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Ca2+-binding protein from human kidney. Purification and properties.

M Staun, O Norén, H Sjöström

    The Biochemical Journal
    |January 1, 1984
    PubMed
    Summary

    Researchers purified a calcium-binding protein (CaBP) from human kidney using heat precipitation and immunoadsorbent chromatography. This CaBP, distinct from intestinal CaBP, has high-affinity calcium-binding sites and a molecular weight of 26,000.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Renal Physiology

    Background:

    • Calcium-binding proteins (CaBPs) play crucial roles in cellular calcium homeostasis.
    • Understanding the specific properties of renal CaBP is essential for elucidating kidney function.
    • Previous characterization of CaBPs has primarily focused on other tissues.

    Purpose of the Study:

    • To purify and characterize a novel calcium-binding protein (CaBP) from human kidney cytosol.
    • To develop specific antibodies for CaBP purification and immunochemical analysis.
    • To investigate the biochemical and biophysical properties of renal CaBP, including its calcium-binding affinity and molecular weight.

    Main Methods:

    • Heat precipitation of kidney cytosol followed by gel filtration and chromatofocusing.

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  • Generation of a specific antibody against purified CaBP and subsequent immunoadsorbent chromatography.
  • Sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE) and gel filtration for molecular weight determination.
  • Crossed immunoelectrophoresis for purity assessment and immunochemical cross-reactivity analysis.

    • Main Results:

    • Two purification procedures yielded homogeneous CaBP with 200-fold and 500-fold increases in specific activity, respectively.
    • Purified CaBP exhibited a molecular weight of 26,000 Da by SDS-PAGE and 28,000 Da by gel filtration under non-denaturing conditions.
    • The protein demonstrated high-affinity calcium-binding (association constant of 0.1 x 10(6) M-1) and Ca2+-dependent electrophoretic mobility.
    • Immunochemical analysis confirmed no cross-reactivity between renal and intestinal CaBP.

    Conclusions:

    • A novel CaBP has been successfully purified from human kidney cytosol.
    • The characterized renal CaBP is biochemically distinct from intestinal CaBP.
    • This purified CaBP provides a valuable tool for further research into renal calcium handling and related physiological processes.