Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets.
The catalytic subunits of bovine platelet protein phosphatases were separated into three distinct forms by chromatography on heparin-Sepharose. Each phosphatase was further purified to apparent homogeneity as judged in sodium dodecyl sulfate-polyacrylamide gel yielding single protein bands of 37, 41, and 36 kDa. The 37-kDa phosphatase was excluded from heparin-Sepharose and preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. It was stimulated by polycations (polybrene or histone H1) and was inhibited by okadaic acid (IC50 = 0.3 nM), but its activity was not influenced by inhibitor-2 or heparin. The 41-kDa phosphatase was eluted from heparin-Sepharose by 0.20-0.25 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was stimulated by polycations and inhibited by okadaic acid (IC50 = 2 nM), but its activity was not affected by inhibitor-2 or heparin. The 36-kDa phosphatase was eluted from heparin-Sepharose by 0.45-0.50 M NaCl and preferentially dephosphorylated the beta-subunit of phosphorylase kinase. It was inhibited by inhibitor-2, heparin, histone H1, and okadaic acid (IC50 = 70 nM). The 37- and 36-kDa phosphatases can be classified as type-2A and type-1 enzymes, respectively. The 41-kDa phosphatase does not precisely fit the criteria of either type, showing only partial similarities to both type-1 and type-2A enzymes and it may represent a novel type of protein phosphatase in bovine platelets.
Erdödi
F
F
Department of Medical Chemistry, University Medical School of Debrecen, Hungary.
Csortos
C
C
Sparks
L
L
Murányi
A
A
Gergely
P
P
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004988:Ethers, Cyclic / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology
D004988:Ethers, Cyclic; D046911:Macromolecular Substances; D010975:Platelet Aggregation Inhibitors; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases