Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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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PMID:Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment. 786 76

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. The alternative behavior of PAI-1 as an inhibitor, a non-inhibitory substrate, or a non-reactive latent form has been shown to be dependent on the initial conformation. In this study, we have evaluated the effect of a substitution outside the reactive site loop (P18) or in the reactive site loop (P6 and P10) on proteinase specificity and conformational transitions in PAI-1. Wild-type PAI-1 (wtPAI-1) revealed the same conformational distribution pattern toward tissue-type plasminogen activator (t-PA) as toward urokinase-type plasminogen activator (u-PA) (i.e. 53 +/- 6. 9% active, 36 +/- 6.8% latent, and 12 +/- 1.9% substrate). Inactivation of wtPAI-1 resulted in the conversion of the labile active form into the latent form while the stable substrate form remained unchanged. PAI-1-P6 (Val --> Pro at P6) revealed a target specificity for t-PA (39 +/- 7% versus 3 +/- 2% of the theoretical maximal value toward t-PA and u-PA, respectively), PAI-1-P10 (Ser --> Pro at P10) was 4-fold more active toward u-PA than toward t-PA, and PAI-1-P18 (Asn --> Pro at P18) exhibited inhibitory properties exclusively toward u-PA (41 +/- 10%). Surprisingly, inactivation of these mutants revealed functional and conformational transitions distinct from those observed for wtPAI-1. Inactivation of PAI-1-P6(Val --> Pro) resulted in a total conversion of the active form into the latent form and in a partial conversion of the substrate form into the latent form. The active forms of both PAI-1-P10(Ser --> Pro) and PAI-1-P18(Asn --> Pro) are also labile but, in contrast to the active form of wtPAI-1, convert into substrate forms. Based on the existence of various conformations of PAI-1, we propose an alternative reaction scheme describing the putative interactions between serpins and their target proteinases. The unusual conformational and functional flexibility of PAI-1 that, according to the current study, appears not to be restricted to the reactive site loop further underlines the importance of potential structural rearrangements (e.g. upon binding to cofactors) in PAI-1 (or serpins in general) for its functional behavior at particular biological sites.
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PMID:Proteinase specificity and functional diversity in point mutants of plasminogen activator inhibitor 1. 913 22

PAI-1, the physiological inhibitor of tissue-type and urokinase-type plasminogen activator, is a unique member of the serpins as it exists in three distinct conformations: an active inhibitory conformation, a non-inhibitory substrate conformation, and a non-reactive latent conformation. Proline substitution of single residues in the P16-P20 region (situated at the proximal hinge of the reactive site loop) of wild-type PAI-1 (wtPAI-1) and a stabilized PAI-1-variant (PAI-1-stab; N150H, K154T, Q301P, Q319L, and M354I, t(1/2)=150), respectively, resulted in two series of PAI-1-variants with different properties. In wtPAI-1 only substitution at P18 resulted in a pronounced u-PA specificity and substrate behaviour towards t-PA. In contrast, in PAI-1-stab substitution at either P18, P19 or P20 resulted in a u-PA specificity and a significantly increased substrate behaviour towards t-PA and u-PA. Importantly, analysis of the kinetics of inhibition did not reveal any differences in the second-order rate constants of inhibition (k approximately 10(7)M(-1)s(-1)). The pronounced differences observed for identical mutations in wtPAI-1 vs PAI-1-stab demonstrate that not merely the sequence of the reactive loop but also intramolecular interactions between the hF/s3A-loop and the main part of the molecule govern the functional and conformational behaviour of PAI-1.
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PMID:Comparative analysis of the proteinase specificity in wild-type and stabilized plasminogen activator inhibitor-1: evidence for contribution of intramolecular flexibility. 1535 69