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Related Experiment Videos

Human factor D of the alternative complement pathway: purification and characterization

J E Volanakis, R E Schrohenloher, R M Stroud

    Journal of Immunology (Baltimore, Md. : 1950)
    |July 1, 1977
    PubMed
    Summary
    This summary is machine-generated.

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    This study purified human plasma protein D, revealing its molecular weight and amino acid composition. Purified protein D demonstrated enzymatic activity and was inhibited by DFP, suggesting its role in biological processes.

    Area of Science:

    • Biochemistry
    • Protein Chemistry
    • Enzymology

    Background:

    • Human plasma contains various proteins with essential biological functions.
    • Understanding the properties and activities of these proteins is crucial for medical and research applications.

    Purpose of the Study:

    • To purify and characterize protein D from outdated human plasma.
    • To investigate the enzymatic activity and substrate specificity of purified protein D.
    • To determine the effect of inhibitors on protein D's activity.

    Main Methods:

    • Protein purification using sequential chromatography (Bio Rex 70, Sephadex G-200, Sephadex G-75).
    • Assay of protein D activity via hemolytic diffusion plate assay.
    • Molecular weight determination using sedimentation equilibrium.

    Related Experiment Videos

  • Amino acid analysis and N-terminal sequencing.
  • Enzymatic assays with synthetic amino acid esters and analysis of protein cleavage using SDS-PAGE and immunoelectrophoresis.
  • Inhibition studies using DFP and reduction/alkylation.

    • Main Results:

    • Protein D was purified to homogeneity with a 4% yield.
    • Established molecular weight of 22,900 daltons and identified Isoleucine as the N-terminus.
    • Demonstrated that protein D cleaves protein B in the presence of Mg++.
    • Identified benzoyl-L-arginine methyl ester (BAME) as a sensitive substrate, with a distinct substrate profile compared to other proteases.
    • Showed irreversible inhibition of both enzymatic and hemolytic activity by DFP and reduction/alkylation.

    Conclusions:

    • Protein D is a distinct enzyme with specific hydrolytic activity.
    • Its enzymatic and hemolytic functions are dependent on specific structural features, as evidenced by inhibition studies.
    • Further research into protein D's biological role and interactions is warranted.