Functional analysis of the plasminogen activator inhibitor-1 reactive center by saturation mutagenesis.
dc.contributor.author | Sherman, Patti Michele | en_US |
dc.contributor.advisor | Ginsburg, David | en_US |
dc.date.accessioned | 2014-02-24T16:17:37Z | |
dc.date.available | 2014-02-24T16:17:37Z | |
dc.date.issued | 1993 | en_US |
dc.identifier.other | (UMI)AAI9409801 | en_US |
dc.identifier.uri | http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9409801 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/103843 | |
dc.description.abstract | Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily, is a specific inhibitor of the serine proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). To systematically examine the roles of the reactive center residues (P$\sb1$Arg-P$\sb1\sp\prime$Met) in PAI-1 function, saturation mutagenesis was performed at the P$\sb1$ and P$\sb1\sp\prime$ positions to construct a library of PAI-1 mutants. Analyses of 177 unique variant proteins demonstrated that a basic residue is required at the P$\sb1$ position of PAI-1 for significant inhibitory activity against uPA. In contrast, a wide range of amino acid substitutions are tolerated at P$\sb1$ for the inhibition of tPA. All amino acid substitutions except proline are allowed at the P$\sb1\sp\prime$ position for activity against either PA when P$\sb1$ is Arg or Lys. A number of variants that were either uPA- or tPA-specific were identified. Kinetic analyses indicated that P$\sb1$Tyr and P$\sb1$His are the most efficient, absolutely tPA-specific inhibitors examined in the panel of variants, with second-order rate constants $(k\sb{i})$ of $\rm 4.0\times10\sp5\ M\sp{-1}s\sp{-1}$ and $\rm 3.6\times10\sp5\ M\sp{-1}s\sp{-1}$, respectively. P$\sb1$Lys-P$\sb1\sp\prime$Ala was identified as the most relatively uPA-specific variant, reacting 40-fold more rapidly with uPA than tPA. Based on these and earlier observations, additional PA-specific PAI-1 variants containing substitutions at P$\sb3$ through P$\sb1\sp\prime$ were engineered. P$\sb3$Tyr-P$\sb2$Ser-P$\sb1$Lys-P$\sb1\sp\prime$Trp had a $k\sb{i}$ value of $\rm 1.7\times10\sp6\ M\sp{-1}s\sp{-1}$ against tPA, but was inactive against uPA. In contrast, P$\sb2$Arg-P$\sb1$Lys-P$\sb1\sp\prime$Ala inhibited uPA 74-fold more rapidly than tPA. Screening of the P$\sb1$-P$\sb1\sp\prime$ variants for inhibitory activity against thrombin in both the absence and presence of heparin identified a number of unique variants which were heparin-dependent thrombin inhibitors. These mutants contained a variety of amino acid substitutions at the P$\sb1$ and P$\sb1\sp\prime$ positions. These findings have important implications for the role of the reactive center in determining serpin function and target specificity, and offer insights into the interactions between PAI-1 and its target proteases. Identification of PA-specific PAI-1 variants provides novel reagents for dissecting the physiological roles of tPA and uPA, and for probing the contribution of PAI-1 to the regulation of plasminogen activation in vivo. | en_US |
dc.format.extent | 153 p. | en_US |
dc.subject | Biology, Molecular | en_US |
dc.subject | Biology, Genetics | en_US |
dc.title | Functional analysis of the plasminogen activator inhibitor-1 reactive center by saturation mutagenesis. | en_US |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Human Genetics | en_US |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/103843/1/9409801.pdf | |
dc.description.filedescription | Description of 9409801.pdf : Restricted to UM users only. | en_US |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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