Purification and characterization of protein phosphatase 2C in rat parotid acinar cells: two forms of Mg(2+)-activated histone phosphatase and phosphorylation by cAMP-dependent protein kinase.
Two forms of Mg(2+)-activated histone phosphatase activities were partially purified from rat parotid acinar cells using Mono Q and gel filtration chromatography. Both enzymes activities were dependent on the presence of Mg2+, showing little activity in the presence of EDTA. The activities fractionated on the Mono Q column into two peaks: the first was a minor peak of histone phosphatase activity; the second was a major peak. These two peaks eluted at distinct positions on the gel filtration column. The molecular masses of the two peak fractions corresponded to 46 and 55 kDa, respectively on SDS-gels. The first 46-kDa peak immunoreacted with anti-PP2Calpha phosphatase antibody and like PP2Calpha phosphatase could be phosphorylated by cAMP-dependent protein kinase. The second 55-kDa peak showed neither reactivity with anti-PP2Calpha phosphatase antibody nor phosphorylability by cAMP-dependent protein kinase, but retained a Mg2+ or Mn2+ dependence for its histone phosphatase activity. Ca2+ showed a strong inhibition on this activity. On the basis of these observations, we have identified the first peak enzyme as PP2Calpha phosphatase and the second peak as a novel PP2C-like phosphatase.
Yokoyama
N
N
Department of Physiolgy, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Kobayashi
T
T
Tamura
S
S
Sugiya
H
H
D000818:Animals; D005591:Chemical Fractionation; D002845:Chromatography; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006657:Histones / Q000378:metabolism*; D015151:Immunoblotting; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010306:Parotid Gland / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D051381:Rats; D029701:Saccharomyces cerevisiae Proteins
D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D017868:Cyclic AMP-Dependent Protein Kinases; C087358:PTC1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D008274:Magnesium