Protein phosphatase 1 catalytic subunit isoforms from alfalfa: Biochemicalcharacterization and cDNA cloning

The catalytic subunit of protein phosphatase 1 (PP1c) was purified from an alfalfa (Medicago sativa) microcallus cell culture. The preparation was inh ibited by rabbit muscle inhibitor-a and okadaic acid and had a molecular ma ss of 35 kDa. Five distinct cDNAs termed IMsPP1 alpha, -beta, -gamma, -delt a, and -epsilon were cloned from a M, sativa somatic embryo library. MsPP1 alpha was identical to a cDNA reported earlier [A Pay, M, Pirck, L, Bogre, H, Hirt, and E, Heberle-Bors Mol. Gen, Genet, 244, 176-182, 1994], while th e others represented novel isoforms encoded by separate genes. The predicte d amino acid sequences of MsPP1 alpha; -beta, -gamma, -delta, and -epsilon were highly similar to each other and to other known PP1c sequences. The GS T-MsPP1 beta fusion protein expressed in Escherichia coli was catalytically active and was inhibited by inhibitor-a and okadaic acid. Affinity-purifie d polyclonal MsPP1antipeptide antibody detected a protein of 36 kDa in crud e cell extracts. These results proved that the cDNA clone encoded an active PP1c which was very similar to the purified enzyme. The mRNA and protein c oncentrations of PP1c as well as the specific activity of protein phosphata se 1 did not change during the cell cycle in a synchronized alfalfa cell cu lture, On the other hand, the isoforms exhibited different steady-state mRN A levels in different plant organs suggesting tissue-specific functions, (C ) 1998 Academic Press.