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[1] A s s a y o f A d e n y l y l C y c l a s e C a t a l y t i c A c t i v i t y

By ROGER A. JOHNSON and YORAM SALOMON Adenylyl cyclase (ATP pyrophosphate-lyase, cyclizing, EC 4.6.1.1, adenylate cyclase) is a family of membrane-bound enzymes that exhibit inactive and active configurations resulting from the actions of a variety of agents, acting indirectly and directly on the enzyme. Enzyme activity may be increased or decreased by stimulatory or inhibitory hormones acting via specific hormone receptors coupled to the catalytic unit of the enzyme by the respective guanine nucleotide-dependent regulatory proteins (G s and Gi, respectively). The G proteins are also activated by aluminum fluoride and are targets for ADP-ribosylation by specific bacterial toxins. The catalytic moiety of adenylyl cyclase from most tissues is stimulated by the diterpene forskolin and is inhibited by analogs of adenosine, the most potent of which is 2'-deoxyadenosine 3'-monophosphate, and the enzyme from some tissues is also stimulated directly by Ca2÷/ calmodulin. The nature of some of these agents and the range of resulting activities can influence the assay conditions used for determining the catalytic activity of the enzyme. The catalytic activity of adenylyl cyclase is determined by methods that rely on the measurement on cAMP formed from unlabeled substrate, with cAMP-binding proteins or radioimmunoassay procedures, or by methods that rely on the use of radioactively labeled substrate followed by isolation and determination of the radioactively labeled product. The two different approaches have different purposes, different sensitivities, and different ease of use. The method of choice will depend in part on the facilities and orientation of a given laboratory. The procedures described here focus on the use of radioactively labeled substrate and isolation of the labeled product. Additional detailed considerations for the assay of adenylyl cyclase by these procedures can be found in the review by Salomon. Considerations for Establishing Reaction Conditions

Requirements for Metal-ATP and Divalent Cations Both ATP and divalent cation (Mg2+ or Mn 2÷) are required for adenylyl cyclase-catalyzed formation of cAMP. 2'3The enzyme conforms to a bireacI y . Salomon, Adv. Cyclic Nucleotide Res. 10, 35 (1979). 2 T. W. Rail and E. W. Sutherland, J. Biol. Chem. 232~ 1065 (1958). s E. W. Sutherland, T. W. Rail, and T. Menon, J. Biol. Chem. 237, 1220 (1962).

METHODS IN ENZYMOLOGY. VOL. 195

Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.

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adenylyl cyclase MgPP i

X ~ g A T P

- -

--

Ino FIG. 1. Reactions involving adenine nucleotides in membrane preparations.

tant sequential mechanism in which metal-ATP2- is substrate and free divalent cation is a requisite cofactor. 4 Thus, adenylyl cyclase requires divalent cation in excess of the ATP concentration, and for determining kinetic constants the concentrations of free Mg2+ or Mn 2÷ should be kept essentially constant by maintaining the total metal concentration at a predetermined fixed concentration above the total ATP concentration. 4,5 Kinetic constants can then be calculated by linear regression analysis of the slopes and intercepts of secondary plots as suggested by Cleland. 5 Buffers that can significantly affect concentrations of free divalent cation (especially Mn 2÷) in the reaction mixture should be avoided. Triethanolamine hydrochloride does not have this problem, whereas Tris-Cl is particularly poor. 4 Examples of approximate K m values for adenylyl cyclases have been reported as follows: detergent-dispersed enzyme from rat brain, Km(MnATP) 7-9 /xM, Km(Mn2+) 2-3 /xM, Km(MgATP) 30-60/.LM, Km(Mg2÷) 800-900/xM4; human platelets, Km(Mg2÷) 1100/xM, Km(MgATP) 50 /~M6; liver, Km(MnATP) and Km(MgATP) were similar (~50/~M). 7

Contaminating Enzyme Activities Adenylyl cyclase is an enzyme that constitutes a very small percentage of membrane-bound protein and as such exists in an environment rich in contaminating enzyme activities, including a number of nucleotide phosphohydrolases, cyclic nucleotide phosphodiesterases, and ATP-utilizing kinases. Thus, adenylyl cyclase in membrane preparations competes with other enzymes for ATP (Fig. I) and the cAMP formed is readily hydrolyzed to 5'-AMP. Analogously, GTP, which is required for hormone-induced 4 D. L. Garbers and R. A. Johnson, J. Biol. Chem. 2,50, 8449 (1975). 5 W. W. Cleland, in "The Enzymes" P. E. Boyer, ed.), 3rd Ed., Vol. 2, p. 1. Academic Press, New York, 1970. 6 R. A. Johnson, W. Saur, and K. H. Jakobs, J. Biol. Chem. 254, 1094 (1979). 7 C. Londos and M. S. Preston, J. Biol. Chem. 252, 5957 (1977).

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activation or inhibition of adenylyl cyclases, is also metabolized. Consequently, the use of regenerating systems to counteract this alternative metabolism of ATP and/or GTP and the use of inhibitors of cAMP phosphodiesterases are nearly unavoidable. Cyclic Nucleotide Phosphodiesterases. cAMP is effectively inactivated through the hydrolysis of its 3'-phosphate bond, yielding 5'-AMP (Step 9 in Fig. 1 above). Since cyclic nucleotide phosphodiesterase activity is substantial in most membrane preparations, these enzymes must be inhibited to measure accurately the rate of formation of cAMP by adenylyl cyclase. This is usually accomplished by the use of unlabeled cAMP in the reaction mixture or by the use of inhibitors of the enzyme, such as papaverine or alkylxanthines (e.g., 3-isobutyl-l-methylxanthine; IBMX). IBMX is also useful through its additional action of effectively blocking adenosine receptors. One must be cautious in the selection of a phosphodiesterase inhibitor, though, because some agents do not block all cAMP phosphodiesterases. For example, the sole use of the imidazolidinone derivative Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] is not recommended. This compound has been useful in the study of adenosine receptor-mediated effects on adenylyl cyclases because it is not an adenosine receptor antagonist as are the alkylxanthines. Although it may substantially suppress the hydrolysis of cAMP in preparations from some tissues (pig coronary arteries), it does so incompletely in others (e.g., platelets). In preparations of human platelet membranes, for example, the addition of a second phosphodiesterase inhibitor is required. In our hands the most effective combinations of agents for inhibition of hydrolysis of labeled cAMP produced by adenylyl cyclase have been the following: 1 mM IBMX; cAMP (100 /~M) with or without papaverine (100 /xM); 100/zM anagrelide [6,7-dichloro- 1,5-dihydroimidazol[2,1-b]quinazolinone monohydrochloride (BL-4162A); or anagrelide (100/xM) plus Ro 20-1724 (500 t~M). 8 ATP-Regenerating Systems. The accurate determination of adenylyl cyclase activities is adversely affected by the hydrolysis of ATP between the/3- and y-phosphates (Step 1 in Fig. 1), due to various membrane-bound ATPases, nonspecific phosphohydrolases, and flux through membranebound kinases (Step 10, Fig. 1) and phosphatases. Cleavage of the bond between the a- and /3-phosphates occurs for ADP (Step 2, Fig. 1) by membrane phosphohydrolases and for ATP by nucleotide pyrophosphatase (Step 6, Fig. I). Whether by (Step 2 or by Step 6) (Fig. 1), the result is 5'-AMP, which is rapidly hydrolyzed by 5'-nucleotidase (Step 3, Fig. 1) to adenosine (Ado). Adenosine can stimulate or inhibit (Step 5, Fig. 1) adenylyl cyclase, either indirectly via inhibitory (A1) or stimulatory (Az) 8 E. A. Martinson and R. A. Johnson, unpublished observations (1986).

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receptors, or inhibit the enzyme directly via the P-site. These reactions can be counteracted and their influence minimized by an ATP-regenerating system. The most commonly used systems have utilized creatine kinase and creatine phosphate or pyruvate kinase and phosphoenolpyruvate to counteract hydrolysis of ATP between the/3- and y-phosphates by catalyzing the conversion of ADP to ATP (Step 7, Fig. 1), but to counteract hydrolysis between the o~-and/3-phosphates myokinase (adenylate kinase) is used to convert 5'-AMP to ADP (Step 8, Fig. 1). Since the action of 5nucleotidase cannot be reversed, the influence of the formed adenosine can be minimized by the use of adenosine deaminase (Step 4, Fig. 1), as the product inosine is without effect on adenylyl cyclase. In adenylyl cyclase reaction mixtures effective concentrations of these enzymes are as follows: myokinase, 100/xg/ml (Boehringer Mannheim, Indianapolis, IN, ammonium sulfate suspension from rabbit muscle); adenosine deaminase, 5 U/ml (Sigma, St. Louis, MO, ammonium sulfate suspension, Type VIII from calf intestinal mucosa). The influence of nucleotide pyrophosphatase and to a lesser extent 5'nucleotidase can be further minimized by pretreatment of membranes with 5 mM EDTA and 3 mM dithiothreitol. 9'~°The necessity for the additions to the assay or the effectiveness of the membrane-pretreatment with chelator and/or dithiothreitol depends on the source and purity of the adenylyl cyclase being studied. An example of the effectiveness of adenosine deaminase and myokinase to enhance hepatic adenylyl cyclase is shown in Table I for an assay conducted at a low (10/.~M) concentration of ATP. Myokinase enhanced adenylyl cyclase activity and helped maintain ATP levels during the reaction, whereas adenosine deaminase did not affect the stability of ATP but enhanced enzyme activity, presumably by the removal of inhibitory levels of adenosine (Table I). Although both creatine kinase and creatine phosphate or pyruvate kinase and phosphoenolpyruvate have been utilized as the basis of ATPregenerating systems, neither is without its pitfalls. Both of the enzymes bind and utilize adenosine phosphates, and both substrates form weak complexes with divalent cations. Moreover, phosphoenolpyruvate has been shown to cause both stimulatory and inhibitory effects on the enzyme from liver and to inhibit the enzyme from heart H and contaminants in creatine phosphate have been found to cause both stimulatory and inhibitory effects on adenylyl cyclases. 9 For these reasons the preferable ATP9 R. A. Johnson, J. Biol. Chem. 255, 8252 (1980). l0 R. A. Johnson and J. Welden, Arch. Biochem. Biophys. 183, 2176 (1977). II R. A. Johnson and E. L. Garbers, in "Receptors and Hormone Action" B. W. O'Malley and L. Birnbaumer, eds.), Vol. 1, p. 549. Academic Press, New York, 1977.

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TABLE I EFFECTS OF MYOKINASE AND ADENOSINE DEAMINASE ON ADENYLYL CYCLASE ACTIVITY AND ON ATP REGENERATION IN LIVER PLASMA MEMBRANESa

NaF (10 mM)

Basal

Glucagon (1 /xM) + GTP (10/zM)

Additions

AC

ATP

AC

ATP

AC

ATP

None Adenosine deaminase Myokinase Adenosine deaminase + myokinase

8.5 11.5 10.5 11

0.57 0.52 0.88 0.74

22 31 33.5 40.5

0.50 0.52 0.81 0.79

35.5 52 57 78.5

0.60 0.62 0.86 0.80

a Adenylyl cyclase activity (AC) is expressed as pmol cAMP (min.mg protein)- i. Activity was determined with 10/zM MnATP, 400/zM excess MnC12, 10 mM creatine phosphate, 100/zg/ml creatine kinase, 1 mM IBMX, and 50 mM glycylglycine, pH 7.5, without additions (basal) or with 10 mM NaF or 1/zM glucagon and 10 ~M GTP. Residual ATP (ATP) is the quantity (ATP with membranes)/(ATP without membranes), determined at the end of the 2-min reaction. ATP concentrations were determined by luciferase luminescence. Adenosine deaminase was 5 U/ml myokinase was 100 ~g/ml. Adapted from Ref. 9. regenerating s y s t e m is creatine kinase (100/xg/ml; Boehringer Mannheim, f r o m rabbit muscle) and creatine phosphate. Creatine phosphate should be used at concentrations low enough (e.g., 2 mM) to minimize the influence of the contaminants, or, preferably, be purified before use, for example, b y anion-exchange c h r o m a t o g r a p h y . 9

Enzyme Concentrations, Reaction Times, and Temperatures T h r e e additional factors that obviously influence adenylyl cyclasecatalyzed formation of c A M P interdependently are the concentration of e n z y m e , the time, and the incubation temperature. Enzyme Concentration. Adenylyl cyclase is m o r e active in crude m e m brane preparations f r o m s o m e tissues than from others, and the levels of the various contaminating e n z y m e s that utilize adenine nucleotides (Fig. 1) also vary. Consequently, under any given set of reaction conditions it is imperative to establish (1) that sufficient e n z y m e is used to catalyze the formation of m e a s u r a b l e amounts of [32p]cAMP; (2) that concentrations of both A T P and creatine p h o s p h a t e are adequate for sustaining stable A T P concentrations; and (3) that formation of c A M P is linear with respect to e n z y m e concentration. F o r m a t i o n of measurable amounts of [32p]cAMP is i m p r o v e d with increasing specific radioactivity of [o~-32p]ATP and increasing e n z y m e concentration. H o w e v e r , increasing concentrations of

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crude membrane preparations of adenylyl cyclase typically also require increased amounts ofcreatine phosphate or proportionally decreased incubation times. Hence, enzyme concentration, ATP specific radioactivity, and incubation times must be adjusted to allow linear formation of cAMP with respect to both protein concentration and time. Useful ranges of specific radioactivity of [ct-32p]ATP are 10 to 200 cpm/pmol, depending principally on enzyme source. For studies of enzyme kinetics with respect to metal-ATP, the range of [a-32p]ATP specific activity will be greater than this. Examples of, but not necessarily upper limits for, protein concentrations yielding linear product formation of 30° with 0.1 mM ATP and 5 mM creatine phosphate, 100/~g/ml creatine kinase, 100 ~g/ml myokinase, 5 U/ml adenosine deaminase, and 10 mM MnCI2 or MgCI 2 would be as follows (in mg/ml): heart, 0.6; liver, 1.2; kidney, 0.7; skeletal muscle, 0.3; adipocytes, 0.2; spleen, 0.5; human platelets, 0.2; bovine sperm particles, 1.0; washed particles from brain, 0.2; detergent-solubilized brain, 0.2. Time. The incubation time for adenylyl cyclase reactions is dictated by a balance between rates of formation of cAMP from ATP, hydrolysis of cAMP to 5'-AMP by contaminating cyclic nucleotide phosphodiesterases, hydrolysis of ATP by a number of membrane-bound phosphohydrolytic enzymes (Fig. 1), inactivation of adenylyl cyclase by regulatory components, and denaturation of the enzyme. It is essential that linearity of product formation with respect to time be established. The reaction is typically linear with respect to time for crude membrane preparations, with the conditions given above, for 2 to 15 min and for purified enzyme for 60 min. Incubation Temperature. Temperature can be used to advantage to exhibit certain characteristics of behavior of adenylyl cyclases as well as to modify rates of alternative substrate utilization and enzyme denaturation. Formation of cAMP is linear with respect to time for a longer period at 30° than at a more physiological 37°. However, the catalytic moiety is readily inactivated in the absence of protective agents by exposure to heat for short periods of time. For example, exposure of adenylyl cyclases from platelets and from $49 lymphoma wild-type and cyc- cells for 8 rain at 35° causes 70 to 75% inactivation. 12,13 Comparable inactivation of adenylyl cyclases from bovine sperm and detergent-dispersed porcine brain occurred by exposure at 45 ° for 8 and 4 min, respectively. ~2Partial protection against thermal inactivation is afforded by forskolin (200/~M), metalATP (submillimolar), the P-site agonist 2',5'-dideoxyadenosine (250/zM), guanine nucleotides (micromolar), and, for the Ca2+/calmodulin-sensitive I2 j. A. Awad, R. A. Johnson, K. H. Jakobs, and G. Schultz, J. Biol. Chem. 258, 2960 (1983). 13 V. A. Florio and E. M. Ross, Mol. Pharmacol. 24, 195 (1983).

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form of adenylyl cyclase, by Ca 2÷/calmodulin (50 ~M/5 lzM). 12-16In addition, adenylyl cyclase reactions conducted at different temperatures can be used to enhance selective regulatory properties of the enzyme. For example, inhibition of adenylyl cyclase mediated by guanine nucleotidedependent regulatory protein (Gi), whether by hormone or stable guanine nucleotide, is more readily shown experimentally at lower temperatures (e.g., 24°), whereas activation, mediated by the stimulatory G protein (Gs), is evident at higher temperatures (e.g., 30°).17

Guanine Nucleotides GTP is required for Gs and Gi mediation of hormone-induced activation and inhibition of adenylyl cyclases. 18-22 The more stable GTP analogs guanosine 5'-(/3,T-imino)triphosphate [GPP(NH)P] and guanosine 5'-0-(3thiotriphosphate) (GTPTS) can substitute for GTP. Effects of these analogs are evident after a distinct lag phase, and preincubation of the enzyme with them will result in persistently activated or inhibited enzyme, depending on incubation conditions. In addition, the effectiveness of Gs and G i in regulating adenylyl cyclase is further influenced by divalent cation, type, and concentration and by membrane perturbants (e.g., Mn 2+ and detergents obliterate Gi-mediated inhibition). Half-maximal stimulation of adenylyl cyclase by hormones is usually observed with 10 to 50 nM GTP, or with 50 to 100 nM GPP(NH)P or GTPTS, with optimal stimulation between 1 and 10/zM GTP, GPP(NH)P, or GTPTS. Half-maximal inhibition occurs with 100 to 500 nM GTP, l0 to 100 nM GPP(NH)P, or 1 to 10 nM GTPyS, with optimal inhibition occurring with GTPTS over I00 nM, GPP(NH)P over l0 nM, and GTP over 1/zM. Consequently, even in relatively pure membrane preparations, enzyme activity may be increased somewhat by stimulatory hormones due to endogenously present GTP (e.g., as a contaminant of ATP; GTP-free ATP can be purchased from Sigma). The addition of GTP enhances stimulation further. By comparison, GTP must 14 M. A. Brostrom, C. O. Brostrom, and D. J. Wolff, Arch. Biochem. Biophys. 191, 341 (1978). 15 R. S. Salter, M. H. Krinks, C. B. Klee, and E. J. Neer, J. Biol. Chem. 256, 9830 (1981). t6 j. p. Harwood, H. LOw, and M. Rodbell, J. Biol. Chem. 248, 6239 (1973). z7 D. M. F. Cooper and C. Londos, J. Cyclic Nucleotide Res. 5, 289 (1979). Is M. Rodbell, L. Birnbaumer, S. L. Pohl, and H. M. J. Krans, J. Biol. Chem. 246, 1877 (1971). ~9 K. H. Jakobs, W. Saur, and G. Schultz, FEBS Left. 85, 167 (1978). 2o C. Londos, D. M. F. Cooper, W. Schlegel, and M. Rodbell, Proc. Natl. Acad. Sci. U.S.A. 75, 5362 (1978). zl D. M. F. Cooper, W. Schlegel, M. C. Lin, and M. Rodbell, J. Biol. Chem. 254, 8927 (1979). 22 E. Perez-Reyes and D. M. F. Cooper, J. Neurochem. 46, 1508 (1986).

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be added for hormonal inhibition of adenylyl cyclase, and GTP-dependent inhibition is often best elicited with enzyme that has been stimulated by forskolin or a stimulatory hormone. In addition, the concentrations of guanine nucleotides necessary for regulation of adenylyl cyclase activity are dependent on enzyme source and incubation temperature and are influenced by the relative activities and abundances of Gs and G i .

Radioactive Substrates: [aH]ATP versus [a-a2P]ATP [3H]ATP and [a-32p]ATP are commonly used as labeled substrate for measuring adenylyl cyclase catalytic activity. The use of each has both advantages and disadvantages, some of which are described below.

[3H]ATPAdvantages The one main advantage to the use of [3H]ATP as labeled substrate is its long half-life (-12.3 years). This allows the nearly complete usage of purchased isotope without regard to loss through decay. Low usage rates may adequately compensate for its being initially substantially more expensive than [a-32p]ATP.

[3H]ATPDisadvantages There are several significant disadvantages to the use of [3H]ATP as substrate in adenylyl cyclase reactions. First, tritium-labeled adenine nucleosides and nucleotides are chemically unstable in that the C-8 tritium exchanges with water, especially under alkaline conditions. This results in a continuous loss of tritium to 3H20 that can occur at the rate of several percent per month. Consequently, for accurate estimations of substrate specific activity the 3H20 must be removed periodically, either chromatographically or by lyophilization. Both procedures necessitate undue handling of and exposure to moderate quantities of isotope and have the potential of major isotope spills in a laboratory environment. The low energy of the beta decay of tritium necessitates the use of scintillation cocktails to detect [3H]cAMP, and the long half-life of tritium means that large volumes of liquid radioactive waste, which necessarily also contains large quantities of organic solvents, must be disposed rather than be allowed to dissipate through radioactive decay as would be the case with 32p. Disposal of radioactive waste, especially mixed with scintillation cocktail, is an expensive and undesirable consequence of the use of tritiumlabeled substrate. The low energy of tritium decay also makes it more difficult to detect if there are inadvertant spills or contamination in a

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laboratory and could thereby lead to undue exposure of laboratory personnel to low-energy radiation. Breakdown products of [3H]ATP or [3H]cAMP include various nucleotides and nucleosides, as well as xanthine, hypoxanthine, and others, that are also labeled. Chromatographic techniques must take this into consideration. These various breakdown products and the continuous formation of 3H20 from tritium-labeled adenine nucleotides contribute to blank values [counts per minute (cpm) for samples in the absence of enzyme], with the Dowex 50/A120 3 column system described below being substantially higher than those obtained with [a-3Zp]ATP as substrate. With adenylyl cyclases of low specific activity in crude membrane preparations, such high blank values may constitute a substantial percentage of the [3H]cAMP formed enzymatically.

[a-32P]ATP Advantages There are several important advantages to the use of [a-32p]ATP as substrate for adenylyl cyclase reactions. The specificity of labeling of [a-32p]ATP, which is dictated by the enzymatic means typically used for its synthesis (as per Ref. 23), means that only a-phosphates are labeled and, since the a-phosphate of ATP is not readily transferred to other compounds, that only nucleotides derivable from ATP will be labeled. Additional products that could result from contaminating activities in crude membrane preparations, for example, [a-3ZP]ADP, [a-32p]AMP, [a-32p]IMP, and [32P]Pi , due to differences in their ionic properties are all readily separated from [3ZP]cAMP. 32p is a high-energy beta emitter that allows detection by Cerenkov radiation and obviates the use of scintallation cocktails, that is, can be detected in aqueous solutions with efficiencies approaching that of tritium in scintillation cocktails but with little influence of agents that typically quench detection of tritium. The high energy of the beta emission also allows easy detection of inadvertant spills with a Geiger/Miiller detector and thereby actually enhances laboratory safety. The short half-life of 32p (-14.3 days) allows waste to be decayed off before disposal, effectively eliminating expensive or awkward disposal of radioactive waste, whether solid or liquid.

[a-32P]ATP Disadvantages The short half-life of 3zp also implies that the usefulness of the isotope is often lost to decay before the [a-32p]ATP is fully utilized. Consequently, if usage rates are low the decay of the isotope may result in [a-32P]ATP actually being more expensive than [3H]ATP. Blank values can depend on

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the quality of substrate, even with the double column procedures described below. The quality can vary substantially among different suppliers and in different batches from a given supplier. Blank values for the adenylyl cyclase assay should be supplied on the product data sheet. The quality of [a-32p]ATP can be assured by its purification before use or through its laboratory synthesis from carrier-free 32pi by published procedures, 23 a process which also includes its purification. Stopping the Reaction There are several good methods for stopping adenylyl cyclase reactions, the choice depending in part on whether [3H]ATP or [a-32p]ATP is used as substrate and on the method used for estimating loss of labeled cAMP during its purification. Some of these considerations are dealt with below.

Stopping with Zinc Acetate/Na2CO3/cAMP or/[3H]cAMP The use of coprecipitation or adsorption of nucleotides with inorganic salts dates from an early assay for adenylyl cyclase developed by Krishna et al. ,24who used a combination of column chromatography on Dowex 50 and precipitation with ZnSO4 and Ba(OH)2, yielding the insoluble salts of BaSO 4 and Zn(OH)E, which adsorb phosphomonoesters and polyphosphates but not cyclic nucleotides. A disadvantage in the use of ZnSO4/ Ba(OH)2 is that cAMP may be formed nonenzymatically from ATP at alkaline pH, especially at elevated temperatures, leading to variable and high blank values. This problem is circumvented by the use of other salt combinations or by the use of acidic inactivation of adenylyl cyclase. The effectiveness of a variety of combinations of inorganic salts, for example, ZnSO4/Na2CO 3, CdCI2/Na2CO 3, ZnSO4/BaC12, BaC12/NazCO 3, to bind labeled ATP, ADP, AMP, cAMP, and adenosine has been cataloged previously in this series. 25 Since comparable separation of ATP and cAMP can be achieved with columns packed with ZRCO325 or A120 3 ,26 it is likely that adsorption to the insoluble inorganic salts, rather than coprecipitation with them, is the basis of the separation of cAMP from the multivalent nucleotides and hence the basis of their usefulness in assays of adenylyl or guanylyl cyclases. It is important to emphasize that since none of these salt combinations will separate cAMP from adenosine, [a-32p]ATP is 23 R. 24 G. 25 p. 26 A.

A. Johnson and T. F. Walseth, Adv. Cyclic Nucleotide Res. 10, 135 (1979). Krishna, B. Weiss, and B. B. Brodie, J. Pharm. Exp. Ther. 163, 379 (1968). S. Chan and M. C. Lin, this series, Vol. 38, p. 38. A. White and T. V. Zenser, Anal. Biochem. 41, 372 (1971).

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preferred to [3H]ATP as substrate, to avoid the possibility that the [3H]cAMP formed through adenylyl cyclase may be contaminated with [3H]adenosine, [3H]inosine, or other labeled compounds deriving from adenine nucleotides. The characteristics of the insoluble inorganic salts are taken advantage of in the following procedure adapted from Jakobs et al. 27 Reagents

Zinc acetate/cAMP: 120 mM Zn(C2H302) 2 • 2H20 (FW 219.49) is prepared in deionized, double-distilled, or Millipore grade water that has been boiled and then cooled to remove dissolved carbon dioxide, and cAMP is then added (165 mg/liter, to 0.5 mM); this is kept refrigerated and tightly capped between uses Zinc acetate/[3H]cAMP: prepare as above except add tritiated cAMP, from which 3 H 2 0 has been removed, to an amount of zinc acetate needed for a given assay to yield approximately 10,000 to 20,000 cpm/ml, when counted in the same volume of eluate (see below) used for samples Sodium carbonate: 144 mM N a 2 C O 3 , anhydrous (FW 106.0) Both zinc acetate and N a 2 C O 3 solutions are stored in and dispensed from glass repipetting dispensers. Procedure

1. Adenylyl cyclase reactions, typically 50 to 200/zl in 1.5-ml plastic Eppendorf tubes, are terminated by the addition of 0.6 ml of 120 mM zinc acetate/cAMP or zinc acetate/[3H]cAMP. Aliquots of these stopping solutions are taken for determining absorbance (A259nm) or radioactivity, as appropriate; the values are to be used for quantitating sample recovery (see below). 2. One-half milliliter of 144 mM Na2CO 3 is added to precipitate Z n C O 3 and multivalent adenine nucleotides. 3. Samples are placed on ice, or they can be kept refrigerated or frozen overnight. The Z n C O 3 precipitate is sedimented by centrifugation in a benchtop centrifuge. Pellets of frozen samples are smaller and heavier than those of unfrozen samples. 4. The supernatant fractions are decanted onto columns for purification of sample cAMP. 5. Assay blanks are prepared by substituting enzyme buffer for enzyme. 6. A potential disadvantage of this method is that if [3H]cAMP is 27 K. H. Jakobs, W. Saur, and G. Schultz, J. Cyclic Nucleotide Res. 2, 381 (1976).

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used, it becomes necessary to use, and hence eventually dispose of, scintillation cocktails for quantitating [32p]cAMP and its recovery. . An advantage of this procedure that has lead to its use in many laboratories is that over 98% of all multivalent nucleotides, namely, substrate [a-32p]ATP, [a-32p]ADP, [32p]AMP, as well as any [32P]Pi, are retained in the capped Eppendorf assay tubes in the ZnCO 3 precipitate. The waste radioactivity is thus highly confined, occupies little volume, and can be allowed to decay off and then be dealt with as normal solid waste. Stopping with ATP/SDS/cAMP with or without [3H]cAMP

The following method, adapted from Salomon et al., 1"~8relies on sodium dodecyl sulfate to inactivate adenylyl cyclase and on unlabeled ATP and unlabeled cAMP to overwhelm adenylyl cyclase and cAMP phosphodiesterases with substrate and thereby effectively prevent the further formation or degradation of [32p]cAMP. Reagents. "Stopping solution" contains 2% (w/v) sodium dodecyl sulfate (SDS), 40 mM ATP, 1.4 mM cAMP, pH 7.5, and approximately 100,000 cpm/ml [3H]cAMP, to monitor recovery of [32p]cAMP. Alternatively, [3H]cAMP could be omitted from the stopping solution and added separately. Procedure

1. Adenylyl cyclase reactions, typically 50 to 200/zl in 13 x 65 mm glass or plastic tubes or in 1.5-ml plastic Eppendorf tubes, are terminated by the addition of 100/xl of the stopping solution. 2. To achieve full membrane solubilization in cases of high membrane content, it is advisable to boil the test tubes for 3 min at this stage. This also facilitates the rate of chromatography. Hence, use heatstable tubes. 3. The mixtures in the reaction tubes are then diluted and decanted onto chromatography columns for purification of sample cAMP. 4. Assay blanks are prepared by omitting enzyme or by adding enzyme after the stopping solution. 5. A disadvantage of this procedure is that all radioactive compounds, including unused substrate [a-32p]ATP, [ct-32P]ADP, [32p]AMP, [32p]p i , as well as degradation products of [3H]cAMP, are passed with the labeled cAMP onto the chromatography column and are typically eluted in a fall through fraction that must be collected and 2s y. Salomon, C. Londos, and M. Rodbell, Anal. Biochem. 58, 541 (1974).

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then dealt with as liquid radioactive waste. To minimize this waste, see below. 6. A disadvantage of either stopping procedure when [3H]cAMP is used to monitor recovery of sample [32p]cAMP is that scintillation cocktails must be used and consequently disposed. Chromatographic Alternatives The characteristic property of alumina and other insoluble inorganic salts to bind multivalent nucleotides but not cAMP is the central feature of a number of variations of assays for adenylyl and guanylyl cyclases. White and Zenser 26 passed reaction mixtures over columns of neutral alumina that were equilibrated and then developed with neutral buffer. However, assay blanks with this procedure were variable and depended highly on the radiochemical purity of the a-a2p-labeled substrate. Salomon et al. 2s and later Wincek and Sweat w showed that sequential chromatography on Dowex 50 and alumina produced an assay for adenylyl cyclase that was more consistent than alumina columns alone, a combination that has also been utilized for the assay of guanylyl cyclase. 3° Additional variations on this procedure have been reported by a number of investigators. For example, nearly quantitative separation of cAMP from ATP was achieved by a combination of precipitation with inorganic salts (zinc acetate/ NaECO3) followed by chromatography on alumina. 27 Another approach to lower blank values has been to inactivate adenylyl cyclase by stopping the reaction with the addition of acid (e.g., 1 ml of 1 M HCI) followed by heating (90 ° for 8-10 min) and then precipitation with ZnSO4/Ba(OH)23~ or, subsequent to acidic heat inactivation (4 min at 95 ° in 0.165 N HC1), chromatography on a selected alumina column. 3a Thus, to minimize the influence of variations in the quality of [a-32p]ATP, the method of choice has become sequential chromatography with Dowex 50 and then alumina columns.l'28 The procedure is as follows. Reagents

Dowex 50, H ÷ form [e.g., Bio-Rad (Richmond, CA) AG50-XS, 100-200 mesh]. Before use the Dowex 50 is washed sequentially with approximately 6 volumes each of 0.1 N NaOH, water, 1 N HCI, and water. The Dowex 50, in an approximately 2 : 1 slurry, is 29 T. J. Wincek and F. W. Sweat, Anal. Biochem. 64, 631 (1975). 30 j. A. Nesbitt III, W. B. Anderson, Z. Miller, I. Pastan, T. R. Russell, and D. Gospodarowicz, J. Biol. Chem. 251, 2344 (1976). 31 C. Nakai and G. Brooker, Biochim. Biophys. Acta 391, 222 (1975). 32 R. Counis and S. Mongongu, Anal. Biochem. 84, 179 (1978).

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then poured into columns ( - 0 . 6 x 4 cm). After each use, Dowex 50 columns are regenerated by washing with 5 ml of 1 N HC1, then stored until reused. Before use the columns are washed 3 times with 10 ml of water. Columns can be reused dozens of times. If flow rates decrease, columns should be regenerated with NaOH, water, and HCI as above. Neutral alumina [e.g., Bio-Rad AG7, 100-200 mesh; Sigma WN-3; ICN (Costa Mesa, CA) Alumina N, Super I]: The source of A1203 is less critical with the two-column procedure than if it is used alone. The alumina (0.6 g) may be poured dry into columns (e.g., with a plastic scoop or a large disposable plastic syringe from which the alumina is allowed to drain). Elution buffers: The original method of Salomon et al. 28 calls for 100 mM imidazole, pH 7.5. An equally effective and less expensive alternative is 100 mM Tris-Cl, pH 7.5. The purpose of the buffer is to elute cyclic nucleotides. Since eluate from the Dowex 50 columns is acidic, which enhances adsorption of cyclic nucleotides to alumina, elution of cyclic nucleotides is achieved principally through an increase in the pH of the buffer rather than through increased ionic strength. Consequently, there is probably wide latitude in buffer choice here. Before initial use alumina columns m u s t be washed once with elution buffer, either 10 ml of 100 mM Tris-Cl or 10 ml of 1 M imidazole, pH 7.5, otherwise the procedure does not work right away. After each use the columns are washed with 10 ml of 100 mM Tris-Cl or 10 ml of 100 mM imidazole, pH 7.5. Once poured both alumina and Dowex 50 columns may be reused virtually indefinitely, though they may need to be topped off occasionally. A p p a r a t u s . Rapid flow rates for the alumina columns, and consequently short chromatography times, are achieved with glass columns with a large cross-sectional area and a coarse sintered glass plug to retain the alumina (Fig. 2). Satisfactory dimensions are alumina to approximately 1 cm in a column 11 mm i.d. by 9 cm attached to a 4-cm glass funnel (24 mm i.d.). [Smaller columns (e.g., - 0 . 6 × 2 cm, alumina) while allowing satisfactory chromatographic performance are slow.) Both Dowex 50 and alumina columns are most conviently used if they are mounted in racks (e.g., plastic) with spacing identical to that of the racks of scintillation vials to be used. The design of the racks supporting the Dowex 50 columns should be such that the columns can be conveniently mounted aboved the alumina columns so that the eluate of the Dowex 50 columns can drip directly onto the alumina columns. Similarly, the design

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.

E E o CO

tion

FIG. 2. Apparatus for column chromatography.

of the racks supporting the alumina columns should allow the eluate from them to drip directly into scintillation vials. Carbon filters for the removal of adsorbable radioactive materials from column eluates are Gelman Ann Arbor, MI (#12011) Carbon Capsules, each containing 100 g activated charcoal. Procedure. Two alternative procedures are described, the choice of which depends on the quality of 32p-labeled substrate. The first procedure should be adequate with all but the poorest quality substrate and the second procedure should lower blank values further if necessary.

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Water elution of Dowex 50 1. Whether reactions are stopped by the zinc acetate/Na2CO 3 or the ATP/SDS/cAMP method, the resulting solutions are decanted directly onto the Dowex 50 columns. 2. The Dowex 50 columns are then washed with about 3 ml water. (The actual volume necessary for this step may vary slightly from batch to batch or with the age of the Dowex 50 and should be determined, i The eluate from this wash contains [32P]Pi, [ct-32p]ATP, and [o~-32pIADP and should be disposed of by the procedure described below. 3. The Dowex 50 columns are then mounted above a comparable number of alumina columns so that the eluate drips directly onto the alumina. Dowex 50 columns are washed with 8 ml water. The eluate from the Dowex 50 columns is slightly acidic and causes cAMP to be retarded on the alumina column. 4. After the eluate from the Dowex 50 columns had dripped onto and through the alumina columns, the alumina columns are placed over scintillation vials. 5. cAMP is eluted from the alumina columns directly into scintillation vials. The volume of elution buffer used depends on whether unlabeled cAMP or [3H]cAMP is used to quantitate sample recovery and on the types of vials used in the scintillation counter. It is important to use sufficient buffer to elute all the cAMP as well as to optimize counter efficiency, which is dictated by the geometry of the counter phototubes. For example, if recovery is monitored with unlabeled cAMP and [32p]cAMP is determined by Cerenkov radiation in 20 ml counting vials, [32p]cAMP is eluted from alumina columns with 8 ml of 100 mM Tris-C1. Smaller volumes do not give optimal counting efficiency. Following counting, the absorbance at 259 nm is determined on an aliquot of the sample to quantitate recovery of unlabeled cAMP. If recovery is monitored with [3H]cAMP, both [3H]cAMP and [32p]cAMP are eluted with 4 ml of 100 mM imidazole into 10-ml vials to which 5 ml scintillation cocktail is then added. The smaller vials spare expensive cocktail. Sample recovery is determined from dual channel counting. 6. Scintillation counting of 32p by Cerenkov radiation is achieved with a single channel with wide open windows. 7. Counting of samples containing both 3H and 32p can be achieved in a two-channel scintillation counter with windows adjusted such that there is zero crossover of 3H into the 32p window and small but measureable crossover of 32p into the 3H window.

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Acid elution of Dowex 50. The following procedure is adapted from White and K a r r . 33 1. Before use the Dowex 50 columns are washed with 10 ml of 10 mM HCIO 4 .

2. Whether reactions are stopped by the zinc acetate/Na2CO3 or the ATP/SDS/cAMP method, the resulting solutions are decanted directly onto the Dowex 50 columns. 3. The Dowex 50 columns are then washed with 6 ml of 10 mM H C I O 4 . (The actual volume necessary for this step may vary from batch to batch or with the age of the resin and should be determined.) The eluate from this wash contains [32p]Pi, [ a - 3 2 p ] A T P , and [ot-32p]ADP and should be disposed of by the procedure described below. 4. The Dowex 50 columns are mounted directly above a comparable number of alumina columns and then washed with 8 ml of l0 mM H C I O 4 that is allowed to drain through both columns. 5. The alumina columns are then washed with l0 ml of water. This eluate is discarded. 6. The alumina columns are mounted above a rack of scintillation vials and the cAMP is eluted as above. Disposal of waste isotope. To avoid contamination of wastewater, all radioactive waste from the above procedures is collected and pooled. It is poured onto a large Btichner funnel, containing perhaps 250 g alumina (e.g., Fisher, AI203 , anhydrous), attached in series to two parallel 100-g carbon filters and a flask attached to a water aspirator. (The purpose of the flask is to allow an aliquot of the filtered waste to be monitored for radioactivity before the waste is discarded down the drain.) By use of alumina and carbon filters, virtually n o 32p radioactivity is discarded in wastewater, though s o m e 3 H 2 0 will be lost if tritiated nucleotides are used ([3H]ATP or [3H]cAMP), and any radioactive waste can be treated as compact solid waste. This is especially useful for tritiated nucleosides and nucleotides. The 32p-labeled solid waste can be allowed to decay off. The alumina can be used almost indefinitely, whereas the carbon filters tend to clog with prolonged use and need to be replaced periodically (e.g., annually). Data Analysis Calculation of adenylyl cyclase activities determined with radioactive substrates is straightforward but must take into consideration the inevitable loss of sample cAMP that occurs during chromatographic purification. 33 A. A. White and D. B. Karr, Anal. Biochem. 85, 451 (1978).

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For this reason sample recovery is determined either with unlabeled cAMP or with cAMP that is labeled with a second isotope. In addition, since many adenylyl cyclases exhibit low activities, especially under basal assay conditions, the radioactivity measured in the sample in the absence of enzyme (no enzyme blank) can represent sizable percentage of that measured with enzyme. Consequently, it becomes important to consider how this value is to be treated in the calculation of activity. If there is measurable nonenzymatic formation of cAMP from ATP, as may be the case under alkaline assay conditions, especially in the presence of manganese, the labeled cAMP must be corrected for sample loss during purification. However, if it can be established that the sample radioactivity in the absence of enzyme is due to 32p-labeled contaminants in the sample, that is, 32p-labeled compounds not adsorbed by alumina33 or as determined through alternative chromatographic techniques, the blank value should not be corrected for sample recovery. Such a correction would give rise to an erroneously high blank value, and the apparent enzyme activity would be lower than it should be. Both sample recovery and assay blank adjustments to the determination are readily made with programmable calculators, though they are more conveniently done with a computer since programs can be written to accommodate variable amounts of protein, substrate concentrations, assay times, and volumes, can be extended to the computation of enzyme kinetic constants, and can be interfaced with graphic plotters. In addition, scintillation counters may be attached directly to such computers to enhance data acquisition and processing. Examples of these calculations are given below.

Example 1. For the calculation of adenylyl cyclase activities with [3H]cAMP used for sample recovery, the assumption is made that the windows for the 32p and 3H channels of the scintillation spectrometer have been set so there is zero crossover of 3H cpm into the 32p channel. The calculation compensates for crossover of 32p cpm into the 3H channel. Velocity = (sample 32p cpm - no enzyme 32p cpm) × ATP concentration × reaction volume/fraction of sample counted/([oz-32p]ATP cpm - no enzyme 32p cpm) × [3H]cAMP std cpm/(sample 3H cpm - {(sample 3zp cpm - no enzyme 32p cpm) × 32p cpm in 3H channel/

[a-32p]ATP-cpm)}/time/protein [3H]cAMP std cpm is the value that would represent 100% recovery of the added [3H]cAMP, for example, the total 3H cpm in the 0.6 ml of zinc acetate containing [3H]cAMP used to stop the reaction, counted under comparable quench conditions used to count the samples.

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Example 2. An analogous though simpler calculation is used for activities when unlabeled c A M P is used for sample recovery and is the same whether [a-32p]ATP or [3H]ATP is used as substrate tracer. Velocity = (sample cpm - no e n z y m e cpm) × A T P concentration × reaction volume/fraction of sample counted/(substrate cpm - no enzyme cpm) × cAMP standard Az59/sample A259/time/protein cAMP standard A259 is the optical density at 259 nm that would represent 100% r e c o v e r y of the added unlabeled cAMP. This value usually also includes a factor to compensate for the volume of the final sample. In the example given here for samples chromatographed first on Dowex 50 then on A120 3 columns, samples are 8 ml. For example, the optical density (A259) of the 0.6 ml of zinc acetate containing unlabeled cAMP is typically determined on an aliquot diluted 40-fold in 100 m M Tris-C pH 7.5, and gives a value of approximately 0.2. Hence, in this example, 0.2 × 40 x 0.6 ml/8 ml ---> 0.6 for cAMP standard A259. For either procedure the determinations of velocity assume that 32p cpm observed in the absence of e n z y m e is not cAMP, and no correction is made for loss during purification of those samples. This is an important assumption only in instances when enzyme activity is low and the cpm observed in the absence of e n z y m e represents a sizable percentage of sample cpm. F o r both calculations the value for "fraction of sample c o u n t e d " is usually 1 and would be less than 1 only if an aliquot of the sample were used for some other purpose. The velocities obtained are in nanomoles c A M P formed per minute per milligram protein when substrate concentration is entered as micromolar, time as minutes, protein as micrograms per tube, and reaction volume as microliters. If protein is not known or if it is not desirable to normalize to protein, a value of 1 is used and velocities are picomoles cAMP formed per minute per tube. Acknowledgments Yoram Salomon is the Charles and TillieLubin Professor of Hormone Research. Research in the laboratory of R.A.J. was supported by Grant DK38828from the National Institutes of Health.

Assay of adenylyl cyclase catalytic activity.

[1] ASSAY OF ADENYLYL CYCLASE CATALYTIC ACTIVITY 3 [1] A s s a y o f A d e n y l y l C y c l a s e C a t a l y t i c A c t i v i t y By ROGER A. J...
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