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Related Experiment Videos

Physicochemical characterization of lymphocyte-activating factor (LAF).

S B Mizel

    Journal of Immunology (Baltimore, Md. : 1950)
    |June 1, 1979
    PubMed
    Summary

    Leukocyte Adhesion Factor (LAF) from P388D1 cells is a stable 12-16 kDa protein. Its structure is independent of disulfide bonds and it shares properties with macrophage-derived LAF.

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    Area of Science:

    • Biochemistry
    • Immunology
    • Cell Biology

    Background:

    • Leukocyte Adhesion Factor (LAF) plays a role in immune responses.
    • Understanding the physicochemical properties of LAF is crucial for its functional characterization.

    Purpose of the Study:

    • To characterize the physicochemical properties of LAF produced by the P388D1 mouse macrophage cell line.
    • To compare the properties of P388D1-derived LAF with LAF from normal macrophages.

    Main Methods:

    • Protease digestion (Streptomyces griseus protease, trypsin, chymotrypsin, papain)
    • Concanavalin A-Sepharose binding assay
    • Reduction and alkylation treatment
    • Denaturation with urea and SDS
    • SDS-polyacrylamide gel electrophoresis
    • Gel filtration
    • Isoelectric focusing

    Main Results:

    • P388D1-derived LAF is a single polypeptide chain with a molecular weight of 12,000-16,000 daltons.
    • LAF is sensitive to Streptomyces griseus protease but resistant to trypsin and chymotrypsin.
    • Papain degrades LAF, especially in the presence of 8 M urea.
    • LAF does not bind to concanavalin A-Sepharose, indicating a lack of significant glycosylation.
    • LAF activity is not dependent on disulfide bonds.
    • LAF is stable in 8 M urea and SDS, suggesting resistance to denaturation.
    • SDS-PAGE indicated a molecular weight of 12,000 daltons, while gel filtration yielded 16,000 daltons.
    • The isoelectric point of LAF is between 5.0 and 5.4.

    Conclusions:

    • P388D1 cell line-derived LAF is a relatively stable molecule.
    • LAF shares several physicochemical properties with normal human and mouse macrophage-derived LAF.
    • These findings contribute to the understanding of LAF's molecular nature and potential function in immune processes.

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