8-Azidoadenosine 3',5'-monophosphate (8-N3-cAMP) containing 32P has been used as a photoaffinity label specific for the adenosine 3',5'-monophosphate (cAMP) binding site(s) present in a partially purified preparation of soluble protein kinase from bovine brain. 8-N3-cAMP and cAMP were found to compete for the same binding site(s) in this preparation, as determined by a standard filter assay. When this protein preparation was equilibrated with [32P]-8-N3-cAMP, and then irradiated at 253.7 nm, the incorporation of radioactivity was predominantly into a protein with an apparent molecular weight of 49,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. This labeled protein comigrated in the gel with the only protein which is endogenously phosphorylated by [gamma-32P]ATP, a protein which has been shown to be the regulatory subunit of the protein kinase (H. Maeno, P. L. Reyes, T. Ueda, S. A. Rudolph, and P. Greengard (1974), Arch. Biochem. Biophys. 164, 551). The incorporation of [32P]-8-N3-cAMP into this protein was half-maximal at a concentration of 7 x 10(-8) M. In accordance with a proposed mechanism involving the formation of a highly reactive nitrene intermediate upon irradiation of the azide, the incorporation of radioactivity into protein was maximal within 10 min of irradiation, and was almost eliminated by preirradiation of the photolabile ligand. Moreover, this incorporation was virtually abolished by a 50-fold excess of cAMP, but not by AMP, ADP, ATP, or adenosine. We suggest that 8-N3-cAMP may prove to be a useful molecular probe of the cAMP-binding site in receptor proteins and report its use in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a highly sensitive and selective radiochemical marker for cAMP-binding proteins.

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Synthetic polypeptides were employed as substrates in kinetic analyses of the reaction mechanism for the catalytic subunit of a cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from calf thymus. This enzyme preparation was shown to catalyze the transfer of phosphate from ATP to histone HI from calf thymus, as well as to two synthetic polypeptides, Arg-Lys-Ala-Ser-Gly-Pro (Hi-6) and Arg-Arg-Lys-Ala-Ser-Gly-Pro (Hi-7), corresponding to the amino acid sequence about serine-38 in calf HI. A related, basic heptapeptide corresponding to a sequence from pig liver pyruvate kinase, Leu-Arg-Arg-Ala-Ser-Leu-Gly (K), was also a substrate. The stoichiometry of peptide phosphorylation was established in each case as the transfer of 1 mol of phosphate from the y position of MgATP to the serine hydroxyl of 1 mol of the peptide. Steady-state, initial-velocity, kinetic parameters were determined for each substrate, using various concentrations of ATP. Under the conditions used, all synthetic peptides reacted with greater maximum velocities than whole histone HI. Nevertheless, the Km for HI, 54 ,uM, was lower than the Km values of the synthetic substrates. The most efficient substrate was peptide K, which had a V..l of 50.6,unmol/min per mg of kinase and a Km of 63 ;&M. In the absence of peptide substrate no ATPase activity was detectable at a sensitivity of 0.05% of the rate of peptide phosphorylation, suggesting that ATP is not cleaved to form an unstable phosphoenzyme-complex. The data are consistent with a sequential reaction mechanism involving a ternary complex between enzyme, polypeptide substrate, and ATP.Protein phosphorylation catalyzed by 3':5'-cyclic AMP (cAMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) mediates many, if not all, of the effects of cAMP in eukaryotic cells (1). The use of synthetic polypeptides as substrates for cAMP-dependent protein kinase has thus far provided data concerning structure-efficacy relationships among phosphorylatable amino acid sequences (2-5). In particular, reports have concerned the minimum length of such sequences (2, 3) and the role of multiple basic residues on the NH2-terminal side of the phosphorylated serine in establishing substrate specificity (4, 5). Apart from modeling phosphorylation site specificity for protein kinases at the level of primary structure, these molecules are ideally suited for probing the catalytic mechanism of protein phosphorylation by a given kinase. The defined structure, homogeneity, and ease of manipulation of synthetic peptides allow a more accurate quantitation of catalytic parameters than can be obtained using intact protein substrates.In the present communication we examine the kinetic mechanism of peptide phosphorylation, using a highly purified protein kinase from calf thymus. We have synthesized hexaand heptapeptides corresponding to sequences about serine 38 in calf thymus histone HI, a reported site of cAMP-dependent

A cyclic AMP-like substance has been isolated from higher plant tissues which can be quantitated with the use of a radioimmunoassay similar to that described by A. L.Steiner, D. M. Kipnis, R. Utiger, and C. Parker 1(1969) Proc. Natd Acad. Sci. USA 64, 367-3731. This compound has been extensively purified and is chromatographically distinct from authentic cyclic AMP. This c clic AMP-like compound inhibited beef heart 3':5'-cyclic-nucleotide phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17), with halfmaximal inhibition occurring at a concentration of 7.6 X 10-10 M cyclic AMP equivalents. The compound also inhibited cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase; EC 2.7.1.37) from bovine heart, with halif-maximal inhibition of mixed histone phosphorylation occurring at 8.0 X 10-11 M cyclic AMP equivalents. Equipotent inhibition of phospho ation and associated trace ATPase activity were observed with the purified catalytic subunit of cyclic AMPdependent protein kinase from calf thymus with a synthetic heptapeptide as substrate. Moreover, steady-state kinetic analysis of this inhibition in the latter system showed it to be nonlinear and noncompetitive versus MgATP.Cytokinesin I, a naturally occurring cell-division-promoting compound isolated from crown gall tumor cells, is a potent inhibitor of animal and plant phosphodiesterases (1). Similarly, 8-bromo cyclic AMP, a phosphodiesterase-resistant analogue of cyclic AMP (cAMP), as well as theophylline, an inhibitor of cAMP phosphodiesterase, promote cell division when used in association with an auxin in normal tobacco pith parenchyma cells (1, 2). These findings suggested that cytokinesin I may exert its cell-division-promoting activity as a regulator of cAMP metabolism. By using a radioimmunoassay for cAMP (3), a positive correlation was found between the intracellular levels of a cAMP-like compound and cell enlargement and chromosomal DNA replication in normal tobacco pith cells (4). There nevertheless appears to be no unequivocal evidence for the presence of authentic cAMP in cells of higher plant species, nor any evidence for the occurrence of a cyclic nucleotide-dependent protein kinase in higher plants, although post-translational protein phosphorylation is known to occur (5).Herein we report the isolation and purification of a compound from higher plant species that reacts positively with a radioimmunoassay for cAMP and inhibits bovine heart cAMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase; EC 3.1.4.17) activity but is chromatographically distinct from cAMP. Moreover, we report the efficacy of this compound as an inhibitor of protein phosphorylation catalyzed in vitro either by bovine heart cAMP-dependent protein kinase (ATP:protein phosphotransferase; EC 2.7.1.37) holoenzyme with mixed histones as substrate or by the isolated catalytic subunit of calf thymus cAMP-dependent protein kinase with a synthetic heptapeptide, Ktide, Leu-Arg-Arg-Ala-Ser-Leu-Gly, as substrate (6). We used th...

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