Detection and quantification of protein phosphatase inhibitor-1 gene expression in total rat liver and isolated hepatocytes.
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.
Aleem
E A
EA
Division of Cellular and Molecular Pathology, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Flohr
T
T
Hunziker
A
A
Mayer
D
D
Bannasch
P
P
Thielmann
H W
HW
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins; D003001:Cloning, Molecular; D015870:Gene Expression; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000378:metabolism*; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C021042:protein phosphatase inhibitor-1