The isolation and characterization of phosphatases from the Ehrlich ascites tumor cell line /

Abstract

Peak II was found to have low specificity toward phosphotyrosine but did contain Mg('2+)- or Mn('2+)-dependent p-nitrophenyl phosphate phosphatase activity. Both Peak I and Peak II p-nitrophenyl phosphate phosphatases activities were similar in that they both require essential Mg('2+) or Mn('2+) ion and are similarly inhibited by pyrophosphate, but are not inhibited by levamisole. However, there is evidence that these two enzymes are distinct. Differences between the two fractions with regard to optimal assay pH and reaction rates; fluoride and phosphate inhibition; and susceptibilities to inactivation by either heat, N-ethylmaleimide, or trypsin treatment while in the presence or absence of either Mg('2+), Mn('2+), p-nitrophenylphosphate, dithiothreitol, or phosphate suggest that peak I and Peak II contain distinct Ehrlich ascites tumor cell enzymes which belong in a newly described class of phosphatases.

The presence of soluble Mg('2+) or Mn('2+)-dependent phosphotyrosyl phosphatase and Mg('2+) or Mn('2+)-dependent p-nitrophenyl phosphate phosphatase activity in Ehrlich ascites tumor cell homogenates is reported.

The crude homogenate was fractionated over Sephadex G-150 Gel filtration and DEAE- sephacel anion exchange columns, and two fractions termed Peak I and Peak II were resolved. Peak I p-nitrophenyl phosphate phosphatase and phosphotyrosyl phosphatase activities were found to be similar in that they both require essential Mg('2+) or Mn('2+) ions and dithiothreitol. Optimal activity was observed at neutral pH. It was further found that these two Peak I activities were inhibited by zinc and fluoride but were not inhibited by vanadate and levamisole.