| 1. |
- Bergquist, Jonas, et al.
(författare)
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Structure-activity relationships for unsaturated dialdehydes 8( *). Comparative effects of 10 sesquiterpenoids on the sea urchin gamete fertilization.
- 1993
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Ingår i: Toxicology in Vitro. - 0887-2333 .- 1879-3177. ; 7:3, s. 205-12
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Tidskriftsartikel (refereegranskat)abstract
- The effects of 10 sesquiterpenoids with unsaturated dialdehyde functionalities were studied on fertilization, first cleavage, and calcium permeability of egg membranes of sea urchin gametes. Fertilization was inhibited by nine compounds when sperm was exposed and by five compounds when eggs were exposed (50 mug/ml for 5 min). All compounds except one (9alpha-hydroxymerulidial) inhibited the first cleavage in a dose-response manner. Only one compound (velleral) increased the Ca(2+) permeability of egg membranes at 20 mug/ml. All compounds reduced to a varying extent the ATP-driven Ca(2+) sequestration by non-mitochondrial intracellular compartments. In general, when hydroxylated and non-hydroxylated derivatives of the compounds are compared, the hydroxylated ones present a lower toxicity when measuring fertilization and cleavage inhibition, and reduction of intracellular Ca(2+) sequestration.
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| 2. |
- Lawrence, D A, et al.
(författare)
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Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.
- 1990
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 265:33, s. 20293-301
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Tidskriftsartikel (refereegranskat)abstract
- Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.
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| 3. |
- Strandberg, L, et al.
(författare)
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Fluorescence studies on plasminogen activator inhibitor 1 : reactive centre cysteine mutants remain active after fluorophore attachment.
- 1994
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 76:3, s. 253-67
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Tidskriftsartikel (refereegranskat)abstract
- To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.
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