Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa

Abstract

The catalytic subunit of protein phosphatase 2A (PP2A c) was purified from Neurospora crassa extract by (NH 4) 2SO 4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/ g with a 2% yield. The apparent M r of PP2A c was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2A c was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the a-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2A c is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2A c did not cross-react with N. crassa PP2A o indicating sequence differences outside the catalytic core of the enzyme.