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Detection and quantification of protein phosphatase inhibitor-1 gene expression in total rat liver and isolated hepatocytes

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Abstract

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr ~ 30 kDa) which could be detected with a specific antibody by immunoblotting. This indicates an intact open reading frame of inhibitor-1 in rat liver. Immunoblotting of liver extract yielded a very weak band which comigrated with the inhibitor-1 proteins from muscle and brain. It is concluded that mRNA expression of inhibitor-1 may have implications for the regulation of protein phosphatase-1 (PP1) in rat liver.

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Authors and Affiliations

  1. Division of Cellular and Molecular Pathology, Germany

    Eiman A. Aleem & Peter Bannasch

  2. Division of Interaction of Carcinogens with Biological Macromolecules, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany

    Eiman A. Aleem, Thomas Flohr & Heinz Walter Thielmann

  3. Division of Cell Biology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany

    Andreas Hunziker

  4. Research Group of Hormones and Signal Transduction, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany

    Doris Mayer

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  1. Eiman A. Aleem
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  2. Thomas Flohr
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  3. Andreas Hunziker
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  4. Doris Mayer
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  5. Peter Bannasch
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  6. Heinz Walter Thielmann
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Correspondence to Heinz Walter Thielmann.

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Aleem, E.A., Flohr, T., Hunziker, A. et al. Detection and quantification of protein phosphatase inhibitor-1 gene expression in total rat liver and isolated hepatocytes. Mol Cell Biochem 217, 1–12 (2001). https://doi.org/10.1023/A:1007141514750

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