ANALYTICAL
BIOCHEMISTRY
85,
45 I-460 (1978)
Improved Two-Step Method for the Assay of Adenylate and Guanylate Cyclase ARNOLD A. WHITE AND DALE B. KARR John
M. Dalton University
Research
Center
and
of Missouri-Columbia,
Department Coiumbia,
of Biochemistry, Missouri 65201
Received February 2, 1977; accepted October 24. 1977 A two-step assay for adenylate and guanylate cyclase is described utilizing c@*P-labeled ATP or GTP as substrate and involving purification of the resulting 3PP-labeled CAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 mM HCI for cGMP and 10mM HCIO, for CAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The CAMPor cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the s*P-labeled CAMP or cGMP is eluted with 0.2 M imidazole buffer and counted. The method delivers blanks amounting to .OOOS% of the substrate radioactivity, high recoveries, and excellent reproducibility.
Earlier we described a method for the separation of 3’S’-cyclic nucleoside monophosphates from other nucleotides on aluminum oxide columns and showed how it could be applied to the assay of adenylate or guanylate cyclase (1). In those assays, (Y-32P-labeled ATP or GTP was used as substrate, giving rise to 32P-labeled adenosine 3’,5’-monophosphate (CAMP) or guanosine 3’S’-monophosphate (cGMP), respectively. The 32P-labeled cyclic nucleotides passed through columns of alumina prepared with buffer in the neutral range, while all other nucleotides were retained. The amount of 32P eluting from the columns was therefore a measure of cyclase activity. However, we noted then that certain lots of [Q-~*P]GTP contained radiochemical impurities which were not retained by alumina and therefore gave a high blank. We had no solution for that problem, except for removing the impurities from the substrate by purification on DEAE-cellulose. As later workers began to use this method and with higher and higher levels of radioactivity in the substrate, several modifications were introduced to reduce the blanks. Salomon et al. (2) and also Wincek and Sweat (3) showed that sequential chromatography on Dowex 50 and alumina produced a more consistent assay for adenylate cyclase, and the same combination was utilized for guanylate cyclase assay (4-6). Be451
0003-2697/78/0852-0451$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
452
WHITE
AND
KARR
cause of the inconsistency in the quality of our supply of [cz-~~P]GTP, we also began to use a two-step method for guanylate cyclase assay, originally patterned after that of Nesbitt et al. (5). We here describe an improvement in that methodology and its application to the assay of adenylate cyclase. MATERIALS
AND METHODS
The aluminum oxide used in this work was Type 90, Brockman activity II-III from E-M Laboratories, Inc. The [a-32P]ATP and [cz-~~P]GTP were purchased from ICN, while the [3H]cAMP and [3H]cGMP were from New England Nuclear and Schwarz/Mann, respectively. GTP was from P-L Biochemicals, while Sigma Chemical Co. supplied all other biochemicals, including the Dowex 50 ion-exchange resin (Dowex 5OWX8, ZOO-400 mesh). The 32P-labeled nucleotides were diluted with cold ATP and GTP upon receipt in order to minimize radiochemical decomposition (7). Both [3H]cGMP and [3H]cAMP were purified before use (7). Solutions. Imidazole buffer, pH 7.07, was used in two concentrations: a 0.2 M solution containing 0.1 M HCl and a 0.1 M solution containing 0.05 M HCI. The imidazole was obtained from Pierce Chemical Co., and the HCl, from Fisher Chemical Co. as a 1 N standard solution. The solutions used to regenerate the Dowex 50 columns were prepared by diluting 50% (w/w) NaOH solution (Fisher) lo-fold to give a 5% solution and diluting concentrated (37.8%) HCl (J. T. Baker) sixfold to give a 6.3% solution. Column preparation. Two types of glass columns were used in this work. The alumina was contained in our standard columns which were made of 9-mm-o.d. standard wall tubing (7-mm nominal inside diameter), 15 cm long, with one end drawn down to an opening of about 1.5 mm. The other end was joined to a 65-mm length of 25-mm-o.d. tubing, which acted as a reservoir of about ZO-ml capacity. The Dowex 50 resin was contained in our narrow-inside-diameter columns, which resembled the standard columns except that the column barrels were made from 9-mm-o.d. tubing with a Z-mm-thick wall (Corning Glass Works Code No. 234104) resulting in a 5-mm nominal inside diameter. Since both columns had the same outer dimensions, they could be held in the same racks. The tips of the Dowex 50 columns were packed with glass wool, while in the alumina columns paper toweling was used (Teri, Kimberly-Clark Corp.). The alumina columns were prepared by measuring out approximately 1 g of alumina with a measuring spoon and emptying the spoon into a column filled with water or, in some experiments, 0.1 M imidazole buffer. After the alumina had settled and the columns were drained, they
TWO-STEP
CYCLASE
ASSAY
453
were washed with one additional column volume (about 20 ml) of water or buffer. Alumina columns were used only once. Dowex 50 columns were used in the H+ form. They were cycled before being used and after each subsequent use by the sequential addition of one column volume of each of the following: H,O, 5% NaOH, H,O, 6.3% HCl, H,O. Cyclase assays. Final volume of the reaction mixtures for both the adenylate cyclase and guanylate cyclase assays is 75 ~1, and both contained 50 mM Tris-HCl, pH 7.6,O. 1 mg of bovine plasma albumin, 10 mM 2-mercaptoethanol, 20 mM caffeine, 15 mM creatine phosphate, and 10 units of creatine phosphokinase. The adenylate cyclase reaction mixture contained 1.2 mM [o!-~*P]ATP, 5 mM MgCl*, and 0.2 pmol of CAMP. The guanylate cyclase reaction mixtures contained 1.2 mM [w~~P]GTP, 6 mM MnCl,, and 5 mM cGMP. There were IO-40 cpm/pmol used in both assays. Reactions were initiated by the addition of the enzyme and were incubated at 30°C usually for 10 min. Reactions were stopped by the addition of 0.15 ml of 1 N HCIOI, and after all reactions were completed 0.3 ml of water containing approximately 30,000 cpm of tritiated cyclic nucleotide was added to each tube. After mixing and centrifuging, the supernatant solutions were poured onto Dowex 50 columns. Purification of cyclic GMP. The Dowex 50 columns for this isolation contained 2 ml of resin giving a IO-cm bed height. Before use, each column was washed with 10 ml of 50 mM HCl. After the reaction mixture supernatant solution had drained through the resin, it was followed by the addition of 2.5 ml of 50 mM HCl. This total effluent was discarded. The Dowex 50 columns were then mounted above a rack of alumina columns, and an additional 3.5 ml of 50 mM HCl was added to each Dowex 50 column and allowed to drain through both columns. The Dowex 50 columns were removed and the alumina was washed first with 10 ml of water and then with 1 ml of 0.2 M imidazole buffer. The alumina columns were then mounted above a rack of scintillation vials and the cGMP was eluted from the alumina and into the vials by the addition of 3 ml of 0.2 M imidazole to each column. The vials were counted after addition of 10 ml of scintillation mixture (8). Purl$cation of cyclic AMP. The Dowex 50 columns each contained I ml of resin, which produced a 5-cm bed height. Each column was washed before use with 10 ml of 10 mM HClO,. After the reaction mixture supernatant solution had drained through the resin bed, 2.5 ml of 10 ITlM HC104 was added to each column twice, the first 2.5 ml being allowed to drain through the column before the second was added. All of the column effluent to this point was discarded. The rack of Dowex 50 columns was then mounted above a rack of alumina columns, after which 7 ml of 10 mM HCIO, was added to each Dowex 50 column and allowed to drain
454
WHITE
AND
KARR
through both columns. The remainder of the assay was the same as the cGMP purification. Comparative methods for cyclic nucleotide purification. The one-step method for the purification of CAMP or cGMP using alumina columns has been described (1). In this method, the reaction was terminated by adding 0.5 ml of 0.1 M HCl, containing approximately 30,000 cpm of tritiated cyclic nucleotide, and heating for 2 min at 100°C. The tube contents were neutralized by the addition of 0.5 ml of 0.2 M imidazole (free base) and were applied directly to the alumina columns, which were prepared and eluted with 0.1 M imidazole buffer. .4fter the application volume had drained through the column, CAMP was eluted with 3 ml of imidazole buffer which was collected in a scintillation vial and counted after the addition of 10 ml of scintillation mixture. If cGMP was to be isolated, the application volume was followed by 1 ml of buffer, both of which were discarded. The cGMP was then eluted with 3 ml of buffer and counted as before. The two-step method of Salomon et al. (2) for the purification of CAMP was used as described except that we substituted 1 g of alumina and the 0.1 M imidazole buffer used here (pH 7.07) for the 0.6 g of alumina and 0.1 M imidazole buffer, pH 7.5, used by those authors. The reaction was stopped with 0.1 ml of 2% sodium dodecyl sulfate (SDS) after which 0.3 ml of [3H]cAMP and 0.58 ml of water were added. This was applied to a Dowex 50 column containing 1 ml of resin which had been equilibrated with water. After the application volume had entered the bed, the column was washed twice with 1 ml of water. The CAMP was then washed from the Dowex 50 column onto an alumina column (equilibrated with 0.1 M imidazole) with 3 ml of water. The alumina column was eluted with 4 ml of imidazole buffer, and this was collected in a scintillation vial and counted after the addition of 15 ml of scintillation mixture. The two-step method for the isolation of cGMP, where water was used to elute the Dowex 50 column, was modified from procedures described by Krishna and Krishnan (4), Nesbitt et al. (5), and Craven and DeRubertis (6). This procedure used the same perchloric acid stop, followed by [3H]cGMP, described above under Cyclase assays and the same Dowex 50 columns described under Purification of cyclic GMP. However, these columns were equilibrated with water rather than acid. After application of the reaction mixture supernatant solution, the Dowex 50 columns were washed with two 0.5ml aliquots of water, and those washes were discarded. The Dowex 50 columns were then mounted above a rack of alumina columns equilibrated with 0.1 M imidazole buffer. The cGMP was eluted from the Dowex 50 columns with 1.5 ml of water, and this was allowed to drain onto the alumina columns. The Dowex 50 columns were then removed, and the alumina columns were washed with 1.5 ml
TWO-STEP
CYCLASE
ASSAY
455
of imidazole buffer which was discarded. The cGMP was then eluted from the aluminainto scintillation vials with 3 ml of 0.1 M imidazole buffer. RESULTS
Our first extensive experience with a two-step assay was in an attempt to use a “high-blank” shipment of [a-32P]GTP. We used a separation method much like that later published’ by Nesbitt et al. (5), and it is described under Comparative methods for cyclic nucleotide purification. While we could reduce the blank with this method, in our hands there was considerable variation within the blank replicates. When low levels of guanylate cyclase activity were assayed, such variation was as troublesome as a high blank. The cause of the variability is explained by the results of the experiment diagrammed in Fig. 1A. This experiment shows that the blank radioactivity, that is, the compounds not adsorbed by alumina, elutes from a Dowex 50 column equilibrated with water in a peak very close to the elution position of cGMP. Therefore, comparatively small variations in column size or a less than precise attention to fraction volume will allow some of the blank peak to enter the cGMP peak. It was, therefore, necessary to improve the separation of these peaks, if we were to improve reproducibility. This we were only able to achieve by running the Dowex 50 columns in acid, which retarded the elution of cGMP (9, 10). Since we knew that cGMP would be retained by alumina under acid conditions, but could be subsequently eluted by buffer (ll), we decided to combine such an adsorption step with acidic Dowex 50 chromatography. The results from such a combination are depicted in Fig. IB. This diagram shows a much improved separation of the blank peak from the [3H]cGMP peak. The small tritium peak eluting early contained radiochemical decomposition product(s) of [3H]cGMP. Although roughly equivalent amounts of [a-32P]GTP radioactivity were used in the experiments diagrammed in Figs. 1A and B, the amount of radioactivity appearing in the blank peak in A is much greater than that in B. The explanation for this is that in Fig. 1A any blank radioactivity in each Dowex 50 fraction was counted directly, while in Fig. 1B, each fraction was first adsorbed onto an alumina column and that column was washed with water before the subsequent elution with imidazole buffer. The water wash removed much of the 32P-containing impurities that could later be eluted by the buffer. Although the bulk of these impurities appeared in the blank peak from the Dowex 50, a small amount of 32P also appeared in the [3H]cGMP peak and adsorbed to the alumina. By consecutive l-ml washes with water. we found that the 32P counts leaching from the alumina appeared to reach a minimum after 8 ml. Of the [3H]cGMP adsorbed to the alumina, not more than 3% of the counts ’ We thank Dr. David Wallach for providing us with this method before publication
456
WHITE AND KARR
ELUTION
VOLUME
(ml)
FIG. 1. Chromatographic separation on Dowex 50 columns of rH]cGMP from the 32Plabeled impurities in [oJPP]GTP that are not retained by alumina. Two guanylate cyclase reaction mixtures were “stopped” by the addition of perchloric acid and diluted with water containing [3H]cGMP as described under Cyclase assays. In A, there were 3.04 x 1oPcpm of [aJrZP]GTP and 351,700 cpm of [HlcGMP, while B contained 2.87 x 108 cpm of [a-“*PIGTP and 203,860 cpm of [SH]cGMP. Both samples were mixed, centrifuged, and decanted onto Dowex 50 columns containing 2 ml of resin, however, in A the column was equilibrated with water, while in B the medium was 50 mM HCl. After the application volume of sample A had entered the resin bed, the Dowex 50 column was eluted with 0.2-ml aliquots of water. Each fraction eluting from the column including the application volume was collected directly onto a separate alumina column, which had been equilibrated with 0.1 M imidazole buffer. After each fraction was collected, the tip of the Dowex 50 column was washed with 1.5 ml of the imidazole buffer, which was also collected on the alumina column below. Both the collected fraction and the wash solution drained through the alumina column and were discarded. After all fractions were collected, the rack of alumina columns was placed above a rack of scintillation vials and each column was eluted with 3 ml of 0.1 M imidazole buffer which was collected and counted. In experiment B the Dowex 50 column was eluted with 0.2~ml aliquots of 50 mM HCl. The fractions were collected onto alumina columns equilibrated with water. The tip of the Dowex 50 column was washed with 2.0 ml of 50 mM HCl and this, too, was collected onto the alumina. After all fractions were collected, the alumina columns were washed with water and eluted with 0.2 M imidazole as described under Purification of cyclic GMP.
were removed during the application step and subsequent wash, and this probably represented tritiated impurities. Table 1A shows the blanks given with a single lot of [cx-~~P]GTP when cGMP was purified by the one-
TWO-STEP
CYCLASE
457
ASSAY
step alumina method, as compared to the two-step methods diagrammed in Fig. IA. We were able to greatly reduce the blank given by alumina as previously reported (4-6) by first purifying the [3H]cGMP on a Dowex 50 column eluted with water (compare Methods A. 1 and A.2 in Table 1). The blank was further reduced, with no decrease in [3H]cGMP recovery, by eluting the Dowex 50 with 50 mM HCl (Method A.3). In these experiments we attempted to magnify the blank values by using comparatively large amounts of [Q-~~P]GTP radioactivity. However, it is under just such conditions, used to detect low enzyme activities, that workers have had high blanks with one-step alumina methods. Because of the improvement we achieved in the guanylate cyclase assay, we applied the same approach to the assay of adenylate cyclase. Here we found that, although 50 mM HCl would improve the separation on TABLE A
COMPARISON
OF THREE FROM
METHODS CYCLASE
1
FOR THE REACTION
ISOLATION
OF
CAMP
OR
cGMP
MIXTURES”
3H recovery (%I
(wm)
(%I
(pm4
80.4 (1.3)
1077 (70)
6.8 x 10-Z (4.5 x 10-S)
61.6 (4.0)
2. Dowex 50 eluted with water + alumina
67.3 (5.0)
24 (4)
1.9 x 10-a (3.9 x 10-q
1.7 (0.4)
3. Dowex 50 eluted with HCI + alumina
67.5 (1.8)
7 (1)
4.8 x IO-’ (4.7 x 10-y
0.4 (0.04)
88.6
330 (14)
4.0 x IO-2 (2.8 x lO-3)
18.1
(2.7) 2. Dowex 50 eluted with water + alumina
75.6 (3.5)
36 (31)
5.3 x 10-S (4.8 x 1O-3)
(2.2)
3. Dowex 50 eluted with HCIO, + alumina
74.2
4 (3)
5.5 x 10-d (4.8 x lo+)
(0.2)
Method A. Guanylate cyclase I. Neutral alumina
B. Adenyiate cyclase 1. Neutral alumina
(0.6)
32P blank
(1.2) 2.4
0.2
a Standard reaction mixtures were used without the addition of enzyme. In A each reaction tube contained 2.0 X 1O”cpm of [@*P]GTP and 31,698 cpm of [3H]cGMP. In B each reaction tube contained 9.0 X loj cpm of [&*P]ATP and 38,046 cpm of r3H]cAMP. The tubes were “stopped” and the cyclic nucleotide was purified as described for each method in Materials and Methods. Each value represents the mean of four determinations. Standard deviations are shown in parentheses. The 3H recovery was calculated as percentage of the added PHIcAMP or [3H]cGMP. The “P blanks are shown first as counts per minute minus background (23 cpm). These data were corrected to 100% recovery from the 3H recovery, and then expressed as percentage of the 32P used, and also as apparent picomoles of CAMP or cGMP.
458
WHITE
AND
KARR
Dowex 50 of [3H]cAMP from the blank peak, this acid would not effect a complete retention of CAMP on alumina. However, r3H]cAMP applied in HClO, was retained, even at HClO, concentrations as low as 1 mM. After trials with 1,5,10, and 50 mM HC104, we found that 10 mM appeared to be optimum since only 1.3% of [3H]cAMP applied in 7 ml of acid was not retained, yet 93% was recovered from the alumina in 3 ml of 0.2 M imidazole buffer. Perchloric acid was as effective as HCl in improving the separation of [3H]cAMP from the blank peak, the former eluting in a broad peak from the fifth through the twelfth milliliter, while the latter appeared in the first 2 ml. We found with the elution procedure of Salomon et al. (2) that [3H]cAMP appeared after the third milliliter, in a total volume of 3.5 to 4.0 ml, while the blank peak again eluted in the first 2 ml. Because of the much delayed elution from Dowex 50 of CAMP as compared to cGMP, the separation of [3H]cGMP from the blank peak that we achieved in Fig. 1B was essentially equivalent to the separation of [3H]cAMP from the blank peak achieved by the procedure of Salomon et al. It was questionable therefore, if the HCIOl elution procedure was an improvement over water elution, for CAMP purification. However, when we compared the blanks given by the two procedures under adenylate cyclase assay conditions, the new method did give lower blanks. Table 1, Method B, shows the results of another comparison experiment where, with a single lot of [a-32P]ATP, we determined the blanks given by one-step alumina and by both two-step methods. In order to maximize [3H]cAMP recovery in the procedure of Salomon et al. (Method B.2), we used the modification they mentioned in their Note Added in Proof (2), in which 4 ml of 0.1 M imidazole buffer is collected, rather than 3 ml. As evidenced by the results presented in Table 1, a preliminary purification on Dowex 50 in water decreased the blank nearly lo-fold, as compared with one-step alumina (compare B.l and B.2). If the Dowex SO column was run in 10 mM HC104, the blank was reduced another 10 times (compare B.2 and B.3). It must be noted that Salomon et al. (2) and also Wincek and Sweat, using similar procedures (3), both reported negligible blanks using amounts of [(u-32P]ATP comparable to those used here. We do not view our results with their procedures as contradictory, since the [a-32PJATP we used was obviously “high-blank” material (See Table 1, Method B.l). However, it was just this variation in substrate quality that prompted the development of two-step assays and our subsequent modification. DISCUSSION
We have previously demonstrated that, when chromatographed on DEAE-cellulose, “high-blank” [a-32P]GTP showed additional radioactive peaks when compared to “low-blank” material and also that the puri-
TWO-STEP
CYCLASE
ASSAY
459
fied [a-32P]GTP from those columns gave a zero blank (1). We concluded that the blank found in a one-step guanylate cyclase assay was not due to unadsorbed substrate, but rather to radiochemical impurities which were not retained by alumina. We herein present evidence that the greater part of this blank material can be separated from [3H]cGMP on a Dowex 50 column and is therefore not preformed [32PJcGMP. We have no information on the chemical structure of the blank compound(s). The blank radioactivity is introduced during the synthesis of the substrate, and manufacturers have given insufficient attention to its removal during purification. Since the blank varies with each lot of substrate, the method used for its separation from cGMP must allow for this variation. We find that, if the Dowex 50 chromatography is performed under acid conditions, the separation of cGMP from the blank radioactivity is much improved and provides a greater margin for error and/or for the magnitude of the blank peak. The broadening of the cGMP peak that occurs under acid conditions is compensated for by the trapping of cGMP on alumina under these same conditions. Thus, high recovery of [3H]cGMP is maintained. The original two-step method of Salomon et al. (2) provided a satisfactory separation of blank from CAMP. (It should be noted that the SDS used by those authors in the stop solution also produces a retardation of cAMPelution from Dowex 50). However, the acid elution method improved the blanks as compared to the water elution method (Table I, Method B). This may in part be due to the additional water wash that the alumina received after CAMP adsorption and before elution with buffer. We have not determined the maximum amount of water that may be used, however, we have occasionally doubled the wash volume with no decrease in tritium recovery. A phenomenon worth mentioning is the appearance of turbidity in the effluents from the alumina columns during the water wash. This turbidity occurs subsequent to the application of HCI or HCIOI, in any concentration, and immediately disappears upon application of the first milliliter of imidazole buffer. The next 3 ml of buffer which are collected are perfectly clear. Turbidity will also result if a water wash follows the application of imidazole or Tris-HCl buffer to alumina. Whenever turbidity occurs we have detected in those fractions a drop in pH of 1 to 2 units. We do not know the nature of the turbidity-producing material. The principal problem with the two-step method we describe is the additional time involved as compared to simple alumina chromatography. This is particularly evident in the guanylate cyclase assay, which requires about 1 hr for the Dowex 50 separation. We have attempted to shorten this by using a coarser resin (100-200 mesh); however because of the consequent broadening of the peaks, a larger resin bed (0.7 x 10 cm) had to be used. Nevertheless, these columns ran almost three times faster
460
WHITE AND KARR
than the 200- to 400-mesh resin columns. In order to maximize recovery with the lOO- to 200-mesh resin, we collected the entire cGMP peak (seventh through thirteenth milliliters) and, after the alumina treatment, obtained 51.9% recovery of [3H]cGMP, which was lower than the 67.5% recovery we obtained with the 200- to 400-mesh resin (Table 1, Method A.3). However, since the 32P blanks given by the larger columns were also slightly lower, the corrected blanks from both columns were equivalent. We have not used the larger columns because their more rapid flow rate is offset by the fact that they take more time to make radiochemically clean (the volumes of NaOH and HCl solutions used for recycling have to be doubled.) We have not been able to clean our Dowex 50 columns with a simple HCl wash, as some have reported (2,5). We find that 8% cross-linkage resin gives as good recovery of [3H]cGMP and [3H]cAMP as 4% cross-linkage (2) when eluted with acid, and the 8% is more stable to recycling with base and acid. In our hands sharp peaks and high recoveries from Dowex 50 primarily result from small bead size. ACKNOWLEDGMENTS This research was supported by USPHS Grant 5 ROl Hl 15002-05.
REFERENCES 1. 2. 3. 4. 5.
White, A. A., and Zenser, T. V. (1971) Anal. Biochem. 41, 372~3%. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548. Wincek, T. J., and Sweat, F. W. (1975)Anal. Biochem. 64, 631-635. Krishna, G., and Krishnan, N. (1975) J. Cyclic Nucleotide Res. 1, 293-302. Nesbitt III, J. A., Anderson, V. B., Miller, Z., Pastan, I., Russell, T. R., and Gospodarowicz, D. (1976) J. Biol. Chem. 251, 2344-2352. 6. Craven, P. A., and DeRubertis, F. R. (1976) Anal. Biochem. 72, 455-459. 7. White, A. A., Northup, S. J., and Zenser, T. V. (1972) in Methods in Cyclic Nucleotide Research (Chasin, M., ed.), pp. 125- 167, Marcel Dekker, New York. 8. White, A. A., Crawford, K. M., Patt, C. S., and Lad, P. J. (1976) J. Biol. Chem. 251, 7304-7312.
9. White, A. A., and Aurbach, G. D. (1969) Biochim. Biophys. Acta 191, 686-697. 10. Schultz, G., Biihme, E., and Hardman, J. G. (1974) in Methods in Enzymology (Hardman, J. G., and O’Malley, B. W., eds.), Vol. 38, pp. 9-20, Academic Press, New York. 11. Jakobs, K. H., Biihme, E., and Schultz, G. (1975) Acta Endocrinol. Suppl. 19, 431.
BIOCHEMISTRY
85,
45 I-460 (1978)
Improved Two-Step Method for the Assay of Adenylate and Guanylate Cyclase ARNOLD A. WHITE AND DALE B. KARR John
M. Dalton University
Research
Center
and
of Missouri-Columbia,
Department Coiumbia,
of Biochemistry, Missouri 65201
Received February 2, 1977; accepted October 24. 1977 A two-step assay for adenylate and guanylate cyclase is described utilizing c@*P-labeled ATP or GTP as substrate and involving purification of the resulting 3PP-labeled CAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 mM HCI for cGMP and 10mM HCIO, for CAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The CAMPor cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the s*P-labeled CAMP or cGMP is eluted with 0.2 M imidazole buffer and counted. The method delivers blanks amounting to .OOOS% of the substrate radioactivity, high recoveries, and excellent reproducibility.
Earlier we described a method for the separation of 3’S’-cyclic nucleoside monophosphates from other nucleotides on aluminum oxide columns and showed how it could be applied to the assay of adenylate or guanylate cyclase (1). In those assays, (Y-32P-labeled ATP or GTP was used as substrate, giving rise to 32P-labeled adenosine 3’,5’-monophosphate (CAMP) or guanosine 3’S’-monophosphate (cGMP), respectively. The 32P-labeled cyclic nucleotides passed through columns of alumina prepared with buffer in the neutral range, while all other nucleotides were retained. The amount of 32P eluting from the columns was therefore a measure of cyclase activity. However, we noted then that certain lots of [Q-~*P]GTP contained radiochemical impurities which were not retained by alumina and therefore gave a high blank. We had no solution for that problem, except for removing the impurities from the substrate by purification on DEAE-cellulose. As later workers began to use this method and with higher and higher levels of radioactivity in the substrate, several modifications were introduced to reduce the blanks. Salomon et al. (2) and also Wincek and Sweat (3) showed that sequential chromatography on Dowex 50 and alumina produced a more consistent assay for adenylate cyclase, and the same combination was utilized for guanylate cyclase assay (4-6). Be451
0003-2697/78/0852-0451$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
452
WHITE
AND
KARR
cause of the inconsistency in the quality of our supply of [cz-~~P]GTP, we also began to use a two-step method for guanylate cyclase assay, originally patterned after that of Nesbitt et al. (5). We here describe an improvement in that methodology and its application to the assay of adenylate cyclase. MATERIALS
AND METHODS
The aluminum oxide used in this work was Type 90, Brockman activity II-III from E-M Laboratories, Inc. The [a-32P]ATP and [cz-~~P]GTP were purchased from ICN, while the [3H]cAMP and [3H]cGMP were from New England Nuclear and Schwarz/Mann, respectively. GTP was from P-L Biochemicals, while Sigma Chemical Co. supplied all other biochemicals, including the Dowex 50 ion-exchange resin (Dowex 5OWX8, ZOO-400 mesh). The 32P-labeled nucleotides were diluted with cold ATP and GTP upon receipt in order to minimize radiochemical decomposition (7). Both [3H]cGMP and [3H]cAMP were purified before use (7). Solutions. Imidazole buffer, pH 7.07, was used in two concentrations: a 0.2 M solution containing 0.1 M HCl and a 0.1 M solution containing 0.05 M HCI. The imidazole was obtained from Pierce Chemical Co., and the HCl, from Fisher Chemical Co. as a 1 N standard solution. The solutions used to regenerate the Dowex 50 columns were prepared by diluting 50% (w/w) NaOH solution (Fisher) lo-fold to give a 5% solution and diluting concentrated (37.8%) HCl (J. T. Baker) sixfold to give a 6.3% solution. Column preparation. Two types of glass columns were used in this work. The alumina was contained in our standard columns which were made of 9-mm-o.d. standard wall tubing (7-mm nominal inside diameter), 15 cm long, with one end drawn down to an opening of about 1.5 mm. The other end was joined to a 65-mm length of 25-mm-o.d. tubing, which acted as a reservoir of about ZO-ml capacity. The Dowex 50 resin was contained in our narrow-inside-diameter columns, which resembled the standard columns except that the column barrels were made from 9-mm-o.d. tubing with a Z-mm-thick wall (Corning Glass Works Code No. 234104) resulting in a 5-mm nominal inside diameter. Since both columns had the same outer dimensions, they could be held in the same racks. The tips of the Dowex 50 columns were packed with glass wool, while in the alumina columns paper toweling was used (Teri, Kimberly-Clark Corp.). The alumina columns were prepared by measuring out approximately 1 g of alumina with a measuring spoon and emptying the spoon into a column filled with water or, in some experiments, 0.1 M imidazole buffer. After the alumina had settled and the columns were drained, they
TWO-STEP
CYCLASE
ASSAY
453
were washed with one additional column volume (about 20 ml) of water or buffer. Alumina columns were used only once. Dowex 50 columns were used in the H+ form. They were cycled before being used and after each subsequent use by the sequential addition of one column volume of each of the following: H,O, 5% NaOH, H,O, 6.3% HCl, H,O. Cyclase assays. Final volume of the reaction mixtures for both the adenylate cyclase and guanylate cyclase assays is 75 ~1, and both contained 50 mM Tris-HCl, pH 7.6,O. 1 mg of bovine plasma albumin, 10 mM 2-mercaptoethanol, 20 mM caffeine, 15 mM creatine phosphate, and 10 units of creatine phosphokinase. The adenylate cyclase reaction mixture contained 1.2 mM [o!-~*P]ATP, 5 mM MgCl*, and 0.2 pmol of CAMP. The guanylate cyclase reaction mixtures contained 1.2 mM [w~~P]GTP, 6 mM MnCl,, and 5 mM cGMP. There were IO-40 cpm/pmol used in both assays. Reactions were initiated by the addition of the enzyme and were incubated at 30°C usually for 10 min. Reactions were stopped by the addition of 0.15 ml of 1 N HCIOI, and after all reactions were completed 0.3 ml of water containing approximately 30,000 cpm of tritiated cyclic nucleotide was added to each tube. After mixing and centrifuging, the supernatant solutions were poured onto Dowex 50 columns. Purification of cyclic GMP. The Dowex 50 columns for this isolation contained 2 ml of resin giving a IO-cm bed height. Before use, each column was washed with 10 ml of 50 mM HCl. After the reaction mixture supernatant solution had drained through the resin, it was followed by the addition of 2.5 ml of 50 mM HCl. This total effluent was discarded. The Dowex 50 columns were then mounted above a rack of alumina columns, and an additional 3.5 ml of 50 mM HCl was added to each Dowex 50 column and allowed to drain through both columns. The Dowex 50 columns were removed and the alumina was washed first with 10 ml of water and then with 1 ml of 0.2 M imidazole buffer. The alumina columns were then mounted above a rack of scintillation vials and the cGMP was eluted from the alumina and into the vials by the addition of 3 ml of 0.2 M imidazole to each column. The vials were counted after addition of 10 ml of scintillation mixture (8). Purl$cation of cyclic AMP. The Dowex 50 columns each contained I ml of resin, which produced a 5-cm bed height. Each column was washed before use with 10 ml of 10 mM HClO,. After the reaction mixture supernatant solution had drained through the resin bed, 2.5 ml of 10 ITlM HC104 was added to each column twice, the first 2.5 ml being allowed to drain through the column before the second was added. All of the column effluent to this point was discarded. The rack of Dowex 50 columns was then mounted above a rack of alumina columns, after which 7 ml of 10 mM HCIO, was added to each Dowex 50 column and allowed to drain
454
WHITE
AND
KARR
through both columns. The remainder of the assay was the same as the cGMP purification. Comparative methods for cyclic nucleotide purification. The one-step method for the purification of CAMP or cGMP using alumina columns has been described (1). In this method, the reaction was terminated by adding 0.5 ml of 0.1 M HCl, containing approximately 30,000 cpm of tritiated cyclic nucleotide, and heating for 2 min at 100°C. The tube contents were neutralized by the addition of 0.5 ml of 0.2 M imidazole (free base) and were applied directly to the alumina columns, which were prepared and eluted with 0.1 M imidazole buffer. .4fter the application volume had drained through the column, CAMP was eluted with 3 ml of imidazole buffer which was collected in a scintillation vial and counted after the addition of 10 ml of scintillation mixture. If cGMP was to be isolated, the application volume was followed by 1 ml of buffer, both of which were discarded. The cGMP was then eluted with 3 ml of buffer and counted as before. The two-step method of Salomon et al. (2) for the purification of CAMP was used as described except that we substituted 1 g of alumina and the 0.1 M imidazole buffer used here (pH 7.07) for the 0.6 g of alumina and 0.1 M imidazole buffer, pH 7.5, used by those authors. The reaction was stopped with 0.1 ml of 2% sodium dodecyl sulfate (SDS) after which 0.3 ml of [3H]cAMP and 0.58 ml of water were added. This was applied to a Dowex 50 column containing 1 ml of resin which had been equilibrated with water. After the application volume had entered the bed, the column was washed twice with 1 ml of water. The CAMP was then washed from the Dowex 50 column onto an alumina column (equilibrated with 0.1 M imidazole) with 3 ml of water. The alumina column was eluted with 4 ml of imidazole buffer, and this was collected in a scintillation vial and counted after the addition of 15 ml of scintillation mixture. The two-step method for the isolation of cGMP, where water was used to elute the Dowex 50 column, was modified from procedures described by Krishna and Krishnan (4), Nesbitt et al. (5), and Craven and DeRubertis (6). This procedure used the same perchloric acid stop, followed by [3H]cGMP, described above under Cyclase assays and the same Dowex 50 columns described under Purification of cyclic GMP. However, these columns were equilibrated with water rather than acid. After application of the reaction mixture supernatant solution, the Dowex 50 columns were washed with two 0.5ml aliquots of water, and those washes were discarded. The Dowex 50 columns were then mounted above a rack of alumina columns equilibrated with 0.1 M imidazole buffer. The cGMP was eluted from the Dowex 50 columns with 1.5 ml of water, and this was allowed to drain onto the alumina columns. The Dowex 50 columns were then removed, and the alumina columns were washed with 1.5 ml
TWO-STEP
CYCLASE
ASSAY
455
of imidazole buffer which was discarded. The cGMP was then eluted from the aluminainto scintillation vials with 3 ml of 0.1 M imidazole buffer. RESULTS
Our first extensive experience with a two-step assay was in an attempt to use a “high-blank” shipment of [a-32P]GTP. We used a separation method much like that later published’ by Nesbitt et al. (5), and it is described under Comparative methods for cyclic nucleotide purification. While we could reduce the blank with this method, in our hands there was considerable variation within the blank replicates. When low levels of guanylate cyclase activity were assayed, such variation was as troublesome as a high blank. The cause of the variability is explained by the results of the experiment diagrammed in Fig. 1A. This experiment shows that the blank radioactivity, that is, the compounds not adsorbed by alumina, elutes from a Dowex 50 column equilibrated with water in a peak very close to the elution position of cGMP. Therefore, comparatively small variations in column size or a less than precise attention to fraction volume will allow some of the blank peak to enter the cGMP peak. It was, therefore, necessary to improve the separation of these peaks, if we were to improve reproducibility. This we were only able to achieve by running the Dowex 50 columns in acid, which retarded the elution of cGMP (9, 10). Since we knew that cGMP would be retained by alumina under acid conditions, but could be subsequently eluted by buffer (ll), we decided to combine such an adsorption step with acidic Dowex 50 chromatography. The results from such a combination are depicted in Fig. IB. This diagram shows a much improved separation of the blank peak from the [3H]cGMP peak. The small tritium peak eluting early contained radiochemical decomposition product(s) of [3H]cGMP. Although roughly equivalent amounts of [a-32P]GTP radioactivity were used in the experiments diagrammed in Figs. 1A and B, the amount of radioactivity appearing in the blank peak in A is much greater than that in B. The explanation for this is that in Fig. 1A any blank radioactivity in each Dowex 50 fraction was counted directly, while in Fig. 1B, each fraction was first adsorbed onto an alumina column and that column was washed with water before the subsequent elution with imidazole buffer. The water wash removed much of the 32P-containing impurities that could later be eluted by the buffer. Although the bulk of these impurities appeared in the blank peak from the Dowex 50, a small amount of 32P also appeared in the [3H]cGMP peak and adsorbed to the alumina. By consecutive l-ml washes with water. we found that the 32P counts leaching from the alumina appeared to reach a minimum after 8 ml. Of the [3H]cGMP adsorbed to the alumina, not more than 3% of the counts ’ We thank Dr. David Wallach for providing us with this method before publication
456
WHITE AND KARR
ELUTION
VOLUME
(ml)
FIG. 1. Chromatographic separation on Dowex 50 columns of rH]cGMP from the 32Plabeled impurities in [oJPP]GTP that are not retained by alumina. Two guanylate cyclase reaction mixtures were “stopped” by the addition of perchloric acid and diluted with water containing [3H]cGMP as described under Cyclase assays. In A, there were 3.04 x 1oPcpm of [aJrZP]GTP and 351,700 cpm of [HlcGMP, while B contained 2.87 x 108 cpm of [a-“*PIGTP and 203,860 cpm of [SH]cGMP. Both samples were mixed, centrifuged, and decanted onto Dowex 50 columns containing 2 ml of resin, however, in A the column was equilibrated with water, while in B the medium was 50 mM HCl. After the application volume of sample A had entered the resin bed, the Dowex 50 column was eluted with 0.2-ml aliquots of water. Each fraction eluting from the column including the application volume was collected directly onto a separate alumina column, which had been equilibrated with 0.1 M imidazole buffer. After each fraction was collected, the tip of the Dowex 50 column was washed with 1.5 ml of the imidazole buffer, which was also collected on the alumina column below. Both the collected fraction and the wash solution drained through the alumina column and were discarded. After all fractions were collected, the rack of alumina columns was placed above a rack of scintillation vials and each column was eluted with 3 ml of 0.1 M imidazole buffer which was collected and counted. In experiment B the Dowex 50 column was eluted with 0.2~ml aliquots of 50 mM HCl. The fractions were collected onto alumina columns equilibrated with water. The tip of the Dowex 50 column was washed with 2.0 ml of 50 mM HCl and this, too, was collected onto the alumina. After all fractions were collected, the alumina columns were washed with water and eluted with 0.2 M imidazole as described under Purification of cyclic GMP.
were removed during the application step and subsequent wash, and this probably represented tritiated impurities. Table 1A shows the blanks given with a single lot of [cx-~~P]GTP when cGMP was purified by the one-
TWO-STEP
CYCLASE
457
ASSAY
step alumina method, as compared to the two-step methods diagrammed in Fig. IA. We were able to greatly reduce the blank given by alumina as previously reported (4-6) by first purifying the [3H]cGMP on a Dowex 50 column eluted with water (compare Methods A. 1 and A.2 in Table 1). The blank was further reduced, with no decrease in [3H]cGMP recovery, by eluting the Dowex 50 with 50 mM HCl (Method A.3). In these experiments we attempted to magnify the blank values by using comparatively large amounts of [Q-~~P]GTP radioactivity. However, it is under just such conditions, used to detect low enzyme activities, that workers have had high blanks with one-step alumina methods. Because of the improvement we achieved in the guanylate cyclase assay, we applied the same approach to the assay of adenylate cyclase. Here we found that, although 50 mM HCl would improve the separation on TABLE A
COMPARISON
OF THREE FROM
METHODS CYCLASE
1
FOR THE REACTION
ISOLATION
OF
CAMP
OR
cGMP
MIXTURES”
3H recovery (%I
(wm)
(%I
(pm4
80.4 (1.3)
1077 (70)
6.8 x 10-Z (4.5 x 10-S)
61.6 (4.0)
2. Dowex 50 eluted with water + alumina
67.3 (5.0)
24 (4)
1.9 x 10-a (3.9 x 10-q
1.7 (0.4)
3. Dowex 50 eluted with HCI + alumina
67.5 (1.8)
7 (1)
4.8 x IO-’ (4.7 x 10-y
0.4 (0.04)
88.6
330 (14)
4.0 x IO-2 (2.8 x lO-3)
18.1
(2.7) 2. Dowex 50 eluted with water + alumina
75.6 (3.5)
36 (31)
5.3 x 10-S (4.8 x 1O-3)
(2.2)
3. Dowex 50 eluted with HCIO, + alumina
74.2
4 (3)
5.5 x 10-d (4.8 x lo+)
(0.2)
Method A. Guanylate cyclase I. Neutral alumina
B. Adenyiate cyclase 1. Neutral alumina
(0.6)
32P blank
(1.2) 2.4
0.2
a Standard reaction mixtures were used without the addition of enzyme. In A each reaction tube contained 2.0 X 1O”cpm of [@*P]GTP and 31,698 cpm of [3H]cGMP. In B each reaction tube contained 9.0 X loj cpm of [&*P]ATP and 38,046 cpm of r3H]cAMP. The tubes were “stopped” and the cyclic nucleotide was purified as described for each method in Materials and Methods. Each value represents the mean of four determinations. Standard deviations are shown in parentheses. The 3H recovery was calculated as percentage of the added PHIcAMP or [3H]cGMP. The “P blanks are shown first as counts per minute minus background (23 cpm). These data were corrected to 100% recovery from the 3H recovery, and then expressed as percentage of the 32P used, and also as apparent picomoles of CAMP or cGMP.
458
WHITE
AND
KARR
Dowex 50 of [3H]cAMP from the blank peak, this acid would not effect a complete retention of CAMP on alumina. However, r3H]cAMP applied in HClO, was retained, even at HClO, concentrations as low as 1 mM. After trials with 1,5,10, and 50 mM HC104, we found that 10 mM appeared to be optimum since only 1.3% of [3H]cAMP applied in 7 ml of acid was not retained, yet 93% was recovered from the alumina in 3 ml of 0.2 M imidazole buffer. Perchloric acid was as effective as HCl in improving the separation of [3H]cAMP from the blank peak, the former eluting in a broad peak from the fifth through the twelfth milliliter, while the latter appeared in the first 2 ml. We found with the elution procedure of Salomon et al. (2) that [3H]cAMP appeared after the third milliliter, in a total volume of 3.5 to 4.0 ml, while the blank peak again eluted in the first 2 ml. Because of the much delayed elution from Dowex 50 of CAMP as compared to cGMP, the separation of [3H]cGMP from the blank peak that we achieved in Fig. 1B was essentially equivalent to the separation of [3H]cAMP from the blank peak achieved by the procedure of Salomon et al. It was questionable therefore, if the HCIOl elution procedure was an improvement over water elution, for CAMP purification. However, when we compared the blanks given by the two procedures under adenylate cyclase assay conditions, the new method did give lower blanks. Table 1, Method B, shows the results of another comparison experiment where, with a single lot of [a-32P]ATP, we determined the blanks given by one-step alumina and by both two-step methods. In order to maximize [3H]cAMP recovery in the procedure of Salomon et al. (Method B.2), we used the modification they mentioned in their Note Added in Proof (2), in which 4 ml of 0.1 M imidazole buffer is collected, rather than 3 ml. As evidenced by the results presented in Table 1, a preliminary purification on Dowex 50 in water decreased the blank nearly lo-fold, as compared with one-step alumina (compare B.l and B.2). If the Dowex SO column was run in 10 mM HC104, the blank was reduced another 10 times (compare B.2 and B.3). It must be noted that Salomon et al. (2) and also Wincek and Sweat, using similar procedures (3), both reported negligible blanks using amounts of [(u-32P]ATP comparable to those used here. We do not view our results with their procedures as contradictory, since the [a-32PJATP we used was obviously “high-blank” material (See Table 1, Method B.l). However, it was just this variation in substrate quality that prompted the development of two-step assays and our subsequent modification. DISCUSSION
We have previously demonstrated that, when chromatographed on DEAE-cellulose, “high-blank” [a-32P]GTP showed additional radioactive peaks when compared to “low-blank” material and also that the puri-
TWO-STEP
CYCLASE
ASSAY
459
fied [a-32P]GTP from those columns gave a zero blank (1). We concluded that the blank found in a one-step guanylate cyclase assay was not due to unadsorbed substrate, but rather to radiochemical impurities which were not retained by alumina. We herein present evidence that the greater part of this blank material can be separated from [3H]cGMP on a Dowex 50 column and is therefore not preformed [32PJcGMP. We have no information on the chemical structure of the blank compound(s). The blank radioactivity is introduced during the synthesis of the substrate, and manufacturers have given insufficient attention to its removal during purification. Since the blank varies with each lot of substrate, the method used for its separation from cGMP must allow for this variation. We find that, if the Dowex 50 chromatography is performed under acid conditions, the separation of cGMP from the blank radioactivity is much improved and provides a greater margin for error and/or for the magnitude of the blank peak. The broadening of the cGMP peak that occurs under acid conditions is compensated for by the trapping of cGMP on alumina under these same conditions. Thus, high recovery of [3H]cGMP is maintained. The original two-step method of Salomon et al. (2) provided a satisfactory separation of blank from CAMP. (It should be noted that the SDS used by those authors in the stop solution also produces a retardation of cAMPelution from Dowex 50). However, the acid elution method improved the blanks as compared to the water elution method (Table I, Method B). This may in part be due to the additional water wash that the alumina received after CAMP adsorption and before elution with buffer. We have not determined the maximum amount of water that may be used, however, we have occasionally doubled the wash volume with no decrease in tritium recovery. A phenomenon worth mentioning is the appearance of turbidity in the effluents from the alumina columns during the water wash. This turbidity occurs subsequent to the application of HCI or HCIOI, in any concentration, and immediately disappears upon application of the first milliliter of imidazole buffer. The next 3 ml of buffer which are collected are perfectly clear. Turbidity will also result if a water wash follows the application of imidazole or Tris-HCl buffer to alumina. Whenever turbidity occurs we have detected in those fractions a drop in pH of 1 to 2 units. We do not know the nature of the turbidity-producing material. The principal problem with the two-step method we describe is the additional time involved as compared to simple alumina chromatography. This is particularly evident in the guanylate cyclase assay, which requires about 1 hr for the Dowex 50 separation. We have attempted to shorten this by using a coarser resin (100-200 mesh); however because of the consequent broadening of the peaks, a larger resin bed (0.7 x 10 cm) had to be used. Nevertheless, these columns ran almost three times faster
460
WHITE AND KARR
than the 200- to 400-mesh resin columns. In order to maximize recovery with the lOO- to 200-mesh resin, we collected the entire cGMP peak (seventh through thirteenth milliliters) and, after the alumina treatment, obtained 51.9% recovery of [3H]cGMP, which was lower than the 67.5% recovery we obtained with the 200- to 400-mesh resin (Table 1, Method A.3). However, since the 32P blanks given by the larger columns were also slightly lower, the corrected blanks from both columns were equivalent. We have not used the larger columns because their more rapid flow rate is offset by the fact that they take more time to make radiochemically clean (the volumes of NaOH and HCl solutions used for recycling have to be doubled.) We have not been able to clean our Dowex 50 columns with a simple HCl wash, as some have reported (2,5). We find that 8% cross-linkage resin gives as good recovery of [3H]cGMP and [3H]cAMP as 4% cross-linkage (2) when eluted with acid, and the 8% is more stable to recycling with base and acid. In our hands sharp peaks and high recoveries from Dowex 50 primarily result from small bead size. ACKNOWLEDGMENTS This research was supported by USPHS Grant 5 ROl Hl 15002-05.
REFERENCES 1. 2. 3. 4. 5.
White, A. A., and Zenser, T. V. (1971) Anal. Biochem. 41, 372~3%. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548. Wincek, T. J., and Sweat, F. W. (1975)Anal. Biochem. 64, 631-635. Krishna, G., and Krishnan, N. (1975) J. Cyclic Nucleotide Res. 1, 293-302. Nesbitt III, J. A., Anderson, V. B., Miller, Z., Pastan, I., Russell, T. R., and Gospodarowicz, D. (1976) J. Biol. Chem. 251, 2344-2352. 6. Craven, P. A., and DeRubertis, F. R. (1976) Anal. Biochem. 72, 455-459. 7. White, A. A., Northup, S. J., and Zenser, T. V. (1972) in Methods in Cyclic Nucleotide Research (Chasin, M., ed.), pp. 125- 167, Marcel Dekker, New York. 8. White, A. A., Crawford, K. M., Patt, C. S., and Lad, P. J. (1976) J. Biol. Chem. 251, 7304-7312.
9. White, A. A., and Aurbach, G. D. (1969) Biochim. Biophys. Acta 191, 686-697. 10. Schultz, G., Biihme, E., and Hardman, J. G. (1974) in Methods in Enzymology (Hardman, J. G., and O’Malley, B. W., eds.), Vol. 38, pp. 9-20, Academic Press, New York. 11. Jakobs, K. H., Biihme, E., and Schultz, G. (1975) Acta Endocrinol. Suppl. 19, 431.
