The catalytically active domain in the A subunit of calcineurin.
Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAdelta that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn2+ and Ni2+ ions effeciently activated CaNA. The Km of a/CaNA was about 16 mM, and the k(cat) of a/CaNA was 10.03 s(-1) using pNPP as substrate. With RII peptide as a substrate, the Km of a/CaNA was about 21 microM and the k(cat) of a/CaNA was 0.51 s(-1). The optimum reaction temperature was about 45 degrees C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-subunit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN.
Xiang
Benqiong
B
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, PR China.
Liu
Ping
P
Jiang
Guohua
G
Zou
Ke
K
Yi
Fang
F
Yang
Shujie
S
Wei
Qun
Q
D009597:4-Nitrophenylphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D019703:Calcineurin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002118:Calcium; D020134:Catalytic Domain / Q000235:genetics*; D002413:Cations, Divalent; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese; D009154:Mutation; D009532:Nickel; D051381:Rats; D013696:Temperature
D002413:Cations, Divalent; D008345:Manganese; D009532:Nickel; D019703:Calcineurin; D009597:4-Nitrophenylphosphatase; D002118:Calcium