An in-gel assay for protein tyrosine phosphatase activity: detection of widespread distribution in cells and tissues.
A method is described for the detection of protein tyrosine phosphatase activity in sodium dodecyl sulfate-polyacrylamide gels. A radiolabeled substrate, 32P-labeled poly(glutamic acid-tyrosine) (random copolymer) is incorporated into gels prior to polymerization. Following electrophoresis, the sodium dodecyl sulfate is removed; the proteins are fully denatured by soaking gels in 6 M guanidine hydrochloride and then renatured by incubation in buffers containing 0.04% Tween 40 and high concentrations of reducing agents. Protein tyrosine phosphatase activity is detected in autoradiographs of dried gels as regions from which the 32P has been selectively removed. Electrophoresis of known cytoplasmic protein tyrosine phosphatases indicates activity as the predicted molecular weights. As little as 10 pg of some cytoplasmic phosphatases is detectable. However, transmembrane tyrosine phosphatases, such as CD45, are detected only at very high protein loadings in this assay. Electrophoresis of whole cell lysates indicates multiple bands of tyrosine phosphatase activity, some of which comigrate with known cytoplasmic protein tyrosine phosphatases. The activity is inhibited by sodium orthovanadate or the omission of reducing agents during the renaturation process. The assay has been used to analyze embryonic and adult tissues, as well as whole cell lysates. A similar profile of bands of tyrosine phosphatase activity is seen with many different cells and tissues. However, some that are highly differentiated, such as adult skeletal muscle, erythrocytes, or sperm, reveal either a reduced level of tyrosine phosphatase activity or a simplified profile of bands.
Burridge
K
K
Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Nelson
A
A
D000328:Adult; D000818:Animals; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005347:Fibroblasts; D005973:Glutamine / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D008970:Molecular Weight; D011108:Polymers / Q000378:metabolism; D011233:Precipitin Tests; D011489:Protein Denaturation; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012394:Rosaniline Dyes / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D015007:Yersinia / Q000201:enzymology
D006146:Guanidines; D011108:Polymers; D012394:Rosaniline Dyes; C080178:glutamine tyrosine copolymer; D005973:Glutamine; D012967:Sodium Dodecyl Sulfate; D014638:Vanadates; D014443:Tyrosine; C048139:Coomassie blue; D017027:Protein Tyrosine Phosphatases; D019791:Guanidine