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Lymphocyte-activating factor. I. Generation and physicochemical characterization.

J S Economou, H S Shin

    Journal of Immunology (Baltimore, Md. : 1950)
    |October 1, 1978
    PubMed
    Summary
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    Lipopolysaccharide-activated macrophages produce lymphocyte-activating factor (LAF), a substance that stimulates thymocytes. This study details a method for producing homogeneous LAF and characterizes its physical properties.

    Area of Science:

    • Immunology
    • Biochemistry
    • Cell Biology

    Background:

    • Murine peritoneal macrophages activated by lipopolysaccharide produce lymphocyte-activating factor (LAF).
    • LAF is known to be mitogenic for murine thymocytes, playing a role in immune responses.
    • Previous studies lacked a method for generating a homogeneous molecular species of LAF.

    Purpose of the Study:

    • To develop a method for producing a relatively homogeneous molecular species of lymphocyte-activating factor (LAF).
    • To characterize the physical and chemical properties of purified LAF.
    • To determine the molecular weight and other biophysical parameters of LAF.

    Main Methods:

    • Sucrose gradient ultracentrifugation to determine sedimentation coefficient (S20,w).
    • Gel filtration on Sephadex G-75 to determine Stokes radius.

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  • CsCl isopycnic ultracentrifugation to determine buoyant density.
  • Svedberg equation used for molecular weight calculation.

    • Main Results:

    • LAF was purified to a relatively homogeneous molecular species.
    • Isoelectric point determined to be 4.8.
    • Apparent sedimentation coefficient (S20,w) was 2.0S.
    • Stokes radius was 22 Å, diffusion coefficient (D20,w) was 9.7 x 10(-7) cm2/sec.
    • Buoyant density was 1.30 g/cm3, partial specific volume estimated at 0.72.
    • Calculated molecular weight (m.w.) was 18,000 daltons.
    • Frictional ratio calculated to be 1.25, indicating a compact molecular shape.

    Conclusions:

    • A method for producing homogeneous LAF was established.
    • Detailed biophysical characterization of LAF was achieved, providing insights into its molecular properties.
    • The findings contribute to understanding the nature of LAF and its role in immune cell activation.