The type 2A protein phosphatases in mammalian tissue extracts are inhibited completely and specifically by 1-2 nM okadaic acid. In contrast, type l protein phosphatases are hardly affected at these concentrations, complete inhibition requiring 1 #M okadaic acid. These observations have been exploited to develop an improved procedure for the identification and quantitation of type 1, type 2A and type 2C protein phosphatases in tissue extracts.

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulindependent multiprotein kinase (CaM-MPK) and cyclic AMP-dependent protein kinase were shown to be Ser-19 and Ser-40, respectively, while Ser-40 was also phosphorylated slowly by CaM-MPK. (ii) Type 2A and type 2C phosphatases accounted for ~90% and z lm of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free M@+(l mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromatBn cells with okadaic acid increased TH phosphorylation by 206% and activity by 775, establishing that type 2A phosphatases play a major role in regulating TH in vivo.

Protein phosphatase activities in extracts of the yeast Saccharomyces cerevisiae showed remarkable similarities to the mammalian type 1, type 2A and type 2C enzymes. Similarities included their substrate specificities, including selectivity for the ~t-and fl-subunits of muscle phosphorylase kinase, sensitivity to okadaic acid and to mammalian inhibitor 1 and inhibitor 2, and requirement for divalent cations. The results suggest that the function and regulation of these enzymes has been highly conserved during evolution and indicate that the improved procedure for identifying and quantitating protein phosphatases [(1989) FEBS Lett. 250, 000-000] may be applicable to all eukaryotic cells.

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