Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa.
The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.
Szöör
B
B
Department of Medical Chemistry, University Medical School of Debrecen, Hungary.
Fehér
Z
Z
Bakó
E
E
Erdödi
F
F
Szabó
G
G
Gergely
P
P
Dombrádi
V
V
D000645:Ammonium Sulfate; D000818:Animals; D002193:Cantharidin / Q000494:pharmacology; D011232:Chemical Precipitation; D002845:Chromatography; D003998:Dicarboxylic Acids / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D008387:Marine Toxins; D052998:Microcystins; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D009492:Neurospora crassa / Q000201:enzymology*; D019319:Okadaic Acid; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D054648:Protein Phosphatase 2; D011817:Rabbits; D012969:Sodium Fluoride / Q000494:pharmacology
D003998:Dicarboxylic Acids; D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; C100030:endothall; D019319:Okadaic Acid; D000431:Ethanol; D012969:Sodium Fluoride; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C057862:cyanoginosin LR; D002193:Cantharidin; D000645:Ammonium Sulfate