Enzymatic Synthesis of Unlabeled and β-32P-labeled β-l-2′,3′-Dideoxyadenosine-5′-triphosphate as a Potent Inhibitor of Adenylyl Cyclases and Its Use as Reversible Binding Ligand

β-l-2′,3′-Dideoxyadenosine-5′-triphosphate (β-l-2′,3′-dd-5′-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The β-32P-labeled analog was prepared similarly, but in a two step reaction. β-l-2′,3′-dd-5′-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5′-ATP·Mn2+) and exhibited an IC50 ∼24 nm. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of β-l-2′,3′-dd-[β-32P]5′-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn2+ to a greater extent than by Mg2+. Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2′,5′-dd-3′ATP, a post-transition state noncompetitive inhibitor.

␤-L-2,3-Dideoxyadenosine-5-triphosphate (␤-L-2,3dd-5-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The ␤-32 P-labeled analog was prepared similarly, but in a two step reaction. ␤-L-2,3-dd-5-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5-ATP⅐Mn 2؉ ) and exhibited an IC 50 ϳ24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of ␤-L-2,3-dd-[␤-32 P]5-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn 2؉ to a greater extent than by Mg 2؉ . Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2,5-dd-3ATP, a post-transition state noncompetitive inhibitor.
Adenylyl cyclases (ATP-pyrophosphate lyase (cyclizing); E.C. 4.6.1.1.) are a family of membrane-bound enzymes that catalyze the formation of cAMP from 5Ј-ATP. From several lines of evidence, it has become clear that the adenylyl cyclase catalytic site exhibits specificity for the adenine moiety, enhanced binding via substrate and inhibitor phosphate groups, and tolerance of modifications to the ribose (1)(2)(3)(4)(5)(6)(7)(8). The impact of the phosphate groups, in terms of structure and electronic character, to interaction of nucleotides with adenylyl cyclases is evident in the observed catalytic efficacy of known substrates (2Ј-d-5Ј-ATP Ͼ 5Ј-ATP Ͼ 5Ј-ATP␥S 1 Ͼ 5Ј-APP(NH)p Ͼ 5Ј-APP(CH 2 )P), 2 the competitive but weak inhibition by 5Ј-AP(CH 2 )PP (6), and the enhanced inhibition via the so-called P-site 3 of adenine nucleosides with progressively more phosphates added at the 3Ј-ribosyl position (1-4, 9 -11). That the enzyme tolerates modifications at the ribose was evident in the earliest comparisons of inhibitors (1, 9 -11) and was developed further in a characterization of P-site-mediated inhibition of several adenylyl cyclase isozymes (5). The adenine nucleoside 3Ј-polyphosphates are the most potent P-site ligands (1)(2)(3)(4) and inhibit via a dead-end noncompetitive mechanism implying that they bind to the enzyme in the post-transition state configuration for and at the leaving site(s) of the products, cAMP and inorganic metal⅐PP i (12)(13)(14). Whereas inhibition by P-site ligands has been well characterized biochemically and pharmacologically and potent and specific inhibitors of the enzyme have been synthesized, potent agents targeted to the pretransition state configuration with which substrate interacts have not been identified.
␤-L-Enantiomers of adenosine and its 5Ј-phosphorylated derivatives are modified through a rotation of the ribose relative to the orientations of adenine and the 5Ј-phosphate and have proven useful as analogs of the naturally occurring ␤-D-5Ј-ATP in the study of viral enzymes and pharmacological characterizations of purinergic receptors (15)(16)(17)(18). The attraction of these enantiomers for work with adenylyl cyclases is that the 3Ј-OH is on the opposite side of the ribose relative to the normal substrate and should place it too distant for the cyclizing reaction yielding cAMP to occur. The importance of the position of the 3Ј-OH group for effective product formation is obvious but its placement in the enzyme catalytic cleft becomes evident from studies of the enzyme reaction (12)(13)(14) and its structure (7,8). Because of the possibility that such ligands would be competitive inhibitors of adenylyl cyclases, several ␤-L-adenosine 5Ј-phosphates were synthesized and their inhibition of a native adenylyl cyclase extracted from rat brain has been characterized. A facile enzymatic procedure was developed for the preparation of unlabeled-and 32 P-labeled analogs of the most potent of these compounds, which was then used for evaluating its reversible binding with the enzyme.
1 H and 31 P NMR spectra were recorded at ambient temperature with a Bruker AC 250 (250 MHz) or Bruker AC 400 (400 Mhz) spectrometer. 1 H NMR chemical shifts (␦) are quoted in parts per million (ppm) referenced to the residual solvent peak (dimethyl sulfoxide (Me 2 SO-d 5 ) at 2.49 ppm) relative to tetramethylsilane. 31 P chemical shifts are reported in ppm with phosphoric acid (H 3 PO 4 ) as external reference. FAB mass spectra were recorded in the positive-ion or negative-ion mode with a JEOL DX 300 mass spectrometer with thioglycerol/glycerol (1/1, v/v, G-T) as matrix. UV spectra were recorded on an Uvikon 810 (KONTRON) spectrophotometer. Optical rotations were measured in a 1-cm cell on a Perkin-Elmer model 241 spectropolarimeter.
Following a previously described procedure (23), a solution of tetrasodium pyrophosphate decahydrate (Sigma S-9515) (446 mg, 1.0 mmol) in water was passed through Dowex 50W-X2 (20 ml; pyridine form). The collected fractions were concentrated to dryness and diluted with pyridine (30 ml) and tri-n-butylamine (1.0 ml). The homogeneous solution was evaporated to a syrup and co-evaporated with pyridine and toluene. The material was dissolved into N,N-dimethylformamide (2.0 ml) and tributylamine (0.20 ml) to give the anhydrous pyrophosphate solution, which was used later.
A cooled (0°C) solution of ␤-L-5Ј-adenosine (1) (53 mg, 0.20 mmol) in trimethylphosphate (0.5 ml) was treated with phosphorus oxychloride (27 l, 0.26 mmol). The mixture was stirred at 0°C for 1.5 h, then the pyrophosphate solution was quickly added under vigorous stirring. 1 min later, the trimetaphosphate was hydrolyzed by adding 20 ml of cold TEA⅐HCO 3 (1 M, pH 7.2), and the mixture was concentrated to dryness. The crude material was enriched by anion exchange chromatography (DEAE-Sephadex A-25; equilibrated in 1 mM TEA⅐HCO 3 ), which was developed with a linear gradient of TEA⅐HCO 3 (1 mM to 1 M). A subsequent enrichment by preparative TLC (isopropanol/aqueous ammonia solution 20%/water, 11/7/2, v/v/v) was performed, followed by filtration through a Millex unity HV-4 (0.45 m, Millipore) to afford a solid material, which was again subjected to anion exchange chromatography as above. The appropriate fractions were combined, the solvent was evaporated under reduced pressure at room temperature, and the residue was lyophilized several times from water. The triphosphate derivative was converted to its sodium salt by passing an aqueous solution through a column of Dowex 50W-X2 (Na ϩ form). Lyophilization afforded ␤-L-5Ј-ATP (5, 45 mg) as a white powder.
Enzymatic Preparation of Unlabeled and 32 P-Labeled ␤-L-2Ј,3Ј-dd-5ЈATP-Unlabeled ␤-L-2Ј,3Ј-dd-5Ј-ATP was prepared from ␤-L-2Ј,3Ј-dd-5Ј-AMP (4) by incubation with myokinase, creatine kinase, and creatine phosphate, with a less than stoichiometric concentration of 5Ј-ATP. 4 This 5Ј-ATP is used by myokinase in the primary phosphorylation of ␤-L-2Ј,3Ј-dd-5Ј-AMP but is then regained from creatine phosphate by the action of creatine kinase. Although concentrations of 5Ј-ATP Ͼ100 M will allow the total conversion of ␤-L-2Ј,3Ј-dd-5Ј-AMP to ␤-L-2Ј,3Јdd-5Ј-ATP to occur more rapidly, separation of substrates and products becomes more difficult. The typical phosphorylation of 1 mM ␤-L-2Ј,3Јdd-5Ј-AMP was achieved with a reaction mixture containing 100 M 5Ј-ATP, 100 g of myokinase/ml, 100 g of creatine kinase/ml, 5 mM creatine phosphate, 10 mM MgCl 2 , and 50 mM triethanolamine⅐HCl in 4.4 ml overnight at 30°C; effective reaction times were determined in other experiments. The resulting ␤-L-2Ј,3Ј-dd-5Ј-ATP was separated from the starting 5Ј-monophosphate and 5Ј-ATP by reverse phase HPLC (see Fig. 1, described below). The column (Beckman Ultrasphere; 5 m, 4.6 ϫ 250 mm) was developed with a linear gradient from a buffer containing 100 mM triethylammonium bicarbonate, pH 8, to a buffer containing 0.9 M triethylammonium bicarbonate, pH 8.5 and 10% methanol. 5 The appropriate fractions were pooled, triethylammonium bicarbonate was removed by repetitive roto-evaporation from HPLC grade methanol, the dried product was resuspended in water, and then stored at Ϫ80°C. Under these reaction conditions there is a quantitative conversion of ␤-L-2Ј, 3Ј-dd-5Ј-AMP to the corresponding 5Ј-triphosphate and the only losses incurred are those associated with purification of product.
Labeled ligand was prepared similarly, but with a two-step incubation, first with myokinase and [␥-32 P]5Ј-ATP and then with creatine phosphate and creatine kinase. Because of the enzymes and [␥-32 P]5Ј-ATP used for this preparation, the ␤-L-2Ј,3Ј-dd-5Ј-ATP becomes labeled in the ␤-phosphate. In a typical reaction 10 mCi of [␥-32 P]5Ј-ATP was incubated in a reaction mixture containing 300 M ␤-L-2Ј,3Ј-dd-5Ј-AMP, 1 mg myokinase/ml, 10 mM MgCl 2 , and 50 mM triethanolamine⅐HCl overnight at 30°C in a volume of 680 l. To inactivate the myokinase, the sample was placed in a boiling water bath for 10 min. It was then cooled on ice for 5 min and creatine phosphate and creatine kinase were added to final concentrations of 10 mM and 1 mg/ml, respectively, to a final volume of 800 l. The reaction was allowed to proceed overnight at 30°C. Substrate and product nucleotides were purified by reverse phase HPLC as used before for the unlabeled ligand above. 32 P from [␥-32 P]5Ј-ATP (Fig. 1, top panel) was effectively transferred to ␤-L-2Ј,3Јdd-5Ј-AMP to yield ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ADP (Fig. 1, middle panel). A small amount of an unknown labeled material eluted later than the 5Ј-diphosphate, suggesting that some 5Ј-triphosphate was also formed.
Because this was not seen in other reactions in which the myokinase concentrations was one-tenth of that used here, it was likely due to contamination of the myokinase with an appropriate phosphotransferase activity. The phosphorylation to ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP was essentially quantitative under these conditions (Fig. 1, bottom panel). The 32 P-labeled product was collected, concentrated, and stored as was done for the unlabeled nucleotide above. Unlabeled and labeled ␤-L-2Ј,3Ј-dd-5Ј-ATP co-migrated upon both anion exchange and reverse phase HPLC (not shown). Taking into account losses due to purification and isotope decay, the overall yield was ϳ6 mCi of purified ␤-L-2Ј,3Јdd-[␤-32 P]5Ј-ATP from 10 mC i [␥-32 P]5Ј-ATP. An advantage of ␤-L-2Ј,3Јdd-[␤-32 P]5Ј-ATP as ligand is that any ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ADP formed during incubations with crude enzyme preparations can be rephosphorylated by an ATP-regenerating system comprising creatine phosphate and creatine kinase with no effect on the isotope-specific activity of the nucleotide.
Quantifying Nucleotides by HPLC-Unlabeled nucleotides were quantified after HPLC as areas under peaks determined with a Waters 996 photodiode array detector and the accompanying Millennium software (v.2.10). Radioactivity of HPLC column eluates was quantified with a Flow-Count Detector from Bioscan (Washington, D.C.) with the accompanying Laura software (GC Ram version 1.4a) from LabLogic (Sheffield, UK).
Adenylyl Cyclase Preparation and Assay-Adenylyl cyclase was prepared as a detergent-dispersed extract from rat brain as described previously (1,24). Activity was assayed in a reaction mixture containing 50 mM HEPES buffer, pH 7.5, 5 mM MnCl 2 , 100 M forskolin, 0.1 mM [␣-32 P]5Ј-ATP (2 ϫ 10 5 to 10 6 cpm), in a volume of 100 l, for 15 min at 30°C. The reaction was started by the addition of [␣-32 P]5ЈATP and was ended by the addition of zinc acetate and sodium carbonate (24). [ 32 P]cAMP was purified by sequential chromatography on Dowex 50 and alumina and was quantified in a scintillation spectrometer by Cherenkov radiation. Inhibition kinetics were evaluated as described previously (25) with variable concentrations of substrate Mn 2ϩ ⅐5Ј-ATP and free cation held fixed in excess of the 5Ј-ATP concentration.

RESULTS
Adenine Nucleotide Isomers and Inhibition of Adenylyl Cyclase-Product formation from the normal substrate, ␤-D-5Ј-ATP, was effectively blocked by its L-isomer, but notably at a concentration significantly lower than that of the substrate (Fig. 2); ␤-L-5Ј-ATP exhibited an IC 50 ϳ3 M in the presence of 100 M substrate. This suggested that the ␤-L-configuration of the nucleotide interacted particularly well with the enzyme nucleotide binding site. By comparison ␤-L-5Ј-AMP exhibited an inhibitory potency comparable to that of the naturally occurring ␤-D-5Ј-AMP ( Fig. 2 and Table I), implying that the addition of phosphate groups may influence the orientation of the ribose moiety in the binding site. The observation that ␤-L-2Ј,3Ј-dd-5Ј-AMP was more potent than ␤-L-5Ј-AMP, how- 5 It is important that the triethylammonium bicarbonate be made fresh for the day of use. Over time, oxidation products of triethylamine will form and the loss of CO 2 , causing an increase in pH, and methanol will result in substantial shifts in retention times.  ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP and Adenylyl Cyclases ever, suggested that this unnatural orientation of the 2Ј-and 3Ј-hydroxyl groups of ␤-L-5Ј-AMP impaired its interaction with the enzyme and that their removal from ␤-L-5Ј-ATP would be expected also to increase inhibitory potency. This was borne out for both ␤-Dand ␤-L-isomers of 2Ј,3Ј-dd-5Ј-ATP ( Fig. 2 and Table I). Consistent with the apparent affinity of ␤-L-5Ј-ATP being greater than that of the naturally occurring D-isomer, ␤-L-2Ј,3Ј-dd-5Ј-ATP was Ͼ30-fold more potent than ␤-D-2Ј,3Јdd-5Ј-ATP. With an IC 50 value of ϳ24 nM, ␤-L-2Ј,3Ј-dd-5Ј-ATP exhibited an inhibitory potency comparable to that of 2Ј,5Ј-dd-3Ј-ATP (IC 50 ϳ40 nM), although it was not as potent as the corresponding 3Ј-tetraphosphate ( Fig. 2 and Table I) (4). With such low IC 50 values, enzyme concentration becomes an important consideration. Although the exact concentration of adenylyl cyclase used in experiments with the rat brain preparation was not known, it was estimated to be approximately 0.9 nM, with the assumptions of a mass of 116 kDa and a specific activity of 7 mol/(min⅐mg of protein) for the purified type I adenylyl cyclase (26,27). This would suggest that the IC 50 values are probably good approximations.
Filtration Binding Assay for Adenylyl Cyclases-The high affinity of ␤-L-2Ј,3Ј-dd-5Ј-ATP and the fact that we could readily synthesize it as a 32 P-labeled ligand suggested the possibility that it could be used in a reversible binding assay for adenylyl cyclases. In early experiments we noted that in the absence of enzyme, components of the incubation buffer significantly affected the apparently nonspecific binding of ␤-L-2Ј,3Јdd-[␤-32 P]5Ј-ATP to nitrocellulose membranes (Table II). At pH 8.2 and 50 mM, both HEPES and TEA⅐HCO 3 resulted in substantial binding of this ligand, but this was reduced at the lower pH of 7.5. Sodium phosphate, even at pH 7.5 and a lower concentration (10 mM) caused a more obviously elevated nonspecific binding. Because of this effect of phosphate, TEA⅐HCl, at pH 7.5, was used in subsequent binding assays.
Binding of ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP to adenylyl cyclase was dependent on enzyme concentration (not shown) and was a saturable process (Fig. 4) under conditions with which equilibrium was rapidly reached (Fig. 5). The average half-time for binding was Ͻ1.8 min, as best as could be determined under these conditions and with these procedures, with maximum binding being achieved within approximately 10 min. When 30 M unlabeled ␤-L-2Ј,3Ј-dd-5Ј-ATP was added at 30 min as indicated, 32 P-labeled ligand was rapidly displaced, with a halftime Ͻ 0.8 min, as best as could be measured here (Fig. 5).  50 values for inhibition of adenylyl cyclase by several adenine nucleotides Rat brain adenylyl cyclase (108 g protein/ml) was assayed as described under "Experimental Procedures" with 100 M 5Ј-ATP as substrate and with 100 M forskolin and 5 mM MnCl 2 . IC 50 values were determined graphically from logistic regression plots. C denotes competitive inhibition; NC denotes noncompetitive inhibition; ND denotes that the inhibitory mechanism was not determined.

Nucleotide
Inhibition pattern IC 50 values rat brain Clearly both on and off rates were rapid and these estimates can only be rough approximations. The concentration of unlabeled ligand used here was Ͼ1,000-fold greater that its IC 50 value and sufficient to displace virtually all labeled ligand from these high affinity binding sites (cf. Table I and Figs. 6 and 7).
Displacement of ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP occurred with concentrations of unlabeled ligand consistent with the sensitivity of the enzyme to inhibition by this ligand (cf. Figs. 2 and 6) and allowed estimation of ligand-specific radioactivity to be ϳ460 Ci/mmol in this experiment. From the Scatchard plot (Fig. 6, inset) a concave upwards plot resulted that may be resolved into two lines yielding maximal binding of 21.6 fmol/g protein and 47.4 fmol/g protein. With the assumptions stated above for the activity of the pure enzyme, a binding of 21.6 fmol/g protein, or 1.3 nmol/liter in this experiment, compares with the estimated enzyme concentration of 0.9 nM. From saturation and displacement data the higher affinity site yielded an estimated K d value of ϳ16 nM.
The concentration-dependent displacement suggested that the 32 P-labeled ligand was binding to adenylyl cyclase and was consistent with the behavior of ␤-L-2Ј,3Ј-dd-5Ј-ATP expected from enzyme inhibition studies (cf. Table I and Figs. 2, 3, and  6). This is supported by the competition displacement pattern observed with several nucleosides and nucleotides (Fig. 7). Not surprisingly, the most effective displacement was achieved with substrate 5Ј-ATP and with the competitive inhibitors ␤-L-2Ј,3Ј-dd-5Ј-ATP and 5Ј-AP(CH 2 )PP. The lesser effectiveness of 2Ј,5Ј-dideoxyadenosine and the corresponding 3Ј-polyphosphates was somewhat unexpected, because these are believed to bind in the catalytic cleft as well, albeit with a different configuration of the enzyme. But the increasingly effective displacement with the addition of 3Ј-phosphates was consistent with the increasing potency of these ligands to inhibit the enzyme ( Table I). The lack of effect of 3Ј-GMP or 3Ј-IMP was expected, because these nucleotides are known not to have any effect on adenylyl cyclase activity. The acyclic adenine derivatives, PMEApp and PMEAp(NH)p, which are comparably effective inhibitors of catalysis (IC 50 values of ϳ170 and ϳ180 nM, respectively (28)), also displaced binding of ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP but with different efficacies.

DISCUSSION
The ␤-L-adenosine 5Ј-phosphate ligands presented here represent a new class of potent inhibitors of one of the most important enzymes involved in mediating transmembrane signal transduction. These compounds considerably extend the number and type of compounds known to inhibit the adenylyl cyclase family of enzymes. Our earlier studies focused on inhibition by P-site ligands (1)(2)(3)(4)(5)13), which exhibit classical noncompetitive inhibition, with the most potent compounds in this series being 2Ј,5Ј-dd-3Ј-ATP (IC 50 ϳ40 nM) and its corresponding 3Ј-tetraphosphate (IC 50 ϳ7.4 nM) (Table I). This inhibition is believed to be via a dead-end mechanism in which inhibitor binds to the leaving configuration for products. The studies in which this was most clearly shown used 2Ј-d-3Ј-AMP as inhibitor and implied the participation of inorganic pyrophosphate, formed with cAMP from 5Ј-ATP by the enzyme (14). Presumably the 3Ј-triphosphate or 3Ј-tetraphosphate, which are also noncompetitive inhibitors, bring their own PP i to the catalytic cleft and do not need additional PP i . By contrast, the ␤-Ladenosine 5Ј-polyphosphates, and the most potent of these (␤-L-2Ј,3Ј-dd-5Ј-ATP; IC 50 ϳ24 nM) in particular, are competitive inhibitors. Thus, we have described two classes of inhibitory nucleotides for adenylyl cyclases. One is a post-transition state noncompetitive inhibitor (2Ј,5Ј-dd-3Ј-ATP) and the other is a comparably potent, pretransition state, competitive inhibitor (␤-L-2Ј,3Ј-dd-5Ј-ATP). The former is viewed as interacting with the enzyme configuration from which cAMP and PP i leave and the latter binds to the same site and configuration with which substrate 5Ј-ATP interacts (Fig. 8). Structures of adenylyl cyclase with 2Ј,5Ј-dd-3Ј-ATP and ␤-L-2Ј,3Ј-dd-5Ј-ATP bound at this site should allow the visualization of the transitions that occur during the catalytic cycle and are presently being developed with the VC 1 ⅐IIC 2 chimeric enzyme complex (29).
An often useful characteristic of adenine nucleotides is that they can be readily labeled with 32 P or 33 P. This and the enzymatic selectivities of myokinase and creatine kinase allowed the ready synthesis of both unlabeled and 32 P-labeled forms of ␤-L-2Ј,3Ј-dd-5Ј-ATP from the corresponding monophosphate. Because the initial labeling step is from [␥-32 P]5Ј-ATP with myokinase, the label appears in the ␤-phosphate position of the triphosphate. Moreover, because [␥-32 P]5Ј-ATP is readily purchased or synthesized (30) at specific activities of 3,000 -6,000 Ci/mmol, it should be possible to prepare ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP routinely with a comparable specific radioactivity, although values we have obtained to date have been in the range of 400 -1,000 Ci/mmol.
The availability of ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP makes several experiments with adenylyl cyclases possible that heretofore have not been possible. One that was explored in this paper was the development of a filtration binding assay for adenylyl cyclase. 6 Consistent with expectations ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP exhibited a rapid, specific, and freely reversible binding to adenylyl cyclase. The interaction was consistent with the concentrations of the unlabeled nucleotide to inhibit adenylyl cyclase and with an interaction with the catalytically active conformation of the enzyme. The Scatchard (31) plot suggests more than one binding affinity, though the basis of this is not known. Obvious possibilities include negative cooperativity, the presence of multiple adenylyl cyclase isozymes in this detergent extract from rat brain, or mistaken assumptions regarding the estimation of specific radioactivity of the 32 P-labeled ligand. Nonetheless, the competitive displacement by other nucleotides suggested further that both ␤-L-2Ј,3Ј-dd-5Ј-ATP and 2Ј,5Јdd-3Ј-ATP interact with adenylyl cyclase at the same site, but with different enzyme conformations, fully consistent with enzyme structures solved with these ligands. In principal, this ligand should prove useful for quantitative estimations of enzyme levels, for characterizing the catalytic domains of adenylyl cyclase isozymes, and in screening applications for the identification and quantifying of other ligands which may interact with this domain. ␤-L-2Ј,3Ј-dd-[␤-32 P]5Ј-ATP and Adenylyl Cyclases