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 phosphatas e type 2B (PP2B/calcineurin), an accurate assay method of PP2B in crude tis sue extracts has not been established. By using recombinant protein phospha tase inhibitor-1 as a substrate and ascorbic acid as an antioxidant, we dev eloped an improved assay method for PP2B activity in crude extracts from mo use 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 prote in in mouse brain, heart, spleen, liver, and testis, respectively, and furt hermore 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, hip pocampus, cerebellum, and brain stem, respectively. This is the first paper to demonstrate a close correlation between tissue distributions and conten t of PP2B, These results showed that the present assay method is extremely powerful for precise measurement of a wide range of PP2B activities includi ng not only high PP2B activity in the brain but also low PP2B activities in other tissues. (C) 2000 Academic Press.