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Sökning: L773:0014 2956 OR L773:1432 1033

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1.
  • Renlund, Martin, et al. (författare)
  • Free N-acetylneuraminic acid in tissues in Salla disease and the enzymes involved in its metabolism
  • 1983
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 130:1, s. 39-45
  • Tidskriftsartikel (refereegranskat)abstract
    • Salla disease is a lysosomal storage disorder of unknown etiology, characterized biochemically by increased urinary excretion of N-acetylneuraminic acid. This compound has now been shown to occur in abnormally large amounts in liver and cultured skin fibroblasts from these patients. Quantification of N-acetylneuraminic acid was performed using a new gas-chromatography/mass spectrometric single-ion method which is sensitive and specific. No abnormalities in the activity of several enzymes involved in sialic acid metabolism (N-acetylneuraminate:pyruvate lyase, neuraminidase, CMP-N-acetylneuraminate N-acylneuraminohydrolase and CTP:N-acyl-neuraminate cytidylyltransferase) were demonstrable. A possible explanation for the defect is a malfunctioning active transport of N-acetylneuraminic acid across the lysosomal membrane.
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2.
  • Elbing, Karin, 1974, et al. (författare)
  • Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae
  • 2004
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:23-24, s. 4855-4864
  • Tidskriftsartikel (refereegranskat)abstract
    • The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.
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3.
  • Eriksson, Torny, et al. (författare)
  • Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module
  • 2004
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:7, s. 1266-1276
  • Tidskriftsartikel (refereegranskat)abstract
    • The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.
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4.
  • Florén, Claes-Henrik, et al. (författare)
  • Effects of fatty acid unsaturation on chylomicron metabolism in normal and hepatectomized rats
  • 1977
  • Ingår i: Eur J Biochem. - : Wiley. - 0014-2956. ; 77:1, s. 23-30
  • Tidskriftsartikel (refereegranskat)abstract
    • 1. Hepatectomized rats were injected intravenously with doubly labelled ([14C]linoleic acid and [3H]palmitic acid) thoracic duct lymphs from rats fed cream, triolein or corn oil. The disappearance of the radioactive fatty acids of different molecular triacylglycerol species and of phospholipids from plasma was studied.2. 73–93% of the injected triacylglycerols had been cleared from plasma within 15 min. At all stages of lipolysis the 3H/14C ratio of the plasma triacylglycerol was the same as in the injected material. If the cream chyle had been cooled to 4 °C before use there was, however, an enrichment of [3H]palmitic acid and of fully saturated triacylglycerols in the remnant particles formed.3. Only 38–50% of the radioactive chyle phosphatidylcholine was eliminated from plasma in 30 min. At this time most of the remaining phosphatidylcholine was, however, in other lipo‐protein classes than the chylomicron remnants.4. Also in intact rats data were obtained, indicating that the major portion of chylomicron phospholipids is transferred to other serum lipoproteins by exchange or net movement rather than being hydrolysed in the 1‐position by lipoprotein lipase or taken up intact by the liver.5. More of both the labelled fatty acids appeared in liver triacylglycerols in experiments with cream chyle than in experiments with corn oil chyle. Data were obtained suggesting that this may be due to a higher uptake of intact triacylglycerol as remnant particles.6. When linoleic acid is fed as a tracer dose in cream, a high proportion (16–36%) is incorporated into chyle phospholipids.
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5.
  • GOLOLOBOV, Mikhail Y., et al. (författare)
  • The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis : Kinetic evidence for the interaction
  • 1993
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 217:3, s. 955-963
  • Tidskriftsartikel (refereegranskat)abstract
    • α‐Chymotrypsin‐catalyzed acyl tranfer was studied using three acyl‐group donors (Mal‐l‐Ala‐l‐Ala‐l‐PheOMe, Bz‐l‐TyrOEt and Ac‐l‐TrpOEt; Mal, maleyl; Bz, benzoyl; OMe, methyl ester; OEt, ethyl ester) and a series of amino‐acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl‐enzyme. The α‐chymotrypsin‐catalyzed transfer of the Mal‐l‐Ala‐l‐Ala‐l‐Phe group to the amides of L‐Phe and L‐Tyr showed a linear dependence of the partition constant, p, on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl‐enzyme–nucleophile complex. The α‐chymotrypsin‐catalyzed transfer of the Bz‐l‐Tyr and Ac‐l‐Trp groups to several amino‐acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl‐enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of α‐chymotrypsin‐catalyzed acyl transfer evident in previous studies.
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6.
  • Kang, J, et al. (författare)
  • Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1 -> 3)-alpha-D-GalNAc
  • 2004
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:23-24, s. 4939-4949
  • Tidskriftsartikel (refereegranskat)abstract
    • The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional H-1 homonuclear and C-13-H-1 heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.
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7.
  • Kvassman, Jan, et al. (författare)
  • Mechanism of glyceraldehyde‐3‐phosphate transfer from aldolase to glyceraldehyde‐3‐phosphate dehydrogenase
  • 1988
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 172:2, s. 427-431
  • 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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8.
  • Larsson, Karin M., et al. (författare)
  • Activity and stability of horse‐liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles
  • 1987
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 166:1, s. 157-161
  • Tidskriftsartikel (refereegranskat)abstract
    • Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan‐3ß‐ol‐17‐one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.
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9.
  • Malm, Johan, et al. (författare)
  • Isolation and characterization of the major gel proteins in human semen, semenogelin I and semenogelin II
  • 1996
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 238:1, s. 48-53
  • Tidskriftsartikel (refereegranskat)abstract
    • Semenogelin I and semenogelin II constitute the major gel-forming proteins in human semen. The gel proteins were rapidly solubilized and separated from spermatozoa in ejaculates collected at pH 9.7 in buffer containing 4 mol/l urea and dithiothreitol. This protected the semenogelins from proteolytic degradation by prostate-specific antigen, and allowed their isolation by affinity chromatograph:g on heparin-sepharose. Semenogelins I and II were almost selectively retained and eluted partially separated in 0.25 mol/l NaCl. Further purification was achieved by chromatography on Superose. Approximately 10-20 mg semenogelin I and 2-5 mg semenogelin II were recovered from each sample with a purity exceeding 95% as judged by SDS/PAGE. The molecular mass of semenogelin I (49,958 Da) and the major form of semenogelin II (63,539 Da) measured by mass spectrometry was consistent with the reported cDNA data. The occurrence of a second, larger form of semenogelin II was due to asparagine-linked glycosylation. The amino-termini of the purified proteins were blocked, but digestion with pyroglutamate aminopeptidase enabled the identification of amino-terminal sequences consistent with the reported cDNA data. The amino acid compositions of the purified proteins were also consistent with those derived from cDNA data. The absorption coefficients (280 nm, 1%, 1 cm) for semenoaelins I and II. were 5.5 and 5.4, respectively, and the isoelectric point was above pH 9.5 for both proteins.
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10.
  • Martinez Arias, Wilma, et al. (författare)
  • Mechanism of NADH Transfer between Alcohol Dehydrogenase and Glyceraldehyde-3-Phosphate Dehydrogenase
  • 1997
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 250:1, s. 158-162
  • Tidskriftsartikel (refereegranskat)abstract
    • Steady-state and transient-state kinetic experiments have been performed to test the proposal that there is a direct (channelled) transfer of NADH from glyceraldehyde-3-phosphate dehydrogenase to alcohol dehydrogenase. The results lend no support to this proposal, but can be best explained in terms of a free-diffusion mechanism for NADH transfer between the two enzymes.
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11.
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12.
  • Morimatsu, K., et al. (författare)
  • Roles of Tyr103 and Tyr264 in the regulation of RecA-DNA interactions by nucleotide cofactors
  • 1996
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 240:1, s. 91-97
  • Tidskriftsartikel (refereegranskat)abstract
    • The DNA-binding mode of the RecA protein, in particular its dependence on nucleotide cofactor, has been investigated by monitoring the fluorescence and linear-dichroism signals of a tryptophan residue inserted in the RecA to replace tyrosine at position 103 or 264. These residues are important for cofactor and DNA binding, as evidenced from their fluorescence changes upon binding of cofactor and DNA [Morimatsu, K., Horii, T, & Takahashi, M. (1995) Eur. J. Biochem. 228, 779-785]. The substitution of these residues with tryptophan does not affect the structure or biological function of the complex and can therefore be exploited to gain structural information in terms of the orientation and environment of the inserted reporter chromophore. The fluorescence change upon formation of the ternary cofactor . RecA . DNA complex was much smaller than the sum of the changes induced by cofactor or DNA alone, This difference indicates that the cofactor and DNA interact with RecA via common components. The fluorescence change caused by DNA in the presence of cofactor was almost independent of the base composition of DNA, in contrast to the interaction in the absence of cofactor. Hence, the contact mode between the selected residues and DNA in the complex may depend significantly on the cofactor, Linear-dichroism measurements indicate that the cofactor does not markedly alter the organization of RecA filament. Linear dichroism shows that neither the aromatic moiety of residue 103 nor that of residue 264 is intercalated between the DNA bases. The textural changes reported for the helical pitch and contour length of RecA fiber upon interaction with cofactor and DNA may derive from a subtle change in orientation of the RecA subunits in the filament.
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13.
  • Nicolaes, GAF, et al. (författare)
  • Altered inactivation pathway of factor Va by activated protein C in the presence of heparin
  • 2004
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:13, s. 2724-2736
  • Tidskriftsartikel (refereegranskat)abstract
    • Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'(306)) or after cleavage at Arg506 (k(506)) and subsequent cleavage at Arg306 (k(306)). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k(506) by UFH was 12-fold, with the secondary cleavage at Arg306 (k(306)) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'(306)) two- to threefold. Low molecular weight heparin (Fragmin(R)) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.
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14.
  • Pettersson, Gösta, et al. (författare)
  • A rapid‐equilibrium model for the control of the Calvin photosynthesis cycle by cytosolic orthophosphate
  • 1987
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 169:2, s. 423-429
  • Tidskriftsartikel (refereegranskat)abstract
    • A simple model based on rapid‐equilibrium assumptions is derived which relates the steady‐state activity of the Calvin cycle for photosynthetic carbohydrate formation in C3 plants to the kinetic properties of a single cycle enzyme (fructose bisphosphatase) and of the phosphate translocator which accounts for the export of photosynthate from the chloroplast. Depending on the kinetic interplay of these two catalysts, the model system may exhibit a single or two distinct modes of steady‐state operation, or may be unable to reach a steady state. The predictions of the model are analysed with regard to the effect of external orthophosphate on the steady‐state rate of photosynthesis in isolated chloroplasts under conditions of saturating light and CO2. Due to the possible existence of two distinct steady states, the model may account for the stimulatory as well as the inhibitory effects of external phosphate observed in experiments with intact chloroplasts. Stability arguments indicate, however, that only the steady‐state case corresponding to phosphate inhibition of the rate of photosynthesis could be of physiological interest. It is concluded that chloroplasts under physiological conditions most likely operate in a high‐velocity steady state characterized by a negative Calvin cycle flux control coefficient for the phosphate translocator. This means that any factor enhancing the export capacity of the phosphate translocator can be anticipated to decrease the actual steady‐state rate of photosynthate export due to a decreased steady‐state rate of cyelic photosynthate production.
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15.
  • Pettersson, Gösta, et al. (författare)
  • Dependence of the Calvin cycle activity on kinetic parameters for the interaction of non‐equilibrium cycle enzymes with their substrates
  • 1989
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 186:3, s. 683-687
  • Tidskriftsartikel (refereegranskat)abstract
    • Kinetic model studies and control analyses of the Calvin photosynthesis cycle have been performed to characterize the dependence of the cycle activity on maximum velocities and Kmvalues for the interaction of the non‐equilibrium cycle enzymes and ATP synthetase with their substrates under conditions of light and carbon dioxide saturation. The results show that Km values have no major influence on the cycle activity at optimal concentrations of external orthophosphate. The maximum cycle activity is controlled mainly by the catalytic capacities of ATP synthetase and sedoheptulose‐bisphosphatase, and is close to the maximum cycle flux that can be supported by these two enzymes.
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16.
  • Pettersson, Gösta, et al. (författare)
  • Metabolites controlling the rate of starch synthesis in the chloroplast of C3 plants
  • 1989
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 179:1, s. 169-172
  • Tidskriftsartikel (refereegranskat)abstract
    • The extent to which different stromal metabolites affecting ADPglucose pyrophosphorylase control the rate of photosynthetic starch production in the chloroplast of C3 plants has been examined by kinetic model studies. The results indicate that ATP, glucose 1‐phosphate, 3‐phosphoglycerate, fructose 6‐phosphate, and orthophosphate may provide significant contributions to the starch synthesis rate changes induced by variation of the external concentration of orthophosphate, the detailed control situation being dependent on the actual concentration of the external metabolite.
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17.
  • Pettersson, Gösta, et al. (författare)
  • On the regulatory significance of inhibitors acting on non‐equilibrium enzymes in the Calvin photosynthesis cycle
  • 1989
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 182:2, s. 373-377
  • Tidskriftsartikel (refereegranskat)abstract
    • Control analyses and kinetic model studies have been performed in order to obtain quantitative information on the regulatory significance of 12 experimentally well‐documented inhibitory interactions of Calvin cycle intermediates with the four non‐equilibrium cycle enzymes. Evidence is presented to show that none of these interactions contributes significantly to the cycle flux control over the range of external orthophosphate concentrations where the reaction cycle shows close to optimal activity. Contrary to what has been generally supposed, the examined inhibitions appear to be of little interest for our understanding of the biological regulation of the Calvin photosynthesis cycle under conditions of light and carbon dioxide saturation.
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18.
  • Rasmusson, A. G., et al. (författare)
  • Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria
  • 1991
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 202:2, s. 617-623
  • Tidskriftsartikel (refereegranskat)abstract
    • Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD+-dependent and NADP+-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca2+-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The K(m) for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The V(max) was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 μM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.
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19.
  • RESLOW, Mats, et al. (författare)
  • On the importance of the support material for bioorganic synthesis : Influence of water partition between solvent, enzyme and solid support in water‐poor reaction media
  • 1988
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 172:3, s. 573-578
  • Tidskriftsartikel (refereegranskat)abstract
    • α‐Chymotrypsin was adsorbed on solid support materials and the catalytic activity of the preparations in organic solvents was studied. The activity was highly dependent on the nature of the support material and on the amount of water present in the reaction mixture. There appears to be competition for the water in the system between the enzyme, the support material and the solvent. The support materials were characterized by measuring their ability to absorb water from water‐saturated diisopropyl ether. For the quotient: (amount of water on the support)/(amount of water in the solvent) in the model system the term aquaphilicity was proposed. The activity of adsorbed chymotrypsin in diisopropyl ether decreased with increasing aquaphilicity of the support material. The same trend was observed when the activity of horse liver alcohol dehydrogenase adsorbed on different supports was measured in diisopropyl ether.
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20.
  • Rosén, Stefan, et al. (författare)
  • A multispecific saline-soluble lectin from the parasitic fungus Arthrobotrys oligospora. Similarities in the binding specificities compared with a lectin from the mushroom agaricus bisporus.
  • 1996
  • Ingår i: European journal of biochemistry / FEBS. - : Wiley. - 0014-2956 .- 1432-1033. ; 238:3, s. 830-7
  • Tidskriftsartikel (refereegranskat)abstract
    • Several fungi can express high levels of saline-soluble and low-molecular-mass lectins that bind to glycoproteins such as fetuin and different mucins but not bind to any monosaccharides. In this paper, we report the binding specificities of such a lectin (designated AOL) isolated from the nematophagous fungus Arthrobotrys oligospora. The results show that AOL is a multispecific lectin that interacts with the following ligands: (a) Several sulfated glycoconjugates including sulfatide, dextran sulfate, and fucoidan. The specificity of this binding was indicated by experiments showing that none of the tested neutral- and sialic-acid-containing glycolipids, chondroitin sulfates B and C, heparin, and polyvinyl sulfate bound to AOL; (b) Phosphatidic acid and phospatidylglycerol, two out of several tested phospholipids. (c) N-linked and O-linked sugar chains bound to intact fetuin. The involvement of such sugar structures was demonstrated by analyzing the binding of AOL to chemically deglycosylated (trifluoromethanesulfonic acid) fetuin. Treating fetuin with O-glycosidase and N-glycosidase indicated that AOL bound to Gal beta GaLNAc alpha-Ser/Thr and to some N-linked complex sugars, respectively. Further assays demonstrated that AOL could interact with several other glycoproteins containing O-linked and/or N-linked sugar chains. The observations that AOL did not bind to free N-linked sugars isolated from fetuin, or to fetuin treated with trypsin or pronase, or to any of the tested neoglycoproteins and glycolipids with neutral- or sialic acid-containing sugars, indicated that the sugar chains need to be bound to an intact peptide backbone to interact with AOL. We have recently shown that the deduced primary structure of AOL has a high similarity to the sequence of a saline-soluble lectin isolated from the mushroom Agaricus bisporus (ABL) (Rosén, S., Kata, M., Persson, Y., Lipniunas, P. H., Wikström, M., van den Hondel, C. A. M. J. J., van den Brink, J. M., Rask, L., Hedén L.-O. and Tunlid, A., see companion paper). It is well known that ABL binds to Gal beta 3GaLNAc alpha-Ser/Thr, and in this paper we demonstrate that ABL binds to sulfatide, phosphatidic acid, phospatidylglycerol, and possibly also to the same N-linked complex sugars as AOL. The above data indicate that AOL and ABL are members of a novel family of fungal lectins sharing similar primary structure and binding properties.
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21.
  • Rosén, Stefan, et al. (författare)
  • Molecular characterization of a saline-soluble lectin from a parasitic fungus : Extensive sequence similarities between fungal lectins
  • 1996
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 238:3, s. 822-829
  • Tidskriftsartikel (refereegranskat)abstract
    • It has been proposed that the interactions between several parasite and pathogenic fungi and their hosts are mediated by soluble lectins present in the fungus. We have cloned and analyzed a gene encoding such a lectin (AOL) from the nematophagous fungus Arthrobotrys oligospora (deuteromycete). The deduced primary structure of the AOL gene displayed an extensive similarity (identity 46.3%) to that of a gene encoding a lectin (ABL) recently isolated from the mushroom Agaricus bisporus (basidiomycete), but not to any other fungal, microbial, plant, or animal lectins. The similarities between AOL and ABL were further demonstrated by the observation that an antibody specific for AOL cross-reacted with ABL. Together with data showing that AOL has a binding specificity that is similar to that of ABL [Rosen, S., Bergstrom, J., Karlsson, K.-A., and Tunlid, A. (1996) Eur. J. Biochem. 238, 830-837], these results indicate that AOL and ABL are members of a novel family of saline- soluble lectins present in fungi. Southern blots indicated that there is only one AOL gene in the genome encoding a subunit (monomer) of the lectin. The primary structure of AOL did not show the presence of a typical N-terminal signal sequence. Comparison of the deduced primary structure with the molecular mass of AOL as determined by electrospray mass spectrometry (16153 Da), indicated that AOL has an acetylated N-terminal but no other post- translational modifications, and that a minor isoform is formed by deamidation. Circular dichroism (CD) spectroscopy suggested that the secondary structure of AOl contains 34% β-sheets, 21% α-helix, and 45% turns and coils.
  •  
22.
  • Ryde-Pettersson, Ulf (författare)
  • A theoretical treatment of damped oscillations in the transient state kinetics of single‐enzyme reactions
  • 1989
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 186:1-2, s. 145-148
  • Tidskriftsartikel (refereegranskat)abstract
    • An extension of the available kinetic theory for reactions in the transient state is presented which establishes that single‐enzyme reactions may exhibit damped oscillations under the conditions of standard kinetic experiments performed by stopped‐flow techniques. Such oscillations may occur for reasonable magnitudes of rate constants in the enzymic reaction mechanism and at physiological concentrations of enzyme and substrate. In the simplest reaction systems, the oscillations will be strongly damped and lead to progress curves resembling those of a reaction governed by standard exponential transients; statistical regression methods may then have to be applied for their detection and characterization. The observation that single‐enzyme reactions may exhibit oscillatory behaviour points to a previously unrecognized possible source of the damped oscillations observed in metabolic systems such as the pathways of glycolysis or photosynthesis.
  •  
23.
  • Ryde-Pettersson, Ulf (författare)
  • Identification of possible two‐reactant sources of oscillations in the Calvin photosynthesis cycle and ancillary pathways
  • 1991
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 198:3, s. 613-619
  • Tidskriftsartikel (refereegranskat)abstract
    • A systematic search for possible sources of experimentally observed oscillations in the photosynthetic reaction system has been performed by application of recent theoretical results characterizing the transient‐state rate behaviour of metabolic reactions involving two independent concentration variables. All subsystems involving two independent reactants in metabolically fundamental parts of the Calvin cycle and the ancillary pathways of starch and sucrose synthesis have been examined in order to decide on basis of their kinetic and stoichiometric structure whether or not they may trigger oscillations. The results show that no less than 20 possible oscillators can be identified in the examined reaction system, only three of which have been previously considered as potential sources of experimentally observed oscillations. This illustrates the superiority of the method now applied over those previously used to identify possible two‐reactant sources of metabolic oscillations and indicates that there should be no difficulty in complex metabolic pathways to point to a multitude of interactions that may trigger an oscillatory rate behaviour of the system.
  •  
24.
  • Ryde-Pettersson, Ulf (författare)
  • On the mechanistic origin of damped oscillations in biochemical reaction systems
  • 1990
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 194:2, s. 431-436
  • Tidskriftsartikel (refereegranskat)abstract
    • A generalized reaction scheme for the kinetic interaction of two reactants in a metabolic pathway has been examined in order to establish what minimal mechanistic patterns are required to support a damped oscillatory transient‐state kinetic behaviour of such a two‐component system when operating near a steady state. All potentially oscillating sub‐systems inherent in this scheme are listed and briefly characterized. The list includes several mechanistic patterns that may be frequently encountered in biological system (e.g. involving feedback inhibition, feed‐forward activation, substrate inhibition or product activation), but also draw attention to some hitherto unforeseen mechanisms by which the kinetic interaction of two metabolites may trigger damped oscillations. The results can be used to identify possible sources of oscillations in metabolic pathways without detailed knowledge about the explicit rate equations that apply.
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25.
  • Schmidtchen, Artur, et al. (författare)
  • Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts
  • 1992
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 208:2, s. 537-546
  • Tidskriftsartikel (refereegranskat)abstract
    • 1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmstrom, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.
  •  
26.
  • Takahashi, Masayuki, 1949, et al. (författare)
  • COORDINATION AND INTERNAL EXCHANGE OF 2 DNA-MOLECULES IN A RECA FILAMENT IN THE PRESENCE OF HYDROLYZING ATP - INFORMATION ON ATP-RECA-DNA STRUCTURE FROM LINEAR DICHROISM SPECTROSCOPY
  • 1992
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 210:1, s. 87-92
  • Tidskriftsartikel (refereegranskat)abstract
    • Solution structure of complexes between DNA and recombinase RecA from Escherchia coli, in the presence of the physiological cofactor ATP, is probed by flow linear dichroism (LD) spectroscopy. A problem of ADP accumulation which promotes dissociation of DNA-RecA is circumvented by using an ATP-regenerating system. The LD features indicate that the local structure of the complex is very similar to that found in the presence of the non-hydrolysable analog of ATP, adenosine-5'-O-[gamma-thio]triphosphate (ATP[gammaS]); the DNA bases are oriented with their planes preferentially perpendicular to the long axis of the filament, while the indole chromophores of the two tryptophan residues of RecA are rather parallel to this reference direction. A much smaller overall amplitude of the LD spectrum, compared to ATP[gammaS], is interpreted as a result of fast dissociation of RecA due to hydrolysis of ATP, producing transiently naked DNA regions which act like flexible joints, diminishing the macroscopic orientation of the RecA filaments. However, the ATP hydrolysis is not found to prevent simultaneous accommodation of two non-complementary DNA molecules in the RecA complex, as judged from the LD behaviour upon successive addition of two different polynucleotides or modified DNA strands. A notable difference from corresponding complexes formed with ATP[gammaS] is that, in the presence of ATP hydrolysis, the order in which the two DNA molecules have been added is insignificant as judged from virtually identical resulting structures; this observation indicates that exchange of DNA occurs between the two DNA accommodation sites within the RecA filament.
  •  
27.
  • Takahashi, M., et al. (författare)
  • Locations of functional domains in the RecA protein - Overlap of domains and regulation of activities
  • 1996
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 242:1, s. 20-28
  • Tidskriftsartikel (refereegranskat)abstract
    • `We review the locations of various functional domains of the RecA protein of Escherichia coli, including how they have been assigned, and discuss the potential regulatory roles of spatial overlap between different domains. RecA is a multifunctional and ubiquitous protein involved both in general genetic recombination and in DNA repair: it regulates the synthesis and activity of DNA repair enzymes (SOS induction) and catalyses homologous recombination and mutagenesis. For these activities RecA interacts with a nucleotide cofactor. single-stranded and double-stranded DNAs, the LexA repressor, UmuD protein, the UmuD(2)'C complex as well as with RecA itself in forming the catalytically active nucleofilament. Attempts to locate the respective interaction sites have been advanced in order to understand the various functions of RecA. An intriguing question is how these numerous functional sites an contained within this rather small protein (38 kDa). To assess more clearly the roles of the respective sites and to what extent the sites may be interacting with each other, we review and compare the results obtained from various biological, biochemical and physico-chemical approaches. From a three-dimensional model it is concluded that all sites are concentrated to one part of the protein. As a consequence there are significant overlaps between the sites and it is speculated that corresponding interactions may play important roles in regulating RecA activities.
  •  
28.
  • Tossavainen, H, et al. (författare)
  • NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea
  • 2003
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:11, s. 2505-2512
  • Tidskriftsartikel (refereegranskat)abstract
    • The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.
  •  
29.
  • von Wachenfeldt, Claes, et al. (författare)
  • Physico-chemical characterisation of membrane-bound and water-soluble forms of Bacillus subtilis cytochrome c-550
  • 1993
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033.
  • Tidskriftsartikel (refereegranskat)abstract
    • Cytochrome c-550 of the Gram-positive bacterium, Bacillus subtilis, is a membrane-bound 13-kDa protein encoded by the cccA gene. The cytochrome has been proposed to be comprised of an N-terminal membrane anchor domain (about 30 residues) which spans the cytoplasmic membrane in an alpha-helical conformation and a C-terminal heme domain (about 70 residues) which is located on the outside of the cytoplasmic membrane. Cytochrome c-550 was purified in the presence of Triton X-100 and characterised. In the reduced state it shows absorption maxima at 415, 521, 550 nm and in the oxidised state a Soret band at 408 nm and a weak band at about 695 nm. The latter absorption band, together with data from amino acid sequence comparisons, strongly suggest His64 and Met99 as the fifth and sixth axial ligands to the heme iron in cytochrome c-550. The midpoint redox potential of the cytochrome, +178 mV, was pH-independent in the pH range 6.0-7.9. Oxidised cytochrome c-550 showed an EPR signal at g(max) = 3.41, which is unusual for low-spin cytochromes c with His/Met axial ligation. The heme domain was isolated as a tryptic fragment of 74 residues and as a protein-A-cytochrome-c-550 hybrid protein. Both these forms were water-soluble and showed thermodynamic and spectroscopic properties indistinguishable from the membrane-bound form of cytochrome c-550 and are suitable for structural analysis of the heme domain by X-ray crystallography or NMR techniques. Polypeptide analysis of the membrane-bound and water-soluble tryptic fragment confirmed that B. subtilis cytochrome c-550 in the membrane consists of 120 amino acid residues and has a two-domain structure.
  •  
30.
  • Westergren-Thorsson, Gunilla, et al. (författare)
  • Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts
  • 1992
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 205:1, s. 277-286
  • Tidskriftsartikel (refereegranskat)abstract
    • Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-beta increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400-500 kDa) increased 5-7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3-4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.
  •  
31.
  • Wittung, Pernilla, 1968, et al. (författare)
  • Thermochemical and kinetic evidence for nucleotide-sequence-dependent RecA-DNA interactions
  • 1997
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 245:3, s. 715-719
  • Tidskriftsartikel (refereegranskat)abstract
    • RecA catalyses homologous recombination in Escherichia coli by promoting pairing of homologous DNA molecules after formation of a helical nucleoprotein filament with single-stranded DNA. The primary reaction of RecA with DNA is generally assumed to be unspecific. We show here, by direct measurement of the interaction enthalpy by means of isothermal titration calorimetry, that the polymerisation of RecA on single-stranded DNA depends on the DNA sequence, with a high exothermic preference for thymine bases. This enthalpic sequence preference of thymines by RecA correlates with faster binding kinetics of RecA to thymine DNA. Furthermore, the enthalpy of interaction between the RecA.DNA filament and a second DNA strand is large only when the added DNA is complementary to the bound DNA in RecA. This result suggests a possibility for a rapid search mechanism by RecA.DNA filaments for homologous DNA molecules.
  •  
32.
  •  
33.
  • Henriksson, Maria, et al. (författare)
  • A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo
  • 2002
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:20, s. 4921-4929
  • 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.
  •  
34.
  • Tengel, Tobias, et al. (författare)
  • Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia
  • 2002
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:15, s. 3659-3668
  • Tidskriftsartikel (refereegranskat)abstract
    • To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278−300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic α helix ranging from Phe280 to Tyr291, followed by a type I β turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278−292 and YopD271−292, lacked helical structure, implicating the β turn in helix stability. An interaction between YopD278−300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide–LcrH complex. These effects were not observed for the unstructured peptide, YopD278−292, which confirms that the α helical structure of the YopD amphipathic domain is a critical binding region of LcrH.
  •  
35.
  • Ellouze, C., et al. (författare)
  • Evidence for Elongation of the Helical Pitch of the RecA Filament Upon ATP and ADP Binding Using Small-Angle Neutron Scattering
  • 1995
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 233:2, s. 579-583
  • Tidskriftsartikel (refereegranskat)abstract
    • Structural changes of the RecA filament upon binding of cofactors have been investigated by small-angle neutron scattering. Both ATP and ADP increased the helical pitch of the RecA homopolymer, which is observed to be 7 nm in the absence of any cofactor. The binding of ATP altered the pitch to 9 nm, whereas the binding of ADP only produced a pitch of 8.2 nm. The pitch determined for the RecA complex with the ATP analog adenosine 5'-[gamma-thio]triphosphate was similar to that found with ATP. Thus, at least three, somewhat different, RecA helical filamentous structures may form in solution. The binding of DNA to RecA did not alter the pitch significantly, indicating that the cofactor binding is the determining factor for the size of the helical pitch of the RecA filament. We also found that elongation of the helical pitch is a necessary, but not a sufficient condition, for the coprotease activity of RecA. The presence of acetate or glutamate ions is also required. The pitch of the ADP . RecA filament is in agreement with that found in the crystal structure. This correlation indicates that this structure corresponds to that of the ADP . RecA filament in solution, although this is not the species active in recombination.
  •  
36.
  • Moses, J, et al. (författare)
  • Biosynthesis of the proteoglycan decorin -- identification of intermediates in galactosaminoglycan assembly
  • 1997
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 248:3, s. 767-774
  • Tidskriftsartikel (refereegranskat)abstract
    • Biosynthesis of decorin was investigated by incubating a rat fibroblast cell line with various radiolabelled protein and galactosaminoglycan precursors. The following cell-associated and distinct intermediates were isolated and identified: a pool of non-glycosylated core protein, two pools of decorin with incomplete chains, one with three sulphated disaccharide repeats and another with five or more sulphated disaccharide repeats, as well as decorin with mature chains. Results of pulse/chase experiments indicated that these pools represented discrete stages in chain growth. Treatment with brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi, resulted in accumulation of decorin with an incomplete chain containing six or seven largely unsulphated disaccharide repeats. During recovery from drug treatment, 4-sulfation reappeared earlier than 6-sulfation. The results suggest that the galactosaminoglycan assembly-line consists of separate multienzyme complexes that build only a limited section of the chain. Furthermore, brefeldin A causes segregation of compartments involved in separate stages of the assembly line. In an earlier report [Moses, J., Oldberg. A., Cheng, F. & Fransson, L.-A. (1997) Eur. J. Biochem. 248, 521-526] we took advantage of such segregation to identify and characterize a transient 2-phosphorylation of xylose in the linkage region.
  •  
37.
  • ADLERCREUTZ, Patrick (författare)
  • On the importance of the support material for enzymatic synthesis in organic media : Support effects at controlled water activity
  • 1991
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 199:3, s. 609-614
  • Tidskriftsartikel (refereegranskat)abstract
    • Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the α‐chymotrypsin‐catalyzed alcoholysis of N‐acetyl‐l‐phenylalanine ethyl ester with 1‐butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose‐CPG was a much better support than hexyl‐CPG, and in the α‐chymotrypsin‐catalyzed reactions, glucose‐CPG favored hydrolysis, and hexyl‐CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.
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38.
  • Berggård, T, et al. (författare)
  • Prothrombin, albumin and immunoglobulin A form covalent complexes with alpha1-microglobulin in human plasma
  • 1997
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 245:3, s. 83-676
  • Tidskriftsartikel (refereegranskat)abstract
    • Molecules containing the 33-kDa plasma protein alpha1-microglobulin were isolated from human plasma by anti-(alpha1-microglobulin) affinity chromatography. Five major bands could be seen after electrophoretic separation of the alpha1-microglobulin-containing proteins under native conditions. Immunoblotting demonstrated alpha1-microglobulin in all five bands. Two of these have been described previously: free alpha1-microglobulin and alpha1-microglobulin complexed with IgA (IgA x alpha1-microglobulin). The other three bands were identified as prothrombin alpha1-microglobulin, albumin x alpha1-microglobulin and dimeric alpha1-microglobulin. Prothrombin x alpha1-microglobulin were 1:2 and 1:1 complexes which carried approximately 1% of total alpha1-microglobulin, had molecular masses of about 145 kDa and 110 kDa upon SDS/PAGE and dissociated completely to free alpha1-microglobulin and prothrombin (72 kDa) when reducing agents were added, suggesting that the complexes were stabilized by disulfide bonds. The alpha1-microglobulin molecules did not inhibit cleavage of prothrombin by factor Xa and were bound to the peptides which were released upon activation of prothrombin. Albumin x alpha1-microglobulin, corresponding to 7% of total plasma alpha1-microglobulin, was a mixture between 1:1 and 1:2 complexes, with masses upon SDS/PAGE of approximately 100 kDa and 135 kDa, respectively. Both these complexes dissociated only partially to free alpha1-microglobulin and albumin when reducing agents were added. The albumin x alpha1-microglobulin complexes carried a yellow-brown chromophore similar to free alpha1-microglobulin. The complex-binding to alpha1-microglobulin did not block the fatty-acid-binding ability of albumin. The plasma concentrations of albumin x alpha1-microglobulin and prothrombin x alpha1-microglobulin were estimated to 5.2 mg/l and 1.1 mg/l, respectively.
  •  
39.
  • CHRISTENSSON, Anders, et al. (författare)
  • Complex formation between protein C inhibitor and prostate‐specific antigen in vitro and in human semen
  • 1994
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 220:1, s. 45-53
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein C inhibitor (PCI), a serine‐proteinase inhibitor first purified from human blood plasma, occurs at high concentrations (3–4 μM) in seminal fluid in both a high‐molecular‐mass and low‐molecular‐mass form. Immunochemical data have previously suggested that PCI in seminal plasma forms complexes with the most abundant serine proteinase in semen, prostate‐specific antigen (PSA). To provide a structural characterization of the PCI target, immunodetected as PSA, a procedure was developed to isolate low‐molecular‐mass and high‐molecular‐mass‐forms of PCI from seminal fluid. The high‐molecular‐mass form of PCI, recognized by monoclonal antibodies against PSA, was dissociated by alkaline treatment into the low‐molecular‐mass form of PCI and a 33‐kDa protein identified as PSA by 25 conclusive steps of N‐terminal sequence analysis. We developed a sensitive immunofluorometric assay (IFMA) to measure PCI‐PSA complexes in body fluids and investigated the rate at which purified PSA may form complexes with purified PCI. Formation of complexes detected by this IFMA and the appearance of SDS‐stable approximately 90‐kDa complexes paralleled loss of PSA activity recorded with chromogenic substrates. The rate of complex formation was slow compared to that reported for PCI and activated protein C, but was enhanced up to sixfold in the presence of heparin. Less than 10% of the initial PSA activity remained after 3 h incubation with a sevenfold molar excess of PCI and in the presence of heparin. In freshly collected ejaculates, the rate of PCI‐PSA complex formation measured by IFMA was similar to that observed between the purified proteins, and paralleled the appearance of SDS‐stable complexes by immunoblotting. During gel dissolution in freshly collected ejaculates, approximately 40% of immunodetected PCI becomes complexed to PSA. Although PCI is a slow inhibitor of PSA, complexes between PCI and PSA are detected at levels that correspond to an inactivation of up to 5% of the PSA activity in the ejaculate.
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40.
  •  
41.
  • Fridén, H, et al. (författare)
  • Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558
  • 1987
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 168, s. 695-701
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His-113 excludes this residue as an axial heme ligand. A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B. subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.
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42.
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43.
  • Hansson, Mats, et al. (författare)
  • Purification and characterisation of a water-soluble ferrochelatase from Bacillus subtilis
  • 1994
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 220:1, s. 201-208
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacillus subtilis ferrochelatase is encoded by the hemH gene of the hemEHY gene cluster and catalyses the incorporation of Fe2+ into protoporphyrin IX. B. subtilis ferrochelatase produced in Escherichia coli was purified. It was found to be a monomeric, water-soluble enzyme of molecular mass 35 kDa which in addition to Fe2+ can incorporate Zn2+ and Cu2+ into protoporphyrin IX. Chemical modification experiments indicated that the single cysteine residue in the ferrochelatase is required for enzyme activity although it is not a conserved residue compared to other ferrochelatases. In growing B. subtilis, the ferrochelatase constitutes approximately 0.05% (by mass) of the total cell protein, which corresponds to some 600 ferrochelatase molecules/cell. The turnover number of isolated ferrochelatase, 18-29 min-1, was found to be consistent with the rate of haem synthesis in exponentially growing cells (0.2 mol haem formed/min/mol enzyme). It is concluded that the B. subtilis ferrochelatase has enzymic properties which are similar to those of other characterised ferrochelatases of known primary structure, i.e. ferrochelatases of the mitochondrial inner membrane of yeast and mammalian cells. However, in contrast to these enzymes the B. subtilis enzyme is a water-soluble protein and should be more amenable to structural analysis.
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44.
  • Lake, Vanessa, et al. (författare)
  • ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo
  • 2004
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:11, s. 2182-2188
  • Tidskriftsartikel (refereegranskat)abstract
    • During biosynthesis of chlorophyll, Mg2+ is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximate to 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called 'ATPases associated with various cellular activities', known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg2+ and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis.
  •  
45.
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46.
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47.
  • Pettersson, Gösta, et al. (författare)
  • A mathematical model of the Calvin photosynthesis cycle
  • 1988
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 175:3, s. 661-672
  • Tidskriftsartikel (refereegranskat)abstract
    • A mathematical model is presented for photosynthetic carbohydrate formation in C3 plants under conditions of light and carbon dioxide saturation. The model considers reactions of the Calvin cycle with triose phosphate export and starch production as main output processes, and treats concentrations of NADPH, NAD+, CO2, and H+ as fixed parameters of the system. Using equilibrium approximations for all reaction steps close to equilibrium, steady‐state and transient‐state relationships are derived which may be used for calculation of reaction fluxes and concentrations of the 13 carbohydrate cycle intermediates, glucose 6‐phosphate, glucose 1‐phosphate, ATP, ADP, and inorganic (Ortho)phosphate. Predictions of the model were examined with the assumption that photosynthate export from the chloroplast occurs to a medium containing orthophosphate as the only exchangeable metabolite. The results indicate that the Calvin cycle may operate in a single dynamically stable steady state when the external concentration of orthophosphate does not exceed 1.9 mM. At higher concentrations of the external metabolite, the reaction system exhibits overload breakdown; the excessive rate of photosynthate export deprives the system of cycle intermediates such that the cycle activity progressively approaches zero. Reactant concentrations calculated for the stable steady state that may obtain are in satisfactory agreement with those observed experimentally, and the model accounts with surprising accuracy for experimentally observed effects of external orthophosphate on the steady‐state cycle activity and rate of starch production. Control analyses are reported which show that most of the non‐equilibrium enzymes in the system have a strong regulatory influence on the steady‐state level of all of the cycle intermediates. Substrate concentration control coefficients for cycle enzymes may be positive, such that an increase in activity of an enzyme may raise the steady‐state concentration of the substrate is consumes. Under optimal external conditions (0.15–0.5 mM orthophosphate), reaction flux in the Calvin cycle is controlled mainly by ATP synthetase and sedoheptulose bisphosphatase; the cycle activity approaches the maximum velocity that can be supported by the latter enzyme. At lower concentrations of external orthophosphate the cycle activity is controlled almost exclusively by the phosphate translocator. At high external orthophosphate concentrations the phosphate translocator resumes predominant control, but also other non‐equilibrium enzymes gain strong flux control with one notable exception: ribulosebisphosphate carboxylase has no significant regulatory influence on the cycle activity under conditions of light and CO2 saturation, nor does it control the concentration of any cycle intermediate other than its substrate.
  •  
48.
  • Ramström, Margareta, et al. (författare)
  • A novel mass spectrometric approach to analysis of hormonal peptides in extracts of mouse pancreatic islets
  • 2003
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:15, s. 3146-3152
  • Tidskriftsartikel (refereegranskat)abstract
    • Liquid chromatography mass spectrometry (LC-MS) is a valuable tool in the analysis of proteins and peptides. The combination of LC-MS with different fragmentation methods provides sequence information on components in complex mixtures. In this work, on-line packed capillary LC electrospray ionization Fourier transform ion cyclotron resonance MS was combined with two complementary fragmentation techniques, i.e. nozzle-skimmer fragmentation and electron capture dissociation, for the determination of hormonal peptides in an acid ethanol extract of mouse pancreatic islets. The most abundant peptides, those derived from proinsulin and proglucagon, were identified by their masses and additional sequence-tag information established their identities. Interestingly, the experiments demonstrated the presence of truncated C-peptides, des-(25-29)-C-peptide and des-(27-31)-C-peptide. These novel findings clearly illustrate the potential usefulness of the described technique for on-line sequencing and characterization of peptides in tissue extracts.
  •  
49.
  • Schmidtchen, Artur, et al. (författare)
  • Analysis of heparan-sulphate chains and oligosaccharides from proliferating and quiescent fibroblasts. A proposed model for endoheparanase activity
  • 1994
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 223:1, s. 211-221
  • Tidskriftsartikel (refereegranskat)abstract
    • Human skin fibroblasts in different growth states were incubated with [3H]glucosamine and/or Na(2)35SO4 and extracted with Triton X-100 for various periods of time. Free heparan-sulphate oligosaccharides and protein-bound heparan-sulphate chains were separated by chromatography on octyl-Sepharose and analyzed. A pool of endogenously produced oligosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7-10 kDa corresponding to approximately 14-20 disaccharides). Analysis by heparinase I and heparinase III degradations followed by electrophoretic separation (oligosaccharide mapping) showed that the oligosaccharides were rich in glucuronic acid but had a few sulphated iduronic acid residues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages between N-sulphated glucosamine and sulphated iduronic acid, generating fragments that comprise a major portion of the unmodified segments and a minor portion of the highly modified segments. Prolonged extraction (24-48 h) of cells with Triton X-100 at 4 degrees C in the presence of proteinase inhibitors resulted in further degradation. There was an increase in the amount of heparan-sulphate oligosaccharides and a concomitant decrease in the amount of protein-bound heparan-sulphate chains present in the same extract. The heparan-sulphate oligosaccharides obtained after prolonged extraction were more heterogeneous in size comprising, in addition to the major species of approximately 7-10 kDa, intermediate and larger fragments of approximately 17 kDa and 30-40 kDa. This observation suggests that endoheparanase acted at periodically appearing, specific regions in the intact heparan-sulphate chain. Furthermore, the enzyme and substrate should remain closely associated during cold Triton X-100 extraction. To determine if the endogenously produced heparan-sulphate oligosaccharides were derived from a particular heparan-sulphate species degraded during the growth phase, proteoglycan-derived heparan-sulphate chains obtained from proliferating or quiescent fibroblasts were also examined. These chains showed similar oligosaccharide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restricted specificity may generate slightly different oligosaccharides in the various growth states.
  •  
50.
  • Svensson, Birgitta, et al. (författare)
  • Low spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis
  • 1996
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; , s. 287-295
  • Tidskriftsartikel (refereegranskat)abstract
    • Synthesis of heme A from heme B (protoheme IX) most likely occurs in two steps with heme O as an intermediate. Bacillus subtilis CtaB, an integral membrane protein, functions in farnesylation of heme B to form heme O. CtaA, also a membrane protein, is required for heme A synthesis from heme O and appears to be a monooxygenase and/or a dehydrogenase. Wild-type ctaA and ctaB expressed together from plasmids in B. subtilis resulted in CtaA containing equimolar amounts of low-spin heme B and heme A; this form of CtaA was named cyt ba -CTA. A mutant ctaB gene was identified and characterised. It encodes a truncated CtaB polypeptide. Wild-type ctaA and the mutant ctaB gene on plasmids resulted in CtaA containing mainly low-spin heme B; this variant was named cyt b -CTA. The heme B component in cyt ba -CTA and cyt b -CTA showed identical properties; a mid-point redox potential of +85 mV, an EPR gmax signal at 3.7, and a split α-band light absorption peak. The heme A component in cyt ba -CTA showed a mid-point potential of +242 mV, an EPR gmax signal at 3.5, and the α-band light absorption peak at 585 nm. It is suggested that the CtaA protein contains two heme binding sites, one for heme B and one for substrate heme. The heme B would play a role in electron transfer, i.e. function as a cytochrome, in the monooxygenase and/or dehydrogenase reaction catalysed by CtaA whereas heme O/heme A would be substrate/product.
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