Nonradioactive technique to measure protein phosphatase 2A-like activity and its inhibition by drugs in cell extracts.
A nonradioactive assay has been developed that can be used to measure serine/threonine protein phosphatase (PP) activity, especially PP2A, in crude extracts from different cell lines. For this technique commercially available casein is used as an already phosphorylated substrate. The prerequisite for reliable measurements is the removal of free phosphate from cell extracts and substrate preparations with desalting columns. The use of different nonspecific or specific inhibitors as well as inhibition characteristics observed after extract dilution suggests that in the absence of magnesium, PP2A-like activity in the extracts is measured by this technique. Inclusion of magnesium allowed the detection of a protein phosphatase activity that is activated by magnesium, which is presumably PP2C. The use of structurally different as well as structurally related inhibitors of PP2A gave results comparable to those of reports from the literature that were obtained with radioactive assays. Thus, our data supported our hypothesis that this nonradioactive assay can be used for the identification of newly synthesized PP inhibitors as well as for performing structure-activity analysis within groups of such new agents.
Fathi
Ali-Reza
AR
Institute of Pharmacology and Toxicology, University of Göttingen, Robert-Koch-Strasse 40, D-37075, Göttingen, Germany.
Krautheim
Andrea
A
Lucke
Susanne
S
Becker
Klaus
K
Juergen Steinfelder
Hans
H
D000818:Animals; D002193:Cantharidin / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism; D002457:Cell Extracts / Q000032:analysis* / Q000494:pharmacology; D002478:Cells, Cultured; D003124:Colorimetry / Q000379:methods*; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D008982:Molybdenum / Q000737:chemistry; D019319:Okadaic Acid / Q000494:pharmacology; D010710:Phosphates / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010756:Phosphoric Acids / Q000737:chemistry; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D051381:Rats; D012394:Rosaniline Dyes / Q000737:chemistry
D002364:Caseins; D002457:Cell Extracts; D004791:Enzyme Inhibitors; D010710:Phosphates; D010756:Phosphoric Acids; D012394:Rosaniline Dyes; C005095:malachite green; D019319:Okadaic Acid; D008982:Molybdenum; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D008274:Magnesium; D002193:Cantharidin; C003125:phosphomolybdic acid