244-253 (1979)

The Measurement of Cyclic GMP and Cyclic AMP Phosphodiesterasesl JOYCE G.~ARTER,$OSAMMA Department of Pharmacology



and the Beaumont-May Institute of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110 Received March 22, 1979

Methods are described for measuring phosphodiesterases for cGMP and CAMP in the range of activity yielding lo-** to 1OwBmol of product. The 5’-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than IO+’ mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD+ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified lOOO-fold with the succinic thiokinasepyruvate kinase cycle. In the case of CAMP diesterase, larger amounts of Y-AMP are measured in the same way as 5’-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5’-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either CAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5’-nucleotidase, this is prevented by adding GMP to the CAMP reagent, AMP to the cGMP reagent, or 5’-UMP to either reagent.

A need arose to measure the cyclic nucleotide phosphodiesterases in individual retinal layers. From some layers it is difficult to obtain samples much larger than 20 ng dry weight. New methods with the required sensitivity have therefore been developed which it is hoped may also be useful in other situations. Enzyme activities vary several hundredfold among the retinal layers, consequently three methods are presented for each enzyme: (A) a simple method for high activities, (B) a somewhat more sensitive and more flexible procedure which requires an extra step, and (C) a slightly more complicated procedure for low to very low activities. Likewise two variants of each method are offered: one for carrying out all steps in test tubes, the other for carrying out

the first steps in “oil wells.” The oil well procedures permit the use of submicroliter volumes (with still further increase in sensitivity), and are ideal for assays of small frozen dried tissue samples. The 5’-GMP methods are based on enzyme reactions used by Goldberg ef al. (1) as steps in the measurement of cGMP2 itself. The cyclic reaction, 4, was first proposed by Cha and Cha (2,3):

1 This paper is dedicated to the memory of Dr. Alvin Nason. We deeply regret our inability to complete it in time for inclusion in the issue dedicated to his memory.

0003-2697/79/180244-10$02.00/O Copyright All tights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.


* Abbreviations used: cGMP, cyclic GMP; glucose6-P, glucose &phosphate; P-diesterase, phosphodiesterase; Reagent 1, containing 50 mM Tris-HCl buffer, pH 8, 1 mM cGMP, 5 mM MgC&, 0.02% bovine plasma albumin, 1 mglml protamine sulfate (salmine), 5 PM guanylylimidodiphosphate; Reagent 2, containing 50 mM imidazole-HCl buffer, pH 7.0, 100 FM ATP, 100 PM P-pyruvate, 75 mM KCl, 2 mhi MgCl,, 2- 10 j&M NADH (according to need), and shortly before use 3 &ml rabbit muscle pyruvate kinase (EC and 0.5 @ml heart lactic dehydrogenase (EC 2.7. I. 11); Reagent 3, containing 50 mM imidazole-HCl buffer, pH 7.0,2 mM MgCI,, 25 PM ATP, 0.02% bovine plasma albumin, 1 &ml guanylate kinase; Reagent 4, con-









+ 2 P-pyruvate +



GTP + ATP + 2 pyruvaie + NAD+



For method A, only reactions l-3 are used; these are combined into a single step and the decrease in NADH fluorescence is measured. Fertel and Weiss (4) also assayed cGMP diesterase with the aid of reaction 1, but they then measured the decrease in ATP (determined with luciferase). Method B differs from A only in that after completion of reaction 3, the NADH is destroyed with acid and the NAD+ is converted to a highly fluorescent product with strong alkali. This not only increases sensitivity but permits much greater latitude in the amount of GMP measured. For the most sensitive Method C, reaction 1 (carried out separately) is followed by cycling reaction 4 in which roughly 1000 mol of NAD+ is produced per mole of GDP. (The yield can be decreased or increased (up to at least 5000 cycles) by varying time, temperature, and enzyme concentration.) Finally the NADH is destroyed with acid and the NAD+ converted to the fluorescent alkaline product as in Method B. The lower-sensitivity Methods A and B taming 50 mM imidazole-HCI, pH 7.5, 20 mM KCl, 2 mM MgCl,, 1 mM sodium succinate, 500 pM coenzyme A, ~KI~LM P-pyruvate, lOOp~~~~~,O.~%bovine plasma albumin, 25 CLg/ml pig heart succinic thiokinase (EC, 100 kg/ml pyruvate kinase, 1 @ml beef heart lactic dehydrogenase; Reagent 3A, containing 100 mM imidazole-HCI, pH 7.4, 200 pM P-pyruvate, 2 mM MgCl,, 50 mM KCl, 0.02% bovine plasma albumin, 0.02 FM ATP, 5 wg/ml pyruvate kinase, 20 &ml adenylate kinase; Reagent 4A, same as Reagent 3A, except that glucose is added at 1 mM concentration, pyruvate kinase is increased to 100 &ml, 25 &ml hexokinase is added, and adenylate kinase and ATP are omitted; Reagent 5, containing 25 mM Tris-HCl, pH 8, 50 pM NADP, 2 mhr EDTA, 0.5 kg/ml baker’s yeast glucose-6-P dehydrogenase (EC

for 5’-AMP are identical to those for 5’GMP except that adenylate kinase is substituted for guanylate kinase in reaction 1. For the high-sensitivity Method C, 5’-AMP is measured as originally described by Breckenridge (5). The AMP is first converted to ATP with pyruvate kinase, adenylate kinase, and a trace of ATP: pyruvate P-pyruvate


After conversion is complete, the ATP is used to catalyze the formation of glucose6-P from glucose in lOO- to 2000-fold yield: pyruvate P-pyrUvate3





Finally the glucose-6-P is measured fluorometrically with glucose-6-P dehydrogenase and NADP+. (Weisset al. (6), in their diesterase method, converted AMP to ATP as above, but measured the ATP directly with luciferase.) With tissues of low diesterase activity, there may be significant loss of AMP or GMP by 5’-nucleotidase action. This can be largely prevented by adding a relatively high level of GMP in the CAMP diesterase assay, or of AMP in the cGMP diesterase assay. Alternatively, 5’-UMP can be substituted in either case for the same purpose. MATERIALS


Enzymes were obtained from Sigma Chemical Company or Boehringer Corporation. Other biochemicals were from Sigma.

246 Cyclic GMP P-Diesterase 10 nmol of Product)




A (1 to

Reagent 1. Reagent 1 contains 50 mM Tris-HCI buffer, pH 8, 1 mM cGMP, 5 mM MgCL 0.02% bovine plasma albumin, 1 mg/ ml protamine sulfate (salmine), and 5 PM guanylylimidodiphosphate. The reagent can be varied according to the requirements for the particular diesterase. The reagent described is adapted from Miki et al. (7) and gave optimal results with whole retina. When there is danger of significant loss of GMP through the action of tissue 5’-nucleotidase this may be prevented by supplementing Reagent 1 with 5’-AMP or 5’-UMP (see section on 5’-nucleotidase below). Reagent 2. Reagent 2 contains 50 mM imidazole-HCl buffer, pH 7.0, 100 PM ATP, 100 PM P-pyruvate, 75 mM KCl, 2 mM MgCl,, 2 to 10 PM NADH (according to need), and shortly before use 3 &ml rabbit muscle pyruvate kinase (EC and 0.5 C.Lg/ml heart lactic dehydrogenase (EC Step I. The sample is added to 50 ~1 of Reagent 1 in a volume of 10 ,ul or less. (For high precision, the total volume should be the same to within 1 ~1, any differences being made up with HzO. Step 2. After 60 min at 25”C, 10 ~1 of 0.25 N NaOH is added and the samples are heated 10 min in a 60°C water bath. Step 3. Five to 50 ~1 from Step 2 is transferred to a fluorometer tube containing 1 ml of Reagent 2. After an initial reading, 1 pg of beef brain guanylate kinase (EC is added in a volume of 10 ~1 or less. A second reading is made when the reaction is complete (15-30 min). GMP standards are carried through the entire procedure. The guanylate kinase stock solution is stabilized by preparing it in 50% glycerol containing 50 mM imidazole-HCl buffer, pH 7, and 0.02% bovine plasma albumin. A I-mg/ml solution has proved to be indefinitely stable at -20°C.



A more sensitive variation of Method A (0.2 to 1 nmol) has been used for frozen dried tissues. Three microliters of Reagent 1 is placed under oil in an oil well rack (8). The sample is added through the oil to start the reaction. The reaction is stopped with 10 ~1 of 0.04 N NaOH. After heating 20 min in an oven at 8O”C, a lo-p1 aliquot is added to 1 ml of Reagent 2 of the same composition as above except that ATP is reduced to 25 PM and NADH to 2 PM. The guanylate kinase for the last step is increased to 2 pg. The ATP reduction is to reduce ADP contamination. (ATP preparations need not contain more than 0.5% ADP.) cGMP P-Diesterase



The sensitivity is increased 5- to IO-fold compared to either the test tube or the oil well variant of Method A. Reagent 2 is modified by adding 1 &ml guanylate kinase and infreasing NADH to 20 PM. The NADH is prepared as a 5 mM stock solution in 0.1 M carbonate buffer, pH 10, and either stored at -40°C or below, or prepared fresh. It is heated 3 min at 95°C before use to destroy traces of NAD+. Steps 1 and2. Steps 1 and 2 are unchanged. (Either the test tube procedure or the oil well variant may be followed.) Step 3. To 50 fi of Reagent 2 in a fluorometer tube is added 1 to 10 ~1 of the sample. Step 4. After 20 min, 10 ~1 of 1 N HCl is added. Step 5. After at least 5 min, 1 ml of 6 N NaOH containing 10 mM imidazole is added with immediate vigorous mixing, and the tubes are heated 10 min at 6O”C, cooled exactly to room temperature, and read. (The imidazole greatly decreases the sensitivity of the fluorescent product to light (9).) For the oil well variant of Method B, Reagent 1 is reduced to 1 ~1 or less, the NaOH to stop the reaction is reduced to 5 ~1, and a 4- or 5-yl aliquot is transferred into the SO-p1 volume of Reagent 2.


cGMP P-Diesterase (IO-250 pmol)





Reagent 3. Reagent 3 contains 50 mM imidazole-HCl buffer, pH 7.0,2 mM MgC12, 25 PM ATP, 0.02% bovine plasma albumin, and 1 CLg/ml guanylate kinase. Reagent 4. Reagent 4 contains 50 mM imidazole-HCl, pH 7.5, 20 mM KCl, 2 mM MgC12, 1 mM sodium succinate, 500 @I coenzyme A, 200 PM P-pyruvate, 100 PM NADH, 0.02% bovine plasma albumin, 25 &ml pig heart succinic thiokinase (EC, 100 @ml pyruvate kinase, and 1 &ml beef heart lactic dehydrogenase. The succinic thiokinase stock solution is prepared in 50% glycerol as described for guanylate kinase above, and is stable for at least many weeks at -20°C. The pyruvate kinase is freed of most of the accompanying (NH&SO, by centrifugation before use. The P-pyruvate could be seriously contaminated with pyruvate, but most preparations are nearly pyruvate-free. Step 1. Samples are added to 25 ~1 of Reagent 1 as for Method A. Step 2. After incubation for 60 min, 5 ~1 of 0.25 N NaOH is added and the tubes are heated 10 min at 60°C. Step 3. One hundred microliters of Reagent 3 is added. Step 4. After 20 to 30 min at room temperature, a l- to lo-p1 aliquot is added to a fluorometer tube in an ice bath containing 100 ~1 of Reagent 4. After all the samples have been added, the rack of tubes is transferred to a water bath at room temperature (or 38°C if a higher cycling rate is needed). Step 5. After 60 min the rack is returned to the ice bath and 10 ~1 of 1 N HCl is added to each tube. Step 6. Step 6 is the same as Step 4 of Method B. The size of the aliquot used in Step 4 is chosen to provide a GDP concentration in the 0.01 to 0.05 PM range. Since GMP standards are carried through the entire




procedure, the incubation time and temperature at Step 4 are not critical. Useful sensitivity can be increased as with Methods A and B by reducing the volume of Reagent 1. However, because of the amplification in Step 4, much smaller samples can be analyzed, in which case, to keep the blank within bounds, volumes for the early steps are reduced even further. The following is a variation for measuring GMP formation in the l- to 25-pmol range. This has been extensively used for assaying nanogram frozen dried tissue samples (e.g., from individual retinal layers). Steps 1 to 4 are carried out in oil wells (see above). Step 1. Reagent is reduced to 0.2 ~1. Step 2. Add 0.5 ~1 of 0.05 N NaOH and heat 20 min at 80°C. Step 3. Add 5 ~1 of Reagent 3. Step 4. After 30 min add 5 ~1 of 0.05 N NaOH. Step 5 -7. Steps 5-7 are the same as Steps 4-6 above except that Reagent 4 is reduced to 50 ~1 and the sample aliquot should not exceed 5 ~1. The assay can be interrupted overnight if necessary at Step 4. If so, it is desirable to add 5 mM EDTA to the NaOH to protect the GDP from danger of hydrolysis by long exposure to Mg*+ in alkaline solution. If there is not to be a long delay, Step 4 (addition of NaOH) can be omitted. CAMP P-Diesterase


A and B

The specific reagent (i.e., Reagent 1) will differ according to the properties of the particular diesterase investigated, but for the present purpose we will assume the use of a reagent identical to that described for cGMP diesterase except that 5 mM CAMP is substituted for 1 mM cGMP. If necessary, 5’-GMP or 5’-UMP can be added to protect the 5’-AMP formed from 5’-nucleotidase (see below). The procedures are otherwise identical to those of Methods A and B for cGMP dies-






terase, with rabbit muscle adenylate kinase substituted in equal quantity (1 Ccg/ml) for the guanylate kinase in Reagent 2. Modifications for reduced volumes in oil wells are made in a similar manner.

The aliquots for Step 4 are chosen to give an ATP concentration of 0.01 to 0.1 PM, i.e., 1 to 10 pmol. Oil well variations of Method C are made as described for the corresponding cGMP diesterase method.

CAMP P-Diesterase (10-250 pmol)

Prevention of Loss of AMP or GMP by S’-Nucleotidase Action



Reagent 3A. Reagent 3A contains 100 mM imidazole-HCI, pH 7.4,200 pM P-pyruvate, 2 mM MgC&, 50 mM KCl, 0.02% bovine plasma albumin, 0.02 PM ATP, 5 pg/ ml pyruvate kinase, and 20 &ml adenylate kinase. Reagent 4A. Reagent 4A is the same as Reagent 3A except that glucose is added at 1 mM concentration, pyruvate kinase is increased to 100 pg/ml, 25 I.Lg/ml hexokinase is added, and adenylate kinase and ATP are omitted. (Both enzymes, as well as the adenylate kinase in Reagent 3A, should have most of the (NH&SO, removed by centrifugation, followed by solution in dilute neutral buffer.) Reagent 5. Reagent 5 contains 25 mM Tris-HCl, pH 8, 50 /.LM NADP+, 2 mM EDTA, and 0.5 CLg/ml baker’s yeast glucose6-P dehydrogenase (EC Steps 1-3. The first three analytical steps are the same as for cGMP diesterase Method C with 5 mM CAMP substituted for cGMP in Reagent 1 and Reagent 3A substituted for Reagent 3. Step 4. At least 20 to 30 min after Reagent 3A has been added, a l- to lo-p1 aliquot is transferred to a fluorometer tube at room temperature which contains 100 ~1 of Reagent 4A. The samples are added at regular intervals. Step 5. Sixty minutes after the first sample was transferred to the fluorometer tube, 1 ml of Reagent 5 is added in the same time sequence as the sample additions. The fluorescence is read when the reaction is complete (5 to 10 min). The EDTA stops any further cycling action.

When 5’-nucleotidase activity is high enough, relative to P-diesterase, to cause significant loss of the AMP (or GMP) produced, this loss can be prevented by adding another 5’-mononucleotide to Reagent 1, GMP or UMP for the CAMP reagent, AMP or UMP for the cGMP reagent. A 0.5 to 1 mM concentration of either nucleotide will achieve a 95 to 98% inhibition of the nucleotidase in brain or retina with little or no reduction of diesterase activity (see Results and Discussion). When measuring CAMP diesterase, 1 mM GMP can be added to the reagent without appreciable increase in the blank or otherwise affecting the assay by any of the three methods. However, when measuring cGMP diesterase, a problem arises with Methods A and B if AMP is added, because the guanylate kinase now commercially available contains adenylate kinase activity plus an activity which catalyzes the reaction GTP + AMP + GDP + ADP. Whether these are side reactions of guanylate kinase or contaminating enzymes has not been determined. Consequently, (a) the ATP required for the guanylate kinase step reacts to some extent with the AMP to give ADP and (b) the GTP formed from GMP, by combined guanylate kinase and pyruvate kinase action, reacts slowly with AMP as shown above to give ADP and regenerate GDP. Neither side reaction matters in Method C, since it is only GDP which is finally measured, but with Methods A and B both ADP and GDP are measured; therefore there is an increase in the blank. Consequently, unless Method C is to be used, it is recommended that 5’-









Activity (mmol kg(prot)-1 min-I)

Coefficient of variation (%I

6 day 7 day Adult

7.8 f 0.76 (4) 21.8 2 0.85 (4) 766 2 23 (8)

1.9 2.9 1.8

a Activities were measured at 25°C by Method A with 100 ~1 of Step 1 reagent. For the adults, homogenate containing about 1.2 pg of protein was used with 9% of the sample carried into the final step. For the 6- and 7-day retinas the amount of homogenate was doubled and 6% of the sample was carried into the final step. Interanimal standard errors are shown (Column 2) for retinas from the number of mice shown in parentheses. Analytical coefficients of variation (Column 3) were obtained by pooling the results from the replicate assays of these retinas.

UMP be employed as the nucleotidase blocker (see also Results and Discussion). RESULTS AND DISCUSSION

Proportionality of Enzyme

with Time and Amount

What is presented here is primarily the means for measuring the GMP or AMP produced by the respective cyclic nucleotide TABLE REPRODUCIBILITY

AMP, Method A



P-diesterases. It is not a study of the properties of the diesterases themselves. There are many kinds of diesterases with different Michaelis constants, different sensitivities to ions, activators, and inhibitors, and presumably different stabilities. Therefore, there can be no general statement made about such things as linearity with time and amount of tissue. With the cGMP reagent described, applied to retina, there was satisfactory linearity for incubation times up to an hour at 25°C and for tissue concentration up to levels that consumed 20% of the substrate; clearly with different tissues or different reagents the linearity parameters would have to be retested. Reproducibility

and Illustrative


With each of the procedures it is possible to restrict the coefficient of variation to 2-3% or less. Table 1 illustrates this with Method A for cGMP diesterase in homogenates of whole mouse retinas (kindly supplied by Dr. Adolf I. Cohen). The extraordinary increase with age has been observed before (10). Standards carried through the entire procedure by each of the methods give highly reproducible and proportional results down to levels of a few picomoles (Table 2). 2


AMP, Method B

AMP, Method C

GMP, Method C









262 262 262 469 469 469 785 785 785

264 268 271 447 461 482 781 785 790

93 93 93 164 164 164 274 274 274

93 93 95 160 160 160 271 271 271

1.94 1.94 1.94 4.07 4.07 4.07 10.8 10.8 10.8

1.71 1.97 1.86 4.20 4.10 4.03 10.3 10.6 11.0

3.8 3.8 3.8 8.3 8.3 8.3 12.8 12.8 12.8

3.2 3.7 3.5 8.1 7.7 7.8 13.0 13.4 13.2

D All values are in picomoles. The oil well variants of Methods A and B were used.




The application of the methods to a histochemical problem is illustrated by measurements of the distribution of cGMP and CAMP diesterase among the several layers of the retina (Table 3). Most of the cGMP diesterase activity is in the outer segment layer and there is a nearly 500-fold range from one extreme to the other. Method A was used for the outer segments (the oil well variant), whereas the much more sensitive Method C was required for the other layers. Activity toward CAMP was much lower than that toward cGMP in the outer segments, but in the nonphotoreceptor layers the reverse was true. Note that Ca2+ enhanced activity in some layers and inhibited activity in others. Protection of AMP and GMP Produced from Enzymatic Destruction

Two enzymes are known which destroy 5’-AMP: AMP aminohydrolase (EC and 5’nucleotidase (EC which also attacks 5’-GMP. Aminohydrolase would not ordinarily be a problem because it has a requirement for a monovalent cation and/or ATP. 5’-Nucleotidase, however, could cause significant loss when P-diesterase levels are low. In rabbit brain and mouse retina the 5’nucleotidase attacks 5’-AMP about twice as fast as it does 5’-GMP. The K, is about the same for both nucleotides (in the range of 20 PM). Whole mouse retina has about four times the activity of whole rabbit brain (on the order of 80 and 20 mmol kg-l (wet) h-l, respectively). Because both AMP and GMP are substrates for the nucleotidase, each, when added at a level much higher than the K,, can effectively protect the other from destruction by competition for the enzyme. Table 4 illustrates this for GMP protection of AMP. The protection approximates that predicted from the mutual K,, but the actual kinetic analysis



is somewhat complicated by the fact that both GMP and AMP decrease during the reaction. Note that with GMP added well above the K,, the loss of AMP will become essentially first order at all levels. For example, 500 PM GMP should inhibit the enzyme %% and the (small) loss of AMP should be almost identical in percentage for 50 and 5 PM AMP even though one is above and the other is below the K, (20 PM). Table 4 also shows that 1 or 2 mM GMP causes minimal inhibition of rabbit brain CAMP P-diesterase. In addition, it will be seen that in this instance, when GMP was omitted the yield of AMP was only diminished about 10% (25% in another experiment). This is because the brain diesterase activity was higher than the maximum nucleotidase activity by a factor of 5 or more. Similar results were obtained with brain for AMP protection of GMP and for lack of serious inhibition of cGMP diesterase by AMP. In the case of whole retina, the diesterase activity is so great that it is unnecessary to inhibit the nucleotidase; however, addition of 0.5 mM GMP did not effect the yield of AMP from 5 mM CAMP. These experiments were made with very high levels of cGMP (1 mM) and CAMP (5 mM). Interference would be more likely to occur with low cyclic nucleotide levels. However, in further experiments with rabbit brain and CAMP reduced to 20 pM (in the presence of EGTA) no inhibition was observed with 0.1 or 1 mM GMP or UMP. Similarly, with 20 PM cGMP (in the presence of 0.1 mM Ca2+) no inhibition was observed with 0.1,0.5, or 1 mM UMP or 0.5 mM AMP. Because of the known multiplicity of Pdiesterases in the body, and the possibility that 5’-nucleotidases in other tissues may differ from those in brain, it is recommended that in assays of nonneural tissues, before using one S-nucleotide to preserve another, it be tested both for protective capacity and




cGMP diesteraseb 148 7.3 1.08 3.28 0.65 0.93 0.40 0.32

Outer segment Inner segment Outer nuclear Outer plexiform Inner nuclear Inner plexiform Ganglion cell Fiber

* f f k + Ii t 2


8 1.4 0.13 0.23 0.05 0.15 0.05 0.02



CAMP diesteraseb with Ca*+ 10.9 1.92 0.78 1.93 1.80 2.05 1.33 1.22

+- 3.5 2 0.20 2 0.12 + 0.15 k 0.22 ir 0.37 2 0.15 ? 0.10


CAMP diesterase” with EGTA 27.8 2.58 0.88 3.03 1.08 1.85 1.30 1.42

2 it k 2 + f f 2

12.8 0.25 0.08 0.25 0.67 0.05 0.08 0.10

a All activities are mmokkg dry wt/min (nmoYmg/min) for (usually) 5 samples f SEM. Samples weighing lo-40 ng were dissected from frozen dried retinas (8) of one Macaca rhesus monkey (for CAMP diesterase) and one Mucaca fasciculnra monkey (for cGMP diesterase). Incubations were for 1 h at 20°C. Method A (oil well variant) was used for the cGMP diesterase analyses of the outer segment layer, and all the other analyses were made with Method C. Reagent 1 for cGMP diesterase was that given under Materials and Methods. Reagent 1 for CAMP diesterase had the same buffer and albumin composition with 1 mM CAMP, 0.5 mM dithiothreitol, 2 mM MgCl,, and either 0.5 mM EGTA or 0.1 mM CaCl, plus 100 units/ml of Ca*+-dependent activator protein (kindly supplied by Dr. Louise Greenberg). b These names are operationally defined, i.e., they are merely the activities observed with the three reagents described in footnote a. Actually most cyclic nucleotide diesterases attack both CAMP and cGMP but usually with different kinetic constants. For example, the diesterase in the outer segments attacks both cyclic nucleotides with about the same V but the K, for cGMP is smaller than that for CAMP by a factor of about 50. Thus the “CAMP diesterase” and “cGMP diesterase” activities shown for the outer segment layer reflect the differences among the reagents and do not signify that different enzymes are being measured.





Brain dilution @v&4



AMP added (PM)

AMP found (PM)

2 2 2 2 0.5 0.5 1 1 1 1 1

0 0 0 0 0 0 5000 5000 5000 5000 5000

0 96 236 959 0 236 0 96 553 955 1844

20 20 20 20 20 20 0 0 0 0 0

2.7 15.0 17.6 19.6 12.6 19.4 127 136 138 135 131

a The assays were conducted by Method B. Reagent 1 contained 50 mM Tris-HCI, pH 8.0, 2 mM MgCI,, 0.02% bovine plasma albumin, and 0.5 mM dithiothreitol. Brain and nucleotide concentrations refer to Step 1.

for possible inhibition of the diesterase, especially if low substrate levels are to be employed. Conversion

of GMP to GDP

Commercial beef brain guanylate kinase in the assay reagent was found to have a K, for ATP of 65 PM and for GMP of 6 PM. The first is somewhat lower and the second identical to those found by Miech and Parks for pork brain (11). The V was about 8 Fmol mg-’ min-I. From these constants the calculated half-time for conversion of low levels of GMP to GDP with 1 &ml of the kinase and saturating ATP would be 0.69 x 6/8 = 0.5 min (8). With 100 PM ATP(Method A) or 25 PM ATP (Method C) the respective calculated half-times would be 0.8 and 2 min (0.5 (100 + 65)/100 and 0.5 (25 + 65)/25). As mentioned earlier, commercial brain



guanylate kinase contains another kinase ioor % conversion activity which is just its converse, i.e., a 75 t GTP-AMP kinase. The following shows why this can cause interference when measuring GMP in the presence of AMP (GK, PK, and GAK refer to guanylate kinase, pyruvate kinase, and the GTP-AMP kinase, respectively):

exo-Y+---O ,x~:O’“,O~,cl~ /50




OH ,0.5nM








Clearly pyruvate production will not stop when GMP is gone, although the sum of GDP plus GTP will be unaffected (Method C). Fortunately, the V for the GTP-AMP kinase was only 5% of that for guanylate kinase. Half-maximal activity was obtained with about 6 mM GTP and 50 mM AMP. In spite of this problem, GMP can be accurately measured by pyruvate formation (Methods A or B) in the presence of much larger amounts of AMP if (a) the guanylate kinase is kept to a minimum, (b) the same amount of AMP is present in samples and standards, and (c) the time of reaction is kept constant. Conversion of AMP to ATP

The time for conversion of AMP to ATP depends on the initial concentrations of both ATP and AMP, as well as the adenylate kinase level (see last cyclic reaction shown in introduction). (Under assay conditions, the rate of reaction is almost entirely limited by the adenylate kinase step.) Both ATP and AMP will be far below their MichaelisMenton constants (about 100 and 50 ,SM, respectively). The bimolecular rate constant (VIKATP KAMP) is about 2 PM-~ min-‘, for the recommended adenylate kinase concentration of 20 mg/liter. (This assumes 500 IU/mg (500 pmol mg-’ min-‘) which is equal to 500 PM min-’ with 1 mg/liter of enzyme, from which k = 20 X 500 PM min-750 pM X lc)o FM.)

FIG. 1. Theoretical rate curves for conversion of AMP to ATP with adenylate kinase, pyruvate kinase, and P-pyruvate. In the upper panel initial AMP concentration is held constant at 1 PM and initial ATP is varied from 0.5 to 50 nM as indicated. In the lower panel initial ATP concentration is held constant at 10 nM and initial AMP is varied from 0.1 p,~ (righthand curve) to 5 pM (left-hand curve). (The use of different symbols is merely to distinguish the curves at the extremes.)

The rate equation for this reaction is d(ATP)/dt = k (ATP)(AMP), where (AMP) = (AMP& + (ATPJ - (ATP). The integrated form is t=


k[@MPo) + (A’WI

1(AMP,)’ 1

(AMRJ + (ATf’o) _ 1 (AT&J (A’W Since ATP,, is small compared this can be reduced to:

to AMP,,

The equation for 99% conversion and a 100: 1 ratio of AMP,,:ATP, is t = ll.S/k(AMPJ, i.e., 11.5 min when k = 1 PM-~ mitt-l and (AMP,) = 1 PM. The time to completion is only slightly affected by changes in initial ATP concentration: A lOO-fold increase in ATP,, would only cut the time in half (Fig. 1). In contrast, a reduction in initial AMP paradoxically greatly prolongs the time to com-


pletion (Fig. 1). With initial AMP concentrations less than 0.2 PM, incubation time would need to be increased beyond that recommended. The Breckenridge (ATP) Cycle

In the ATP cycle, the adenylate shuttles back and forth between ADP and ATP (see introduction). However, as applied here, adenylate kinase is carried over from the previous step. This creates a potential steady state situation in which a significant amount of the adenylate could be sequestered as AMP with less than the expected amplification. (Although adenylate kinase might be destroyed before proceeding, it is a very stable enzyme, and it would seem preferable to avoid this necessity.) At equilibrium, (AMP) = K,,(ADPYI (ATP). K,, varies with Mg2+ concentration, but is in the neighborhood of 0.5. Assuming this value, with ADP to ATP ratios of 2, 1, 0.5, and 0.2, the equilibrium percentage of total adenylate present as AMP would be 40, 20, 4, and 0.3, respectively. Thus to avoid substantial AMP accumulation, the (ADP):(ATP) ratio should be 0.5 or less. This means that the first-order activity of pyruvate kinase should be at least twice that of hexokinase. On a weight basis, the fustorder activity (V/K,) of pyruvate kinase under assay conditions is slightly higher than that of hexokinase. Therefore a 2: 1 or greater ratio on a weight basis is satisfactory. The overall precision of AMP assay has proved to be highly satisfactory. In a test with AMP in the range of lo-900 pmol, in which only 1 to 10 pmol of ATP was actually used in the cycling part of the assay, the coefficient of variation was 2%. Assays with Low CAMP or cGMP Concentrations

The procedures described are for measuring total diesterase activity and therefore


specify high CAMP or cGMP levels. The methods are nevertheless completely satisfactory with much lower substrate levels and have been used in kinetic studies and attempts to discriminate among the several isozymes, with CAMP or cGMP concentrations down to 5 PM. And this is not the limit. GMP or AMP production in the 0.1 PM range (final concentration at the first step) can easily be measured with the amplification procedures (Method C) inasmuch as 0.01 PM GDP or ADP concentrations at the enzymatic cycling step give good precision. The need to block 5’-nucleotidase may become more important in assays with low substrate levels, especially if the K, of the diesterase is greater than that of the S’-nucleotidase (about 20 PM in nervous tissue), or ifalarge fraction of the substrate is consumed. ACKNOWLEDGMENTS This work was supported in part by grants from the American Cancer Society (BC-4s) and the U. S. Public Health Service (NS-08862 and EY-02294).

REFERENCES 1. Goldberg, N. D., Dietz, S. B., and O’Toole, A. G. (1%9) J. Biol. Chem. 244, 4458-4466. 2. Cha, S., and Cha, C. M. (1965) Mol. Pharmacol. 1, 178- 189. 3. Cha, S., and Cha, C. M. (1970) Anal. Biochem. 33, 174-192. 4. Fertel, R., and Weiss, B. (1974) Anal. Biochem. 59, 386-398. 5. Breckenridge, B. M. (1964) Proc. Nat. Acad. Sci. USA 52, 1.580-1586. 6. Weiss, B., Lehne, R., and Strada, S. (1972)Anal. Biochem. 45, 222-235. 7. Miki, N., Baraban, J. M., Keims, J. J., Boyce, J. J., and Bitensky, M. W. (1975) J. Biol. Chem. 250, 6320-6327. 8. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York. 9. Lowry, 0. H., and Carter, J. G. (1974) Anal. Biochem. 59, 639-642. 10. Farber, D. B., and Lolley, R. N. (1976) J. Cyclic Nucleotide Res. 2, 139- 148. 11. Miech, R. P., and Parks, R. E., Jr. (1965) J. Biol. Chem. 240, 350-357.

The measurement of cyclic GMP and cyclic AMP phosphodiesterases.

ANALYTICAL BIOCHEMISTRY 100, 244-253 (1979) The Measurement of Cyclic GMP and Cyclic AMP Phosphodiesterasesl JOYCE G.~ARTER,$OSAMMA Department of...
847KB Sizes 0 Downloads 0 Views