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Calmodulin‐Dependent Phosphatases of PC12, GH 3 , and C 6 Cells: Physical, Kinetic, and Immunochemical Properties
Author(s) -
Farber Len H.,
Wilson Frank J.,
Wolff Donald J.
Publication year - 1987
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1987.tb02880.x
Subject(s) - egta , calmodulin , isoelectric point , microbiology and biotechnology , phosphatase , biochemistry , divalent , protein subunit , biology , chemistry , enzyme , calcium , organic chemistry , gene
Calmodulin‐dependent phosphoprotein phosphatase (CaMDP) activity has been found in each of three cultured cell lines: rat pheochromocytoma (PC 12), glioma (C 6 ), and pituitary adenoma (GH 3 ) cells. These CaMDP activities bind to immobilized calmodulin in the presence of Ca 2+ and are eluted by EGTA. Sucrose density centrifugation revealed that the phosphatase activities exhibited sedimentation coefficients of 4.37, 4.23, and 4.59 for proteins derived from C 6 , GH 3 , and PC 12 cells, respectively. The Stokes radii measured for the PC 12 and C 6 activities were 41.8 and 40.0 A, respectively. The estimated molecular weights calculated for the enzymes from these data are 79, 100 and 72, 200. The phosphatase activities required the presence of divalent cations such as Ca 2+ or Mn 2+ for expression of activity, which was optimal only in the presence of calmodulin. The apparent K m for phosphorylated myelin basic protein substrate was 8 μ M. Affinity‐purified antibodies to the B subunit of bovine brain CaMDP were found by immunoblot (Western blot) to cross‐react with a single protein among proteins extracted from PC 12, C 6 , and GH 3 cells that had been resolved by two‐dimensional electrophoresis. In each case, the cross‐reacting protein exhibited an M r of 16,000 and an isoelectric point of 4.7, values virtually identical to those reported previously for the B subunit of bovine brain CaMDP (sometimes called calcineurin). This cross‐reacting protein was found among cellular proteins eluted from immobilized calmodulin by EGTA. Immuno‐cytochemical localization of the cross‐reacting protein in undifferentiated PC 12 cells or in cells differentiated in response to nerve growth factor revealed its presence diffusely throughout the cytoplasm. These experiments support the contention that each of these cell lines contains a calmodulin‐regulated phosphatase homologous physically and kinetically, and immunologically related to bovine brain CaMDP.