| 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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