PURIFICATION AND CHARACTERIZATION OF A NOVEL XYLULOSE 5-PHOSPHATE-ACTIVATED PROTEIN PHOSPHATASE CATALYZING DEPHOSPHORYLATION OF FRUCTOSE-6-PHOSPHATE,2-KINASE - FRUCTOSE-2,6-BISPHOSPHATASE

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K, (1 994) (J, Biol, Chem, 269, 26100-26106) that the administration of high concentrations of glu cose stimulates dephosphorylation of Fru-6-P,a kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5- P activates the dephosphorylation reaction, To characterize the protei n phosphatase, we have purified the Xu Ei-P-activated protein phosphat ase to homogeneity from livers of rats injected with high glucose, Sev eral protein phosphatases in the livers were separated by DEAE cellulo se chromatography, but only one peak of the enzyme was activated by Xu 5 P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1- 3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A, The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa), catalytic (C, 36 kDa), and regulatory (B, 52 M)a) subunits, Amino acid sequences of five tryptic peptides derive d from the B subunit showed similarity with those of the B alpha isofo rm of rat protein phosphatase 2A, but five out of 73 residues were dif ferent, The protein phosphatase catalyzed dephosphorylation of Fru-6-P ,2-kinase:Fru-2,6-Pase, phosphorylase K-m and pyruvate kinase, and the K, values were 0.8 mu M, 3.7 mu M, and 2.2 mu M, respectively, Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K-a value for Xu 5-P was 20 ELM. Xu 5 P was the only sugar phosphate which activated the PP2A among all the su gar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liv er which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-P ase and was activated specifically by Xu 5-P, The Xu 5-P-activated pro tein phosphatase 2A explains the increased Fru 2,6-P-2 level in liver after high glucose administration.