Activity and molecular weight of protein tyrosine phosphatases in cell lysates determined by renaturation after gel electrophoresis.
A procedure for renaturing and detecting the activity of protein tyrosine phosphatases (PTPases) after sodium dodecyl sulfate (SDS)-gel electrophoresis with greatly improved sensitivity and resolution is described. Epidermal growth factor receptor-kinase, c-src kinase, and focal adhesion kinase were phosphorylated on tyrosine with 32PO4 and incorporated into gels prior to electrophoresis. These proteins are dephosphorylated when cellular proteins are electrophoresed and the separated PTPases are renatured in the gel by removing SDS with extensive washing. With whole cell lysates, at least eight separate bands of decreased radioactivity corresponding to PTPase activity with molecular weights between 110 and 34 kDa are seen in autoradiographs of the dried gels. PTPases detected are similar with different cell types and with the three 32P-labeled protein substrates, although they are different in cytosolic and membrane-associated fractions. A PTPase detected above 200 kDa in wheat germ agglutinin eluates from solubilized cells suggests that some receptor PTPases can be renatured. While microgram levels of recombinant PTP-1C are required for detection, nanogram levels of recombinant PTP-1B are easily detected. Assaying the activity of renatured PTPases after they have been separated by molecular weight in SDS gel electrophoresis provides a simple and rapid means of determining the activity of individual PTPases in cell fractions.
Gates
R E
RE
Research Service, Department of Veterans Affairs Medical Center, Nashville, Tennessee, 37212, USA.
Miller
J L
JL
King
L E
LE
D002458:Cell Fractionation; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology; D008722:Methods; D008970:Molecular Weight; D011489:Protein Denaturation; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification
D017027:Protein Tyrosine Phosphatases