PURIFICATION AND CHARACTERIZATION OF A PROTEIN PHOSPHATASE THAT DEPHOSPHORYLATES PYRUVATE-KINASE IN AN ANOXIA TOLERANT ANIMAL

A protein phosphatase that dephosphorylates pyruvate kinase (PK) in vi tro was purified and characterized from the foot muscle of the anoxia tolerant gastropod mollusc Busycon canaliculatum. Purification involve d three steps: negative chromatography through Blue Dextran and CM Sep hadex, affinity chromatography on DEAE Sephadex and gel exclusion chro matography on Sephacryl S-400. Pyruvate kinase phosphatase (PK-Pase) a ctivity was monitored by following changes in PK I-50 values for L-ala nine that had previously been linked to changes in the degree of PK ph osphorylation. The purified PK-Pase gave a single band on SDS-polyacry lamide gel electrophoresis with a molecular weight of 41 +/- 1 kdalton s. Isoelectric focusing analysis showed that the PK-Pase had an isoele ctric point of 4.2 +/- 0.1. Kinetic analysis showed that the enzyme wa s a Type 2C protein phosphatase with a pH optimum of 6.5. Maximal acti vity required the presence of magnesium ions (K-M = 7.9 +/- 0.6 mu M) although high concentrations of Mg2+ were inhibitory (I-50 = 23 +/- 0. 4 mM). The protein phosphatase activity was not affected by either spe rmine, cAMP, cGMP, potassium phosphate, tartrate, NaF, HgCl2, citrate or concentrations of CaCl2 less than 10 mM. The enzyme could also use ATP, ADP, and GTP as substrates.