Development of an assay method for activities of serine/threonine protein phosphatase type 2B (calcineurin) in crude extracts.
Despite the physiological importance of serine/threonine protein phosphatase type 2B (PP2B/calcineurin), an accurate assay method of PP2B in crude tissue extracts has not been established. By using recombinant protein phosphatase inhibitor-1 as a substrate and ascorbic acid as an antioxidant, we developed an improved assay method for PP2B activity in crude extracts from mouse tissues and investigated tissue distribution of its activity. Under the assay conditions, the PP2B activities were stable for at least 30 min with more than 100-fold higher sensitivity than those previously reported. The specific activities of PP2B were 22.3, 0.85, 2.9, 0.36, and 1.5 mU/mg protein in mouse brain, heart, spleen, liver, and testis, respectively, and furthermore in each region of the brain they were 26.1, 13.7, 42.8, 40.5, 15.1, and 8.6 mU/mg protein in cerebrum, midbrain plus interbrain, striatum, hippocampus, cerebellum, and brain stem, respectively. This is the first paper to demonstrate a close correlation between tissue distributions and content of PP2B. These results showed that the present assay method is extremely powerful for precise measurement of a wide range of PP2B activities including not only high PP2B activity in the brain but also low PP2B activities in other tissues.
Mitsuhashi
S
S
Section of Biochemistry, Institute of Immunological Science, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo, 060-0815, Japan.
Shima
H
H
Kikuchi
K
K
Igarashi
K
K
Hatsuse
R
R
Maeda
K
K
Yazawa
M
M
Murayama
T
T
Okuma
Y
Y
Nomura
Y
Y
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019703:Calcineurin / Q000032:analysis*; D002645:Chickens; D051379:Mice
D019703:Calcineurin