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3' :5'-Cyclic Monophosphate Alfred G Proceedings of the National Academy of Sciences Vol. 67, No. 1, pp. 305-312, September 1970 A Protein Binding Assay for Adenosine 3' :5'-Cyclic Monophosphate Alfred G. Gilman LABORATORY OF BIOCHEMICAL GENETICS, NATIONAL HEART AND LUNG INSTITUTE, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND 20014 Communicated by Marshall Nirenberg, June 24, 1970 Abstract. A simple and sensitive assay for adenosine 3':5'-cyclic monophos- phate (cAMP) has been developed that is based on competition for protein binding of the nucleotide, presumably to a cAMP-dependent protein kinase. The nucleotide-protein complex is adsorbed on a cellulose ester filter. Assay conditions are such that a binding constant approaching 10-9 M\1 is obtained, and the assay is thus sensitive to 0.05-0.10 pmol of cAMP. While assays for adenosine 3' :5'-cyclic monophosphate (cAMIP) are proliferat- ing almost as rapidly as are newly discovered actions of the nucleotide, the tremendous current interest in the biological role of cAMP dictates the need for a sensitive, accurate, and readily performed procedure for estimation of its cellular levels. The methods currently availablel-8 do not have the extreme sensitivity required by the low tissue levels of the compound, or else they are laborious to perform, or both. The present study was initiated with the thought that the binding of [5H]- cAMP to a cAMP-dependent protein kinase9-12 would form the basis for a sensitive assay, if a simple means could be found to isolate the protein-nucleotide complex. Further impetus was -provided by the discovery of Walsh et al.3 that a heat-stable protein, an inhibitor of the cAMP-dependent protein kinase,14"15 increased the affinity of the cyclic nucleotide for the enzyme. 'A protein kinase from muscle was chosen for investigation because of its favorable binding con- stant for cAMP.16 It was readily determined that cAMP-dependent protein kinase and cAMP-binding activities from muscle extracts could be quantitatively adsorbed on cellulose ester (Millipore) filters. The binding of cAMP to specific proteins from Escherichia coli and the adrenal cortex has recently been studied on Millipore filters.17'18 This simple means for estimating cAMP binding may be utilized for assay of unknown quantities of the cyclic nucleotide in deproteinized tissue extracts. As little as 0.05-0.10 pmol of cAMP can be detected; thus, less than milligram quantities of many tissues are sufficient for assay. Materials and Methods. cAMP-dependent protein kinase: Two purification schemes have been employed-a more extensive one to characterize the protein and a simplified method for routine use. Procedures are patterned after those of Walsh et at.9 and Miyamoto et al."' Fresh bovine muscle was prepared as described" through the ammonium sulfate precipitation step. This fraction, from 250 g of muscle, was applied to a column of DEAE-cellulose (Whatinan DE 11, 1 meq/g; 32 X 2.6 cm), previously equilibratcd with 5 mMI potassium phosphate, pH 7, and tile columni was 305 Downloaded by guest on September 30, 2021 306 BIOCHEMISTRY: A. G. GILMAN PROc. N. A. S. s A 30 I -300'7 *2R43E II - -430 I FIG. 1. DEAE-cellulose chroma- K20 200 I tography of protein kinase and cAMP xOOZEg O DO < binding activities: gradient elution lo- HO'Ewasperformed4> it as described under c. k Methods. Binding activity was as- B o sayed at pH 4 with 40 nM cAMP. 4 I l81E Fractions were 20 ml. (A): 0, pro- s03 . \ 6 ° tein kinase activity; --- potassium E , -; phosphatei gradient. (B): 0, cAMP l X4o binding activity; --- optical density C,,, 0~~~~~~at 280 nm. I0 20 30 40 50 60 70 Fraction washed with the same buffer. Elution was with a linear gradient of potassium phos- l)hate, pH 7, from 5 to 400 mM, as shown in Fig. 1. Two prominent peaks of kinase and binding activity are seen.i6 Fractions 39-51, the second major peak of kinase and bind- ing activity (DEAE II), were pooled and dialyzed against 5 mM potassium phosphate, pH 7. A portion of this preparation was concentrated by precipitation with ammonium sulfate (0.33 g/ml), dialyzed, and applied to a column of Sephadex G-200 (2.5 X 100 cm), equilibrated and eluted with 5 mM potassium phosphate, pH 7. For routine purposes a satisfactory preparation was obtained by applying the am- monium sulfate fraction1' to a column of D)EAE-cellulose aiid eluting the early peak(s) of activity with 100 mM potassium phosphate, pH 7. The last l)eak (D)EAE II) was theft collected with 300 mM potassium phosphate and was dialyzed against 5 mM potassium phosphate, pH 7. Such a preparation was utilized in this work and had an enzymatic activity of 24 pmol of 32P/,gg protein per 10 mil, and bound 0.3 l)nol of cAMP/Jug protein under standard assay conditions. Up to 200 /Ag protein of this l)relparatioii could be applied to a single Millipore filter with resultant quantitative removal of kinase, binding activity, and protein. When larger amounts were filtered, the kinase, binding activity, and protein were removed to a very similar extent. Both kinase and binding activity are stable for several months at -400C. Ptotein-kinase inhibitor: The inhibitor preparation was modeled after that of Appleman et al.', Bovine muscle was homogenized in 10 mM Tris chloride, pH 7.5 and was boiled for 10 min. After removal of particulate material by filtration, activity was precipitated with 1/9 volume of 50% trichloroacetic acid. The precipitate was col- lected at 15,000 X g and dissolved in water, and pH was adjusted to 7 with 1 N NaOH. This fraction was dialyzed against distilled water at room temperature, and the pre- cipitate which formed was discarded. The preparation was used at this stage of purity, although it was verified that the kinase-inhibitory activity fractionated on Sephadex G-75 as previously described." Cyclic 3' :5'-nucleotide phosphodiesterase: This enzyme was purified from bovine cardiac muscle through the dialyzed acid-supernatant fraction of Butcher and Suther- land.19 Protein kinase assay: cAMP-dependent protein kinase was assayed essentially as described,ii although phosphorylated protein was precipitated with 8% trichloro- acetic acid, and precipitates were collected on glass fiber filters (Millipore). cAMP binding and cAMP assay: The standard binding reaction was conducted in a volume of 50 or 200 ,l in 50 mM sodium acetate/acetic acid, pH 4.0 and incubated for longer than 60 min at 0C. The only other components of the incubation were [3H]- cAMP (Schwarz BioResearch, 16.3 Ci/mmole), sufficient binding protein to bind less than 30% of the nucleotide, and, where indicated, a maximally effective concentration of Downloaded by guest on September 30, 2021 VOL. 67, 1970 ASSAY FOR CYCLIC AMP 307 the protein-kinase inhibitor preparation. Reactions were initiated by addition of bind- ing protein. At equilibrium, the mixtures were diluted to 1 ml with cold 20 mM potas- sium phosphate, pH 6; 4-5 min later they were passed through a 24-mm cellulose ester (Millipore) filter (0.45 Am) previously rinsed with the same buffer. The filter was then washed with 10 ml of this buffer and placed in a counting vial with 1 ml of Cellosolve, in which the filter readily dissolves. A scintillation mixture of toluene-Cellosolve (3:1) plus fluors was utilized, and efficiency was approximately 30% (10,750 cpm/pmol). Bowud counts were independent of filtration speed and the volume of rinse from 3 to 20 ml. The blank in the absence of binding protein was 20-50 cpm. For the assay of cAMP, [(H]cAMP was utilized at 10-20 nM (0.5-1.0 pmol/50 Al) in the presence of the inhibitor or 40 nM in its absence. These are saturating concentra- tions of cAMP, and the effect of added unknown or standard cAMP solutions could thus be evaluated from a linear, and nearly theoretical, decrease in the total bound [3H]- cAMP. Protein-kinase inhibitor assay: The inhibitor preparation may be assayed in the cAMP-dependent protein kinase reaction or by its enhancement of [3H]cAMP binding at submaximal concentrations of the nucleotide. Tissue extracts: Tissue samples were homogenized in 1 ml of 5% trichloroacetic acid, and supernatants were extracted 5 times with 2 volumes of ether after the addition of 0.1 ml of 1 N HCl. The eltracts were then dried and redissolved in 50 mM sodium acetate, pH 4. Results. The choice of the DEAE peak (Fig. 1) of binding and protein kinase activity to use in this assay (DEAE II) was made because of its apparent greater purity and because of a slightly greater affinity for cAl\IP (data not shown). The first peak of activity (DEAE I) had the advantage of little de- pendence of binding affinity and total binding sites on pH. An additional small peak(s) of binding and kinase activity between DEAE peaks I and II may be seen in Fig. 1; it was not investigated further. Proof that the binding protein is in fact the cAl\IP-dependent protein kinase must await further purification of the protein. However, cochromatography of the two activities on DEAE-cellulose and Sephadex G-200 (data not shown) supports the hypothesis that they are properties of a single molecule. Cochro- matography on Sephadex G-200 was also apparent when the binding protein was labeled with [3H ]cAMP before gel filtration. During initial purification (Table 1), the specific activity of the protein kinase increased somewhat more than that for binding, perhaps because of removal of inhibitors of kinase activity" or additional cAMP-binding proteins.
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