Concurrent purification of type-1 and type-2A protein phosphatase catalytic subunits.
We describe a simple purification scheme for the active catalytic subunits of both protein phosphatases type-1 and type-2A. The advantage of this procedure over others is that it produces intact proteins with high yield and specific activity and is suitable for either kilograms of tissue or a dish of cells. Type-1 and type-2A phosphatases from rabbit skeletal muscle were resolved on polylysine-agarose and subsequently obtained in homogeneous form. The phosphatases demonstrated characteristic properties. The phosphatase-1 catalytic subunit was inhibited by inhibitor-2 and phosphoinhibitor-1 whereas phosphatase-2A was not. The phosphatase activities were stable for years at -20 degrees C when stored in the presence of Mg2+ and glycerol. Based on the predicted sequence of the carboxyl terminus of each phosphatase, antibodies specific for phosphatases-1 and -2A were produced in rabbits using synthetic peptides as immunogens. Immunoblots showed complete specificity of these antibodies for their respective phosphatases and confirmed that the purified phosphatases has intact carboxyl termini. The purified catalytic subunits and antibodies will be useful for examining the regulation and the physiological roles of these protein phosphatases in cellular physiology.
Martin
B L
BL
Section of Biochemistry, Brown University, Providence, Rhode Island 02912.
Shriner
C L
CL
Brautigan
D L
DL
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D011817:Rabbits
D007527:Isoenzymes; D046911:Macromolecular Substances; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2