Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit.
A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/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 Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.
Nagase
T
T
Department of Biochemistry, Hiroshima University School of Medicine.
Murakami
T
T
Nozaki
H
H
Inoue
R
R
Nishito
Y
Y
Tanabe
O
O
Usui
H
H
Takeda
M
M
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004912:Erythrocytes / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions; D014018:Tissue Distribution
D007106:Immune Sera; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2