| 1. |
- Hegardt, Cecilia, et al.
(författare)
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Rapid caspase-dependent cell death in cultured human breast cancer cells induced by the polyamine analogue N-1,N-11-diethylnorspermine
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:3, s. 1033-1039
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Tidskriftsartikel (refereegranskat)abstract
- The spen-nine analogue N-1,N-11-diethylnorspermine (DENSPM) efficiently depletes the cellular pools of putrescine, spermidine and spermine by down-regulating the activity of the polyamine biosynthetic enzymes and up-regulating the activity of the catabolic enzyme spermidine/spermine N-1-acetyltransferase (SSAT). In the breast cancer cell line L56Br-Cl. treatment with 10 muM DENSPM induced SSAT activity 60 and 240-fold at 24 and 48 h after seeding. respectively, which resulted in polyamine depletion. Cell proliferation appeared to be totally inhibited and within 48 h of treatment, there was an extensive apoptotic response. Fifty percent of the cells were found in the sub-G(1) region, as determined by flow cytometry, and the presence of apoptotic nuclei was morphologically assessed by fluorescence microscopy. Caspase-3 and caspase-9 activities were significantly elevated 24 h after seeding, At 48 h after seeding, caspase-3 and caspase-9 activities were further elevated and at this time point a significant activation of caspase-8 was also found. The DENSPM-induced cell death was dependent on the activation of the caspases as it was inhibited by the general caspase inhibitor Z-Val-Ala-Asp fluoromethyl ketone. The results are discussed in the fight of the L56Br-Cl cells containing mutated BRCA1 and p53, two genes involved in DNA repair.
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| 2. |
- Karlsson, Johan, et al.
(författare)
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Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 268:24, s. 6498-6507
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Tidskriftsartikel (refereegranskat)abstract
- There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.
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| 3. |
- Lönn, Anna, et al.
(författare)
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Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:1, s. 157-163
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Tidskriftsartikel (refereegranskat)abstract
- Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.
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| 4. |
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| 5. |
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| 6. |
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| 7. |
- Månsson, Martin, et al.
(författare)
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Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:3, s. 808-818
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Tidskriftsartikel (refereegranskat)abstract
- Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS. capillary electrophoresis coupled to ESI-MS. composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. It was found that the LPS contains the common structural element of H. influenzae, L-alpha-D-Hepp-(1 --> 2)-[PEtn --> 6]-L-alpha-D-Hepp-(1 --> 3)-[beta-D-Glcp-(1 --> 4)]-L-alpha-D-Hepp-(1 --> 5)-[PP Etn --> 4]-alpha-Kdop-(2 --> 6)-Lipid A. in which the beta-D-Glcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4. A second acetyl group is located at O-3 of the distal heptose residue (HepIII). HepIII is chain elongated at O-2 by either a beta-D-Glcp residue (major), lactose or sialyllactose (minor, i.e. alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-beta-D-Glcp), where a third minor acetylation site was identified at the glucose residue. Disialylated species were also detected. In addition. a minor substitution of ester-linked glycine at HepIII and Kdo was observed.
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| 8. |
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| 9. |
- Achen, M G, et al.
(författare)
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Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.
- 2000
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 267:9
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.
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| 10. |
- Ademark, Pia, et al.
(författare)
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Cloning and characterisation of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 268:10, s. 2982-2990
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Tidskriftsartikel (refereegranskat)abstract
- α-Galactosidase (EC 3.2.1.22) and β-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an α-galactosidase (AglC) and a β-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. nigerα-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.
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| 11. |
- ADLERCREUTZ, Patrick
(författare)
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On the importance of the support material for enzymatic synthesis in organic media : Support effects at controlled water activity
- 1991
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 199:3, s. 609-614
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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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| 12. |
- Akerstrom, B., et al.
(författare)
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Structural relationship between α1-microglobulin from man, guinea-pig, rat and rabbit
- 1988
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Ingår i: European Journal of Biochemistry. - 0014-2956. ; 170:1-2, s. 143-148
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Tidskriftsartikel (refereegranskat)abstract
- Rabbit α1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Sephadex G-100 affinity chromatography on concanavalin-A - Sepharose and ion-exchange chromatography on DEAE-Sephadex. Rabbit α1-microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. α1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of α1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human α1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit α1-microglobulin, with a gap between each band of 2.6-2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig α1-microglobulin. Our results indicate that human α1-microglobulin is substituted with two N-linked oligosaccharides, while only one is attached to each of the other α1-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N-linked oligosaccharides carry the immunosuppressive activity of α1-microglobulin.
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Andersson, Dick, et al.
(författare)
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Contribution of tryptophan residues to the CD spectrum of the extracellular domain of human tissue factor : Application in folding studies and prediction of secondary structure
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 268:4, s. 1118-1128
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Tidskriftsartikel (refereegranskat)abstract
- The contribution to the circular dichroism (CD) spectrum made by each of the four Trp residues in the extracellular domain of human tissue factor, sTF (s designates soluble), was determined from difference CD spectra. The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated (180-305 nm). The sum of the individual spectra of each Trp residue in the near-UV region was qualitatively identical to the wild-type spectrum, clearly demonstrating that the Trp residues are the major contributors to the spectrum in this wavelength region. Trp CD bands interfere with the peptide bands in the far-UV region, leading to uncertainty in the predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp-free CD spectrum obtained after subtraction of all individual Trp spectra from the wild-type spectrum. The mutated Trp residues were also exploited as intrinsic probes to monitor the formation of local native-like tertiary structure by kinetic near-UV CD measurements. The global folding reaction was followed in parallel with a novel functional assay that registered the recovery of cofactor activity, i.e. stimulation of the amidolytic activity of Factor VIIa. From these measurements, it was found that sTF appears to regain FVIIa cofactor activity before the final side-chain packing of the Trp residues. The combined kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.
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| 17. |
- Andersson, Emma, et al.
(författare)
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Antimicrobial activities of heparin-binding peptides.
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 271:6, s. 1219-1226
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Tidskriftsartikel (refereegranskat)abstract
- Antimicrobial peptides are effector molecules of the innate immune system. We recently showed that the human antimicrobial peptides alpha-defensin and LL-37 bind to glycosaminoglycans (heparin and dermatan sulphate). Here we demonstrate the obverse, i.e. structural motifs associated with heparin affinity (cationicity, amphipaticity, and consensus regions) may confer antimicrobial properties to a given peptide. Thus, heparin-binding peptides derived from laminin isoforms, von Willebrand factor, vitronectin, protein C inhibitor, and fibronectin, exerted antimicrobial activities against Gram-positive and Gram-negative bacteria. Similar results were obtained using heparin-binding peptides derived from complement factor C3 as well as consensus sequences for heparin-binding (Cardin and Weintraub motifs). These sequence motifs, and additional peptides, also killed the fungus Candida albicans. These data will have implications for the search for novel antimicrobial peptides and utilization of heparin-protein interactions should be helpful in the identification and purification of novel antimicrobial peptides from complex biological mixtures. Finally, consensus regions may serve as templates for de novo synthesis of novel antimicrobial molecules.
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| 18. |
- Andersson, H.O., et al.
(författare)
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Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors
- 2003
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:8, s. 1746-1758
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Tidskriftsartikel (refereegranskat)abstract
- HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 Å resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the d-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.
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| 19. |
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| 20. |
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| 21. |
- Antuch, W, et al.
(författare)
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The NMR solution structure of a Kunitz-type proteinase inhibitor from the sea anemone Stichodactyla helianthus.
- 1993
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 212, s. 675-684
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Tidskriftsartikel (refereegranskat)abstract
- The solution structure of a 55-amino-acid Kunitz-type proteinase inhibitor, ShPI, purified from the Caribbean sea anemone Stichodactyla helianthus, was determined by NMR spectroscopy. Nearly complete sequence-specific 1H-NMR assignments were obtained at pH 4.6 and 36 degrees C, and stereo-specific assignments were determined for 23 pairs of diastereotopic substituents. A data set of 666 upper distance limit constraints and 122 dihedral angle constraints collected on this basis was used as input for a structure calculation with the program DIANA. Following energy minimization with the program OPAL, the average root-mean-square diviation (RMSD) of the 20 DIANA conformers used to represent the solution structure relative to the mean structure is 61 pm for all backbone atoms N, C alpha and C', and 106 pm for all heavy atoms of residues 2-53. This high-quality solution structure of ShPI has a nearly identical molecular architecture as the bovine pancreatic trypsin inhibitor (BPTI), despite a mere 35% of sequence similarity between the two proteins. Exchange rates measured for 48 out of the 51 backbone amide protons showed that the positions of 20 slowly exchanging amide protons correlate well with hydrogen bonds involving these protons in the energy-minimized solution structure. The solution structure of ShPI is compared to the four homologous proteins for which the three-dimensional structure is also available.
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| 22. |
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| 23. |
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| 24. |
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| 25. |
- Avliakulov, NK, et al.
(författare)
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Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli
- 2000
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:6, s. 1723-1731
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity. In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro. L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe. The cloned gene was overexpressed and purified as a his-tag fusion protein in E. coli. Radioligand binding experiments, using [?-35S]ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity. Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates. Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues. Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of [?-35S]ATP at subclinical levels. Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and bind strongly to L3 in nearly stoichiometric amounts.
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| 26. |
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| 27. |
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| 28. |
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| 29. |
- Baykov, Alexander A., et al.
(författare)
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Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers
- 1999
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Ingår i: European Journal of Biochemistry. - : Wiley-Blackwell Publishing Inc.. - 0014-2956 .- 1432-1033. ; 260:2, s. 308-317
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Tidskriftsartikel (refereegranskat)abstract
- Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg2+-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg2+-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer–trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.
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| 30. |
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| 31. |
- 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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| 32. |
- Ben Nasr, Abdelhakim, et al.
(författare)
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Streptokinase activates plasminogen bound to human group C and group G streptococci through M-like proteins
- 1994
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Ingår i: European Journal of Biochemistry. - 0014-2956. ; 222:2, s. 76-267
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Tidskriftsartikel (refereegranskat)abstract
- An ability to interact with plasminogen or plasmin could provide micro-organisms with a mechanism for invasion. Thus, group A, C and G streptococci secrete streptokinase which binds and activates plasminogen. Some streptococci also express surface structures which bind plasminogen without causing its activation. Plasminogen-binding surface proteins were extracted from one group C and one group G streptococcal isolate. Both proteins were found to bind plasmin, fibrinogen and serum albumin in addition to plasminogen. Gene fragments encoding the streptococcal proteins were amplified by PCR and were subsequently cloned and expressed in Escherichia coli. DNA sequence determination revealed for both genes open reading frames encoding proteins which contained repetitive domains and a carboxyl-terminal unrepeated region that were typical of M and M-like proteins. Though the amino-terminal regions of the group C and G streptococcal proteins demonstrated a rather high overall similarity between themselves, they were not similar to the variable regions of other M-like proteins with one exception: there was a 46% identity between the first 22 amino acids of the group G streptococcal protein and the corresponding sequence of PAM, the plasminogen-binding M-like protein of type M53 group A streptococci. Like the proteins extracted from the streptococci, the recombinant proteins bound plasminogen, fibrinogen and albumin. The three plasma proteins bound to separate sites on the streptococcal M-like proteins. Plasminogen bound by the group C and G streptococcal proteins was readily activated by streptokinase, providing evidence for a functional link between the secreted plasminogen-activator and proteins exposed on the bacterial surface.
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| 33. |
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| 34. |
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| 35. |
- Berggård, T, et al.
(författare)
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Prothrombin, albumin and immunoglobulin A form covalent complexes with alpha1-microglobulin in human plasma
- 1997
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 245:3, s. 83-676
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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.
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| 36. |
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| 37. |
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| 38. |
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| 39. |
- Blomqvist, M, et al.
(författare)
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Identification of defensins in human lymphocyte nuclei
- 1999
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Ingår i: EUROPEAN JOURNAL OF BIOCHEMISTRY. - : BLACKWELL SCIENCE LTD. - 0014-2956. ; 263:2, s. 312-318
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The cell nucleus plays an essential role in all aspects of cell function and regulation. Most of the nuclear proteins/ peptides are synthesized in the cytoplasm. and transported into the nucleus through the nuclear pore complexes. The nuclear proteins/pep
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| 40. |
- Blomqvist, M, et al.
(författare)
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Identification of defensins in human lymphocyte nuclei
- 1999
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 263:2, s. 312-8
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Tidskriftsartikel (refereegranskat)abstract
- The cell nucleus plays an essential role in all aspects of cell function and regulation. Most of the nuclear proteins/peptides are synthesized in the cytoplasm and transported into the nucleus through the nuclear pore complexes. The nuclear proteins/peptides conjugate with each other and interact in transcriptional activation/inactivation. Several of the high molecular mass transcription factors (> 30 kDa) have been identified and characterized. However, the information on the low molecular mass proteins/peptides of the nucleus is limited. We have investigated these low molecular mass proteins/peptides from the nucleus of human peripheral blood lymphocytes using reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC fractions were further analysed by matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, electrospray ionization time of flight (ESI-TOF) mass spectrometry and electrospray ionization fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry for mass determination. Using this combination of mass spectrometry techniques and microsequence analysis, we have shown that human lymphocyte nuclei contain defensins, a mixture of human neutrophil granule peptide 1, 2 and 3.
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| 41. |
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| 42. |
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| 43. |
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| 44. |
- Brodelius, Maria, et al.
(författare)
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Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:14, s. 3570-3577
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Tidskriftsartikel (refereegranskat)abstract
- A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.
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| 45. |
- Brodelius, Peter, et al.
(författare)
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The Synthesis of Three AMP-Analogues: N6-(6-Amino-hexyl)-Adenosine 5'-Monophosphate, N6-(6-Aminohexyl)-Adenosine 2',5'-Bisphosphate, and N6-(6-Aminohexyl)-Adenosine 3',5'-Bisphosphate and Their Application as General Ligands in Biospecific Affinity Chromatography
- 1974
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 47, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- Phosphorylation of 6-chloropurine riboside with phosphorus oxychloride and phosphorus trichloride gave a mixture of the two isomers, 6-chloropurine-riboside 2’,5‘-bisphosphate and 6-chloropurine-riboside 3‘,5‘-bisphosphate. Reaction with Iy6-diaminohexane followed by resolution of the isomeric mixture on a Dowex 1-X2 column yielded N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 3’,5‘-bisphosphate.The inhibition of several NADP+-dependent and NAD+-dependent dehydrogenases by N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, N6-(6-aminohexyl)-adenosine 3’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 5’-monophosphate was examined.These three AMP-analogues were attached to Sepharose 4B by the cyanogen bromide method and the binding of several NAD(P)+-dependent enzymes were investigated. NADP+-dependent enzymes were bound to Sepharose substituted with N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, whereas NAD+-dependent enzymes were not bound under the same conditions. Conversely, when N6-(6-aminohexy1)-adenosine 5‘-monophosphate was used as the immobilised ligand only the NAD+- dependent enzymes were bound, as well as glucose-6-phosphate dehydrogenase showing weak affinity. These observations strongly suggest that these two immobilised analogues represent true biospecific and group-specific adsorbents. The enzymes were eluted with their complementary nucleotides, NAD(H) and NADP(H). These techniques were utilised to purify several NADPf-dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.
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| 46. |
- Brunk, Ulf, 1937-, et al.
(författare)
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The mitochondrial-lysosomal axis theory of aging : Accumulation of damaged mitochondria as a result of imperfect autophagocytosis
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:8, s. 1996-2002
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Tidskriftsartikel (refereegranskat)abstract
- Cellular manifestations of aging are most pronounced in postmitotic cells, such as neurons and cardiac myocytes. Alterations of these cells, which are responsible for essential functions of brain and heart, are particularly important contributors to the overall aging process. Mitochondria and lysosomes of postmitotic cells suffer the most remarkable age-related alterations of all cellular organelles. Many mitochondria undergo enlargement and structural disorganization, while lysosomes, which are normally responsible for mitochondrial turnover, gradually accumulate an undegradable, polymeric, autofluorescent material called lipofuscin, or age pigment. We believe that these changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constituents and autophagocytosed material), but also because of the inherent inability of cells to completely remove oxidatively damaged structures (biological 'garbage'). A possible factor limiting the effectiveness of mitochondial turnover is the enlargement of mitochondria which may reflect their impaired fission. Non-autophagocytosed mitochondria undergo further oxidative damage, resulting in decreasing energy production and increasing generation of reactive oxygen species. Damaged, enlarged and functionally disabled mitochondria gradually displace normal ones, which cannot replicate indefinitely because of limited cell volume. Although lipofuscin-loaded lysosomes continue to receive newly synthesized lysosomal enzymes, the pigment is undegradable. Therefore, advanced lipofuscin accumulation may greatly diminish lysosomal degradative capacity by preventing lysosomal enzymes from targeting to functional autophagosomes, further limiting mitochondrial recycling. This interrelated mitochondrial and lysosomal damage irreversibly leads to functional decay and death of postmitotic cells.
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| 47. |
- 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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| 48. |
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| 49. |
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| 50. |
- Carlsson, Karin, et al.
(författare)
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Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor
- 2003
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:12, s. 2576-2582
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Tidskriftsartikel (refereegranskat)abstract
- Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.
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