title: Phosphoprotein phosphatase activity at the outer surface of intact normal and transformed 3T3 fibroblasts.
abstract: Using 32P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphatase activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile 32P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K+; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkeable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002471:Cell Transformation, Neoplastic / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D007770:L-Lactate Dehydrogenase; D010749:Phosphoprotein Phosphatases