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EXPERT MISSION FOR THE ASSESSMENT OF AIPROJECT IN ASEAN MEMB MEMBER STATES.

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A study is being carried out to determine the relationship between structure and function in plasminogen activator inhibitor-1 (PAI-1), the inhibitor of tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA).

The complementary deoxyribonucleic acid (cDNA) encoding for PAI-1 has been cloned and inserted into a pMa/c plasmid allowing expression of active recombinant PAI-1 in Escherichia coli. This vector system has the advantage that it can serve for both site directed mutagenesis and for expression without the need for recloning. Using fluorescence spectroscopic studies, certain conformational differences were found to exist between latent, active and substrate forms of PAI-1, which were at least partially associated with the occurrence of significant differences in tryptophan environment in the 3 forms. PAI-1 contains 4 tryptophan (Trp) residues (Trp86, Trp139, Trp175, Trp262). Using oligonucleotide directed mutagenesis, 4 PAI-1 mutants will be constructed, each coding for a single amino acid substitution (Trp to phenylalanine, Phe) for 1 of the 4 Trp residues in native PAI-1. The purpose of these mutants is to assess the contribution of each of the 4 Trp residues to the alteration in intrinsic fluorescence associated with the conversion between the different forms. From these data it should be possible to delineate the regions of the molecule that are involved in the conformational changes associated with alterations of the functional properties. Biochemical and functional characterisation of purified PAI-1 and PAI-1 mutants will be performed.

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Riso National Laboratory
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4000 ROSKILDE
Dänemark

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