Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa.
Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.
Szöór
B
B
Department of Medical Chemistry, University Medical School of Debrecen, Hungary.
Dombrádi
V
V
Gergely
P
P
Fehér
Z
Z
D000818:Animals; D005075:Biological Evolution; D002845:Chromatography; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005800:Genes, Fungal; D066298:In Vitro Techniques; D008387:Marine Toxins; D052998:Microcystins; D018482:Muscle, Skeletal / Q000201:enzymology; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D010766:Phosphorylation; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D011817:Rabbits
D004791:Enzyme Inhibitors; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C057862:cyanoginosin LR