PURIFICATION AND CHARACTERIZATION OF TYPE-1 PROTEIN PHOSPHATASE FROM SACCHAROMYCES-CEREVISIAE - EFFECT OF THE R73C MUTATION

Type 1 protein phosphatase encoded by the GLC7 gene was purified from Saccharomyces cerevisiae as a 1:1 complex with mammalian inhibitor 2 f used to glutathione S-transferase. The complex was inactive and requir ed treatment with Co2+ and trypsin for maximal activity. The specific activity toward phosphorylase alpha was about 1.8 units/mg of Glc7p, a nd IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 7.3, 81, and 0.30 nM:, respectively. The complex could be activated by glyc ogen synthase kinase-3 in the presence of Mg2+ and ATP to 20% of the a ctivity seen with Co2+ and trypsin, Thus, the catalytic properties of the yeast type 1 phosphatase are similar to those of the mammalian pro tein phosphatase 1, The R73C mutant phosphatase from the glycogen-defi cient strain, glc7-1, purified as a 1:1 complex with the inhibitor 2 f usion, had a specific activity toward phosphorylase alpha of 0.9 unit/ mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin -LR were 13.1, 113, and 0.37 nM, respectively. The R73C mutation sligh tly decreases the specific activity and sensitivity to inhibitors, sug gesting that changes in biochemical properties may affect glycogen lev els. However, the modest changes are consistent with our previous prop osal (E. M. Reimann et al., 1993, Adv. Protein Phosphatases 7, 173-182 ) and with the results of Stuart et al. (1994, Mol. Cell. Biol. 14, 89 6-905) that the mutation may selectively alter the interaction of Glc7 p with regulatory proteins. (C) 1998 Academic Press.