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Novack,* Harry Charbonneau,* J. Kelley Bentley,' Kenneth A. Walsh,i and Joseph A. Beavo*** Departments zyxwvuts of Pharmacology and Biochemistry, University of Washington, Seattle, Washington 98195 Received December 12, 1990; Revised Manuscript Received April 1, 1991 ABSTRACT: Partial protein sequences from the 59-kDa bovine heart and the 63-kDa bovine brain calmo- dulin-dependent phosphodiesterases (CaM-PDEs) were determined and compared to the sequence of the 61-kDa isozyme reported by Charbonneau et al. [Charbonneau, H., Kumar, S., Novack, J. P., Blumenthal, D. K., Griffin, P. R., Shabanowitz, J., Hunt, D. F., Beavo, J. A. & Walsh, K. A. (1991) Biochemistry (preceding paper in this issue)]. Only a single segment (34 residues) at the N-terminus of the 59-kDa isozyme lacks identity with the 61-kDa isozyme; all other assigned sequence is identical in the two isozymes. Peptides from the 59-kDa isozyme that correspond to residues 23-41 of the 61-kDa protein bind calmodulin with high affinity. The C-terminal halves of these calmodulin-binding peptides are identical to the corresponding 59-kDa sequence; the N-terminal halves differ. The localization of sequence differences within this single segment suggests that the 61- and 59-kDa isozymes are generated from a single gene by tissue-specific alternative RNA splicing. In contrast, partial sequence from the 63-kDa bovine brain CaM-PDE isozyme displays only 67% identity with the 61-kDa isozyme. The differences are dispersed throughout the sequence, suggesting that the 63- and 61-kDa isozymes are encoded by separate but homologous genes. c y c l i c nucleotide phosphodiesterases (PDEs)' catalyze the hydrolysis of cAMP and/or cGMP to their corresponding 5'-nucleoside monophosphates. At least five different enzyme families have been identified, and most of these families contain multiple forms of closely related PDEs. One large family, the CaM-dependent PDEs (CaM-PDEs) is particularly critical for intracellular signaling in that they respond to calcium by decreasing the concentration of cAMP and cGMP [for review see Wang et al. (1990)l. Although a number of 'This work was supported by National Institutes of Health Grants DK 21723 and EY08197 (to. J.A.B.),GM15731 (to K.A.W.),and GM07750 (to J.P.N.). * Address correspondence to this author. 4 Department of Pharmacology. 8 Department of Biochemistry. members of the CaM-PDE family have been described (Beavo, 1988; Beavo & Reifsnyder, 1990), three well-characterized forms are the 59-kDa isozyme isolated from bovine heart and the 61- and 63-kDa isozymes isolated from bovine brain (LaPorte & Storm, 1979; Hansen & Beavo, 1982; Sharma & Wang, 1986, 1987). Sharma and Wang (1986) also de- scribed a 58-kDa CaM-PDE from bovine lung that bound I Abbreviations: PDE, cyclic nucleotide phosphodiesterase; CaM, calcium/calmodulin complex, CaM-PDE, calmodulin-dependentcyclic nucleotide phosphodiesterase; CM, S-carboxymethyl; BNPS-skatole, an acronym for the reagent described by Fontana et al. (1973); HPLC, high-performanceliquid chromatography; TPCK, Nn-p-tosyl-L-phenyl- alanine chloromethyl ketone; EGTA, ethylene glycol bis((3-aminoethyl ether)-N,N,N',N'-tetraacetic acid; SDS-PAGE, sodium dodecyl sul- fate-polyacrylamide gel electrophoresis; Pth, phenylthiohydantoin. 0006-2960/9 1 /0430-7940$02.50/0 zyxwvu 0 1991 American Chemical Society