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

Mol Cell Biochem. 2001 Jan;217(1-2):1-12. doi: 10.1023/a:1007141514750.

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 approximately 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.

MeSH terms

  • Animals
  • Blotting, Western
  • Carrier Proteins*
  • Cloning, Molecular
  • Gene Expression Profiling
  • Gene Expression*
  • Hepatocytes / metabolism*
  • Intracellular Signaling Peptides and Proteins*
  • Liver / metabolism*
  • Male
  • Muscle, Skeletal / metabolism
  • Open Reading Frames
  • Protein Biosynthesis
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / biosynthesis
  • RNA-Binding Proteins / genetics*
  • Rats
  • Rats, Sprague-Dawley
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Carrier Proteins
  • Intracellular Signaling Peptides and Proteins
  • RNA, Messenger
  • RNA-Binding Proteins
  • protein phosphatase inhibitor-1