (EIAV) were investigated. KM (1.1 [plusmn] 0.1 [mu ]M) and kcat (25 s[minus ]1) were found to be independent of pH in the neutral pH range. Above pH 8.0, KM increases slightly. Below pH 6.0, the enzyme is rapidly deactivated. Detergent was found to enhance activity, leaving KM and kcat unaffected. Compared to the Escherichia coli dUTPase, the EIAV enzyme is equally potent in hydrolyzing dUTP, but less specific. Inhibition of the viral enzyme by the nucleotides dTTP, dUMP and a synthetic analogue, 2[prime ]-deoxyuridine 5[prime ]-([alpha ],[beta ]-imido)triphosphate, is stronger by one order of magnitude.
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| 9. |
- Ohlsson, Gabriel, 1982, et al.
(författare)
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Solute transport on the sub 100 ms scale across the lipid bilayer membrane of individual proteoliposomes
- 2012
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 12:22, s. 4635-4643
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Tidskriftsartikel (refereegranskat)abstract
- Screening assays designed to probe ligand and drug-candidate regulation of membrane proteins responsible for ion-translocation across the cell membrane are wide spread, while efficient means to screen membrane-protein facilitated transport of uncharged solutes are sparse. We report on a microfluidic-based system to monitor transport of uncharged solutes across the membrane of multiple (>100) individually resolved surface-immobilized liposomes. This was accomplished by rapidly switching (<10 ms) the solution above dye-containing liposomes immobilized on the floor of a microfluidic channel. With liposomes encapsulating the pH-sensitive dye carboxyfluorescein (CF), internal changes in pH induced by transport of a weak acid (acetic acid) could be measured at time scales down to 25 ms. The applicability of the set up to study biological transport reactions was demonstrated by examining the osmotic water permeability of human aquaporin (AQP5) reconstituted in proteoliposomes. In this case, the rate of osmotic-induced volume changes of individual proteoliposomes was time resolved by imaging the self quenching of encapsulated calcein in response to an osmotic gradient. Single-liposome analysis of both pure and AQP5-containing liposomes revealed a relatively large heterogeneity in osmotic permeability. Still, in the case of AQP5-containing liposomes, the single liposome data suggest that the membrane-protein incorporation efficiency depends on liposome size, with higher incorporation efficiency for larger liposomes. The benefit of low sample consumption and automated liquid handling is discussed in terms of pharmaceutical screening applications.
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