A 74-kDa δ/B' subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (α/C) and 63- and 74-kDa regulatory subunits (β/A and δ/B') in a ratio of 1:1:1. The purified δ/B' was used as an immunogen in mice, to prepare specific antisera against δ/B'. Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 ± 0.3 S and the Stokes radius of 5.5 ± 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10-9 M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The K(m) value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 ± 0.3 μM and 192 ± 2 mol P1 released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (α1β1δ1/ CAB'). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.
|Number of pages||10|
|Journal||Journal of biochemistry|
|Publication status||Published - 1997 Jul|
- Protein phosphatase 2A
- Rat brain
- Regulatory subunit 72-kDa δ/B'
- Tissue and subcellular distributions
ASJC Scopus subject areas
- Molecular Biology