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Related Experiment Videos

Antithrombin-III Denver, a reactive site variant.

A W Stephens, B S Thalley, C H Hirs

    The Journal of Biological Chemistry
    |January 25, 1987
    PubMed
    Summary

    Antithrombin-III Denver is a mutant protein with a Ser394Leu substitution, significantly impairing thrombin binding. This genetic defect reduces the protein

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Genetics

    Background:

    • Antithrombin-III is a crucial inhibitor of serine proteases.
    • Mutations in Antithrombin-III can lead to bleeding disorders.
    • Antithrombin-III Denver is a known variant with impaired function.

    Purpose of the Study:

    • To characterize the molecular defect in Antithrombin-III Denver.
    • To identify the specific mutation responsible for altered protein function.
    • To quantify the impact of the mutation on thrombin binding kinetics.

    Main Methods:

    • Affinity separation using heparin-Sepharose.
    • Gel filtration chromatography.
    • Tryptic peptide mapping and Vydac C18 fractionation.
    • Protein sequencing and synthesis.
    • Kinetic analysis of thrombin binding.

    Main Results:

    • A unique tryptic peptide from Antithrombin-III Denver was identified, corresponding to residues 394-399.
    • The mutation was identified as a Ser394Leu substitution at the reactive site.
    • Thrombin binding to Antithrombin-III Denver showed a drastic reduction in the second-order rate constant (approx. 4000-fold reduction in the presence of heparin).

    Conclusions:

    • The Ser394Leu substitution is the primary defect in Antithrombin-III Denver.
    • This mutation significantly impairs the ability of antithrombin to inhibit thrombin.
    • The findings highlight the importance of the reactive site serine in antithrombin function.

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