ISOLATION AND CHARACTERIZATION OF THE CATALYTIC SUBUNIT OF PROTEIN PHOSPHATASE 2A FROM NEUROSPORA-CRASSA

The catalytic subunit of protein phosphatase 2A (PP2A(c)) was purified from Neurospora crassa extract by (NH4)(2)SO4-ethanol precipitation f ollowed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yiel d. The apparent M(r) of PP2A(c) was estimated to be 35 kDa by gel filt ration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maxi mal inhibition of PP2A(c) was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-la, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitiv e to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical p roperties, N. crassa PP2A(c) is very similar to its mammalian counterp arts. Antipeptide antibodies raised against the N-terminal and C-termi nal ends of human PP2A(c) did not cross-react with N. crassa PP2A(c), indicating sequence differences outside the catalytic core of the enzy me.