Open AccessPurification, characterization and structure of protein phosphatase 1 from the cilia of Paramecium tetraurelia
Author(s) -
FRIDERICH Gerald,
KLUMPP Susanne,
RUSSELL Chris B.,
HINRICHSEN Robert D.,
KELLNER Roland,
SCHULTZ Joachim E.
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17259.x
Subject(s) - paramecium , biology , peptide sequence , biochemistry , cyanogen bromide , microbiology and biotechnology , amino acid , threonine , phosphatase , serine , protein primary structure , enzyme , gene
A type 1 serine/threonine protein phosphatase (PP1) which is mostly localized in the excitable ciliary membranes from the protozoan Paramecium , was purified to homogeneity. Approximately 4 μg enzyme of 37 kDa was isolated from 100 1 axenic culture. The enzymic properties were characterized using phosphorylase a from rabbit skeletal muscle as a substrate and several known effectors of mammalian PP1. The protozoan PP1 was enzymically indistinguishable from its mammalian congener. The amino acid sequence of the Paramecium PP1 was deduced from its cDNA. The full‐length clone was obtained in several steps starting with a pair of degenerate primers made according to the two most conserved peptides of rabbit PP1 and PP2A. The gene encodes a protein of 36 392 Da. The identity of the cloned gene and the isolated ciliary PP1 was unequivocally established by microsequencing of four tryptic and cyanogen‐bromide peptides which were generated from the purified protein. Paramecium PP1 shows 75% amino‐acid‐sequence identity with rabbit PP1α. Areas of major differences are the C‐termini and N‐termini and a sequence between residues 219–242.