Alteration in protein-tyrosine phosphatase of rat epithelial cells by RSV-transformation: application of phospho-tyrosyl glutamine synthetase to the study of protein-tyrosine phosphatase.
We prepared phospho-tyrosyl glutamine synthetase (P-GS) with suppressed activity from a highly adenylylated glutamine synthetase and applied it to the assay of protein-tyrosine phosphatase (PTPase) present in non-malignant rat liver cells (BRL) by RSV-transformation. The maximum PTPase activity toward P-GS was observed at neutral pH (pH 7.5-8.0) in the soluble and particulate fractions prepared from both BRL and RSV-transformed (RSV-BRL) cells. At low activity levels (< about 0.3 U), the PTPase activity in each fraction was proportional to the sample protein concentration (A280) and the specific activity of PTPase in the soluble fraction of BRL cells was about twofold higher than that in the soluble fraction of BRL cells, while those in particulate fractions of BRL and RSV-BRL cells were almost the same as each other. Soluble fractions of BRL and RSV-BRL were subjected to molecular-sieve and anion-exchange chromatographies. One major PTPase activity, with an M(r) of about 40,000 (40k), was detected in the BRL soluble fraction, and two were detected in the RSV-BRL soluble fraction with M(r)s of about 40k and 60k. The 40k PTPases in BRL and RSV-BRL had the same profiles on anion-exchange chromatography, but the 60k PTPase in RSV-BRL cells showed a different profile. We suggest that the RSV-transformation of BRL cells induced the appearance of the 60k PTPase in the soluble fraction.
Mashima
K
K
Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University, Tokyo.
Okajima
Y
Y
Usui
J
J
Shimizu
T
T
Kimura
K
K
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000502:physiology*; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D005591:Chemical Fractionation; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D051381:Rats
D017027:Protein Tyrosine Phosphatases; D005974:Glutamate-Ammonia Ligase