| 1. |
- Ben-Menachem, Gil, et al.
(författare)
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The physico-chemical characteristics of the phosphocholine-containing glycoglycerolipid MfGL-II govern the permeability properties of Mycoplasma fermentans
- 2001
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Ingår i: The FEBS Journal, European Journal of Biochemistry. - : Wiley. ; 268:13, s. 3694-3701
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Tidskriftsartikel (refereegranskat)abstract
- Mycoplasma fermentans seems to be involved in several pathogenic condtions in humans, and is among other things capable of fusing with T-cells and lymphocytes. The choline-containing phosphoglycolipid 6'-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-α-d-glucopyranosyl-(1'→3)-1,2-diacylglycerol (MfGL-II) in the membrane of M. fermentans has been suggested to enhance the fusion process, and the characteristics of MfGL-II were therefore investigated. When a cell culture ages the fraction of MfGL-II increases, and the fraction of the other major membrane lipid, phosphatidylglycerol (PtdGro), decreases concomitantly. Swelling experiments showed that the permeability and osmotic fragility are markedly reduced in aged cells. MfGL-II is selectively released into the surrounding medium when aged M. fermentans cells are incubated in buffer containing EDTA. The physico-chemical properties of MfGL-II were studied by NMR spectroscopy and differential scanning calorimetry, and they can explain the biochemical results. The temperature for the transition between gel and lamellar liquid crystalline (Lα) phases is 35–45 °C higher for MfGL-II than for PtdGro, which most probably gives rise to the reduced permeability in aged cells. At high water contents MfGL-II forms an Lα phase and isotropic aggregates which were interpreted to be vesicles with a radius of ≈ 450 Å. It is proposed that MfGL-II forms vesicles in the surrounding medium when it is released from the cell membrane. Neither EDTA nor Ca2+ ions have a significant influence on the aggregate structures formed by MfGL-II. Our results indicate that MfGL-II has no fusogenic properties. It is more probable that a recently identified lysolipid in the M. fermentans membrane acts as a fusogen.
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| 2. |
- Bugaytsova, Zhanna, et al.
(författare)
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Localization, purification and properties of a tetrathionate hydrolase from Acidithiobacillus caldus
- 2004
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Ingår i: European Journal of Biochemistry. - Hoboken : Wiley-Blackwell. - 0014-2956 .- 1432-1033. ; 271:2, s. 272-280
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Tidskriftsartikel (refereegranskat)abstract
- The moderately thermophilic bacterium Acidithiobacillus caldus is found in bacterial populations in many bioleaching operations throughout the world. This bacterium oxidizes elemental sulfur and other reduced inorganic sulfur compounds as the sole source of energy. The purpose of this study was to purify and characterize the tetrathionate hydrolase of A. caldus. The enzyme was purified 16.7-fold by one step chromatography using a SP Sepharose column. The purified enzyme resolved into a single band in 10% polyacrylamide gel, both under denaturing and native conditions. Its homogeneity was confirmed by N-terminal amino acid sequencing. Tetrathionate hydrolase was shown to be a homodimer with a molecular mass of 103 kDa (composed from two 52 kDa monomers). The purified enzyme had optimum activity at pH 3.0 and 40 degreesC and an isoelectric point of 9.8. The periplasmic localization of the enzyme was determined by differential fractionation of A. caldus cells. Detected products of the tetrathionate hydrolase reaction were thiosulfate and pentathionate as confirmed by RP-HPLC analysis. The activity of the purified enzyme was drastically enhanced by divalent metal ions.
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| 3. |
- Elleby, Björn, et al.
(författare)
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Characterization of carbonic anhydrase from Neisseria gonorrhoeae
- 2001
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Ingår i: FEBS Journal. - : Wiley. ; 268:6, s. 1613-9
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s−1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66→Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO−, SCN− and N3− as HCA II, while CN− and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO−, SCN−, CN− and N3− resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8-2.9 mGdnHCl was observed.
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| 4. |
- Elleby, Björn, et al.
(författare)
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Enhancement of catalytic efficiency by the combination of site-specific mutations in a carbonic anhydrase-related protein
- 2000
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Ingår i: The FEBS Journal, European Journal of Biochemistry. - : Wiley. ; 267:19, s. 5908-15
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Tidskriftsartikel (refereegranskat)abstract
- A single mutation, involving the replacement of an arginine residue with histidine to reconstruct a zinc-binding site, suffices to change a catalytically inactive murine carbonic anhydrase-related protein (CARP) to an active carbonic anhydrase with a CO2-hydration turnover number of 1.2 × 104 s−1. Further mutations, leading to a more ‘carbonic anhydrase-like’ active-site cavity, results in increased activity. A quintuple mutant having His94, Gln92, Val121, Val143, and Thr200 (human carbonic anhydrase I numbering system) shows kcat = 4 × 104 s−1 and kcat/Km = 2 × 107 m−1·s−1, greatly exceeding the corresponding values for carbonic anhydrase isozyme III and approaching those characterizing carbonic anhydrase I. In addition, a buffer change from 50 mm Taps/NaOH to 50 mm 1,2-dimethylimidazole/H2SO4 at pH 9 results in a 14-fold increase in kcat for this quintuple mutant. The CO2-hydrating activity of a double mutant with His94 and Gln92 shows complex pH-dependence, but the other mutants investigated behave as if the activity (kcat/Km) is controlled by the basic form of a single group with pKa near 7.7. In a similar way to human carbonic anhydrase II, the buffer behaves formally as a second substrate in a ping-pong pattern, suggesting that proton transfer between a zinc-bound water molecule and buffer limits the maximal rate of catalysis in both systems at low buffer concentrations. However, the results of isotope-exchange kinetic studies suggest that proton shuttling via His64 is insignificant in the CARP mutant in contrast with carbonic anhydrase II. The replacement of Ile residues with Val in positions 121 or 143 results in measurable 4-nitrophenyl acetate hydrolase activity. The pH–rate profile for this activity has a similar shape to those of carbonic anhydrase I and II. CD spectra of the double mutant with His94 and Gln92 are variable, indicating an equilibrium between a compact form of the protein and a ‘molten globule’-like form. The introduction of Thr200 seems to stabilize the protein.
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| 5. |
- Eneqvist, Therese, et al.
(författare)
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The transthyretin-related protein family
- 2003
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 270:3, s. 518-532
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Tidskriftsartikel (refereegranskat)abstract
- A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.
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| 6. |
- Fa, M, et al.
(författare)
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Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.
- 2000
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:12, s. 3729-34
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Tidskriftsartikel (refereegranskat)abstract
- The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.
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| 7. |
- Hankamer, B, et al.
(författare)
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Isolation and biochemical characterisation of monomeric and dimeric photosystem II complexes from spinach and their relevance to the organisation of photosystem II in vivo
- 1997
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 243:1-2, s. 422-429
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Tidskriftsartikel (refereegranskat)abstract
- Membranes enriched in photosystem II were isolated from spinach and further solubilised using n-octyl beta-D-glucopyranoside (OctGlc) and n-dodecyl beta-D-maltoside (DodGlc(2)). The OctGlc preparation had high rates of oxygen evolution and when subjected to size-exclusion HPLC and sucrose density gradient centrifugation, in the presence of DodGlc(2), separated into dimeric (430 kDa), monomeric (236 kDa) photosystem II cores and a fraction containing photosystem II light-harvesting complex (Lhcb) proteins. The dimeric core fraction was more stable, contained higher levels of chlorophyll, beta-carotene and plastoquinone per photosystem II reaction centre and had a higher oxygen-evolving activity than the monomeric cores. Their subunit composition was similar (CP43, CP47, D1, D2, cytochrome b 559 and several lower-molecular-mass components) except that the level of 33-kDa extrinsic protein was lower in the monomeric fraction. Direct solubilisation of photosystem-II-enriched membranes with DodGlc(2), followed by sucrose density gradient centrifugation, yielded a super complex (700 kDa) containing the dimeric form of the photosystem II core and Lhcb proteins: Lhcb1, Lhcb2, Lhcb4 (CP29), and Lhcb5 (CP26). Like the dimeric and monomeric photosystem II core complexes, the photosystem II-LHCII complex had lost the 23-kDa and 17-kDa extrinsic proteins, but maintained the 33-kDa protein and the ability to evolve oxygen. It is suggested, with a proposed model, that the isolated photosystem II-LHCII super complex represents an in vivo organisation that can sometimes form a lattice in granal membranes of the type detected by freeze-etch electron microscopy [Seibert, M., DeWit, M. & Staehelin, L. A. (1987) J. Cell Biol. 105, 2257-2265].
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| 8. |
- Henriksson, Maria, et al.
(författare)
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A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:20, s. 4921-4929
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Tidskriftsartikel (refereegranskat)abstract
- 14-3-3 proteins play an important role in a multitude of signalling pathways. The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner. Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3. Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.
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| 9. |
- Holmberg, M, et al.
(författare)
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The species-specific differences in the cAMP regulation of the tissue-type plasminogen activator gene between rat, mouse and human is caused by a one-nucleotide substitution in the cAMP-responsive element of the promoters.
- 1995
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 231:2, s. 466-74
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Tidskriftsartikel (refereegranskat)abstract
- In rat ovarian cells tissue-type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP-dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase-type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species-specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP-responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one-nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP-inducing agents forskolin or follicle-stimulating hormone. Following the conversion of the mouse and human CRE-like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE-like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE-like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP-responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle-stimulation hormone seem to be due to the inability of these CRE-like sequences to efficiently bind transcription factor CRE binding protein.
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| 10. |
- Kotova, Irina, et al.
(författare)
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A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 268:16, s. 4527-4536
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Tidskriftsartikel (refereegranskat)abstract
- Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.
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