A microassay for guanosine 3′,5′-monophosphate phosphodiesterase activity

ANALYTICAL

59, 386-398 (1974)

BIOCHEMISTRY

A Microassay

for

Guanosine

3’,5’-Monophosphate

Phosphodiesterase RICHARD Department

FERTEL

of Pharmacology, Philadelphia,

AND

Activity BENJAMIN

Medical Pennsylvania

WEISS

College of Pennsylvania, 19129

Received August 22, 1973; accepted November

16, 1973

The activity of cyclic GMP phosphodiesterase was determined using a three step procedure. In the first step, cyclic GMP phosphodiesterase catalyzes the conversion of cyclic GMP to 5’-GMP. In the second step, a known amount of ATP and guanylate kinase are incubated with the 5’GMP formed in the first step. The amount of ATP which remains is inversely related to the amount of Y-GMP formed. In the third step, the concentration of ATP is measured using the firefly luciferin-luciferase technique. The validity of the assay is confirmed by its ability to show the linearity of the cyclic GMP phosphodiesterase reaction with respect both to time of incubation and concentration of tissue. It is capable of detecting less than 5 pmoles of 5’-GMP in 150 pl, and can be used to measure cyclic GMP phosphodiesterase activity in a supernatant fraction of rat cerebrum which contains less than 25 ng of protein. It has been used to determine the activity and properties of cyclic GMP phosphodiesterase in unpurified supernatant and particulate fractions of several tissues of the rat, as well as in highly purified fractions of rat caudate nucleus.

A physiological role for adenosine 3’,5’-monophosphate (cyclic AMP) has now been firmly established (l-3)) and there is a growing understanding of the importance of guanosine 3’,5’-monophosphate (cyclic GMP) in biological processes(4-6). The intracellular concentration of cyclic AMP and cyclic GMP is governed, in part, by the activity of the phosphodiesterases which hydrolyze them. These enzymes exist in several molecular forms (7-11) which are characteristically distributed according to tissue (7,9) and cell types (12,13). These forms have different stabilities (10) and substrate affinities (8,11), and can be differentially inhibited by structurally unrelated drugs (14). By determining the distribution of these enzymes and the factors which regulate their activities, one can gain an insight into the means by which an organism can control the intracellular concentrations of the cyclic nucleotides. Several methods have been devised to measure the cyclic nucleotide phosphodiesterases. In general, they measure 5’-nucleotide, the product 386 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

CYCLIC

GMP

PHOSPHODIESTERASE

387

ASSAY

of the phosphodiesterase reaction, by hydrolyzing it to the nucleoside plus inorganic phosphate, and determining the concentration of one of these breakdown products. The initial investigators in this area measured inorganic phosphate (15). Subseq~le~lt workers have measured the nucleoside. These latter in~resti~ators add radioa~ti~?e cyclic nu~leotide to a phosp~~odiesterase pre~3aratio~~, and treat the ra~~io-lal~elled 5’-nudeotide which is formed with a 5’-nucleotidasc. The labelled nucleositle which results is chromatographically separated by one of several m&hods: the addition of anion exchange resin (8’1, resin-coated paper (IG) , application to a resin (17) or alumina (18) column, or paper chromatography (19). The purified nucleoside is then counted in a scintillation spectrometer. A simple microassay for cyclic AMP ~~hosphodiesterase has recently been reported (20) which utilizes the firefly luciferin-lu~iferase ~e~hniqne for determi~lation of ATP, the end product, of the assay procedure. Using the same general concepts, we have developed a specific and sensitive assay for cyclic GMP phosphodiesterase which does not require the use of radioactive substrat’c. In this paper we describe the assay and compare some of the properties of the cyclic GMP phosphodiesterase and the cyclic AMP phosphodiesterase in rat, tissues. MATERIALS

AND METHODS

The Cyclic GMP ~~os~~o~~~~~er~s~Assag. Cyclic GMP phosphodiesterase is measured with a three-step procedure (Fig. 1). In the first step, the tissue to be assayed is incubated with cyclic GMP. In the second step, the guanosine 5’-monophosphate (5’-GMP) formed from the hydrolysis of cyclic GMP is measured using the reaction catalyzed by guanylate kinase. In the third step, the remaining ATP is determined by adding firefly luciferin-luciferase (21) and detecting the generated light with the luninescence biometer (22). Under the conditions of our assay, the decline of ATP in the reaction mixture is proportional to, and therefore is a measure of, the initial con~entratio~l of 5’-GXP. The entire assay is carried out in a 6 X 50 mm curette as follows: 50 ,J of phosphodiesterase sample is added to 50 (11of 400 FM cyclic GRIP, 2 m&f NgCl,, and 0.03% bovine serum albumin, all in 50 mM glycylglycine CYCLIC 5’ GMP +

ATP

+

FIG.

LUCIFERIN

1.

3’,5’ ATP

GMP

RIOSPHOO(ESTERASE

C_GUANYLA?E LUCIFERISE

KINASE

>

>

>

5’GMP

GDP +ADP

ADE~YLOXYLUClFE~lN

f

Assay of cyclic GMP pl~ospI~odic~te~ise activity.

LIGHT

388

FEXTEL

AND

WEISS

buffer, pH 8.0. The cuvettes are incubated at 37°C in a Dubnofl’ shaking incubator. At the end of this first incubation period, (usually 60 min) the cuvettes are placed in a boiling water bath for 5 min to prevent furt’her phosphodiesterase activity. Fifty microliters of a reagent (glycylglycine buffer, pH 8.0 (50 DIM) ; bovine serum albumin (0.03%) ; dithiothreitol (45 m&l) ; guanosine 5’-monophosphate kinase (10 mIU/50 ~1) ; MgCl, (2 ml1 J ; and ATP (0.1-5 PM) is then pipetted into each cuvet,te, and the samples are incubated for 60 min at 37°C. Following this second incubation period, 10 ~1 of luciferin-lucifcrase reagent is injected into each cuvette, and the generated light is recorded in digital form by the luminescence biometer. The light can also be measured with other instruments, including a liquid scintillation spectrometer (23). In order to calculate the amount of 5’GMP formed in the first react,ion, a series of 5’-GMP standards is carried through all reaction steps in parallel with the samples. Purification of Cyclic GMP. The cyclic GMP obtained from Sigma Chemical Co. is contaminated with a small (
CYCLIC

GMP

PHOSPHODIESTERASE

ASSAY

389

with the arnol~nt of ATP reordaining in the standard tubes. The st.aIldards consist of varying concentrations of 5’-GMP carried through the entire reaction sequence. The tissue samples are assayed in the absence and presence of cyclic GMP, and the dif’fcrenee bet~wvccnthese values is used to calculate enzyme activity. Mnderiula Guanosine 5’-monophosphate kinasc (EC 2.7.4.8) (guanylate kinase) was obtained from Boehringer hlannheim, firefly lucifcrinluciferase and the Luminescence Biomcter from E. I. DuPont de Xemours & Co., Alumina (grade I, neutral‘) from Waters Assor., and Dowex 50 W-X8 from BioRad Laboratories. Tris (,hydroxymethyl) aminomethane (Tris) and all other chemicals and reagents mere obtaine~~ from Sigms Chemical Co. RESULTS

AX)

DISCXSSION

~en,~i~i~~~~ and Linearity

of the Assay for 5’-G’MP. Under the assay conditions described, the reaction catalyzed by guanylate kinase reaches equ~~ibriul~~ by 60 min. At cq~~i~~bri~~nl~the conversion of ATP to ALIP is not stoi~hiometrically r&ted to the amount of 5’-GXP present, but it does bear a definite inverse relationship to the concentration of 5’-GMP (‘Fig. 2). The se~lsi~ivit~ of the assay ran be altered by varying the anloul~t of ATP in the second reaction step. The lower t.he initial concemration of

FIG+. 2. Sensitivity and linearity of the assay of 5’-CMP. Varying atnounts of 5’GMP and ATP were incubated with S’-GMP reagent in a total volume of 150 ELI for 60 min at 37°C. The amount, of ATP which remained was determined by the firefly luciferin-luciferase technique as described in the text. Each point represents the mean of five determinations.

390

FERTEL

AND

WEISS

ATP, the greater will be the percentage clccrcast~ in ATP with a given amount of 5’-GNP. As can be seen in Fig. 2, the concent,ration of ATP decreases in proportion to the amount of 5’-C:I\IP added, regardless of the amount cf ATP which mas initially present. However, a given amount of 5’-GMP causes a greater percentage dcrwase when a lower concentration of ATP is present. Under the conditions rlcscribed, the assay can be used to measure about one picomole of 5’-GNP per sample, or less than 1O-x M 5’-GMP. Specificity of the Reaction for 5’-GMP. The specificit)y of the assay is conferred by the selectivity of the guanylate kinase reaction for 5’-GMP in the second step of the assay. To demonstrate this selectivity, we added a series of 5’-nucleotides and measured the decline of .4TP. There was no detectable hydrolysis of ATP m-hen the samples were incubated with 5’-TMP, 5’-CUP, 5’-IMP or 5’-CM’. Only 5’-GNP and 5’-AMP had any effect on the concentration of ATP (Fig. 31. The effect of 5’-AMP is

t5’

GMP

/

PICOMOLES

OF

5’

NUCLEOTIDE

FIG. 3. Specificity of the 5’-GMP reaction. Varying concentrations of each of six 5’-nucleotides were incubated for 60 min with 5’-GMP reagent containing 150 pmoles ATP in a final volume of 150 pl. The ATP remaining in each sample is plotted as a percent of the ATP in a sample with no 5’-nucleotide. Each point represents the mean value of two determinations. 5’-CMP = cytidine 5’-monophosphate, 5’-IMP = uridine 5’-monophosphate, 5’-TMP = thymidine 5’-monophosphate, 5’-IMP = inosine 5-monophosphate, 5’-AMP = adenosine 5’-monophosphate, 5’-GMP = guanosine 5’monophosphate.

CYCLIC

GMP

P~iOSPHOD~ESTERASE

391

ASSAY

probably due to the contamination of the guanylate kinase preparation with adenylate kinase. This, however, does not affect the specificity of the cyclic GMP assay for several reasons. First, the guanylate kinase reaction is about 20-fold more sensitive to 5’-GNP than to 5’-AMP. Further, since cyclic GNP is added as a substrate, 5’-GMP is being generated in the initial step while 5’-AMP is not. Finally, under the conditions of the experiment shown in Fig. 3, approsimately 400 pmoles of 5’-AMP must be present to cauxc Cven a 10% decrease in ATP. This is far in excess of the conccntratioll of 5’-ANP normally found in the pg an~ounts of tissue required for this assay. Xerertheless, any effect on the assay due to 5’-AIUP in the tissue can be accoLlnted for by measuring the decrease in ATP caused by a boiled tissue preparation.

OY-

100

200 RAT

300

400

500

CEREBRUM

(ng protevd FI~J. 4. Hydrolysis of cyclic GMP m a function of increasing concentrations of phosphodiesterase. Cyclic GMP (0.2 mM) was incubated for 60 min at 37°C wit.h varyiug amounts of a soluble supernatant fraction of rat cerebrum in 100 ~1 of 50 mM: glycylglycine buffer pH 8.0. The cyclic GMP phosphodiesterase activity was determined as described in the section on methods, using 2 FLM ATP in the Y-GblP reagent step. Each point represents the mean activity of six determinations. Bra&ets indicate the standard error.

392

FERTEL

AND

WEISS

Cyclic GMP Pho~pho~~e~te~ase Activity ns a Fmcfion oj Tissue Concentration. Figure 4 shows the conversion of cyclic GMP to 5’-GMP as a function of increasing concentrations of a soluble supcrnatant fraction of rat cerebral homogenates. The rate of hydrolysis of cyclic GMP was linearly related to the amount of tissue present. The phosphodiesterase activity contained in less than 100 ng of protein was readily detected. Cyclic Gil&’ Phosphodiesterase Activity as a Fmction of Incubation Fime. Figure 5 shows the conversion of cyclic GMP to 5’-GMY as a f~Inction of the time of in~ubatioI1, with the soluble s~Ipernatant fraction of rst cerebra1 homogenate as the phosphodiesterase source. The rate of formatxion of 5’-GMP was linear for at least 60 min. Effect of Dicalent Cations on the Cyclic GMP Phosphodiesterase

IO

FIU. 5. Hydrolysis of cyclic so!uble supernatant fraction of tein) was incubated with cyclic phosphodiesterase activity was using 2 JLM ATP in the 5’-GMP five samples. Brackets indicate

20

30

40

MINUTES

OF

INCUBATION

50

60

GMP as a function of the time of incubation. The rat cerebral homogenate (equivalent to 0.55 pg proGMP (0.2 InM) for varying times. Cyclic GMP determined as described in the section on Methods. reagent, step. Each point represents the mean of t.he standard error.

CYCLIC

GMP

P~OSP~O~~~ST~RASE

393

ASSAS

FIG. 6. Effect of calcium and magnesium ions on the cyclic GMP phosphodiesterase activit,y of rat cerebrum, The soluble supernatant fraction of rat cerebral homogenates (equivalent to 0.55 pg protein) was assayed for cyclic GMP phosphodiesterase activity using 200 PM cyclic GMP as substrate in the presence of varying con~entrat.ions of Ca’+ or Mg’+ (100 pi EDTA was present in all expcr~Inents v&h Mg”“). Each point represents the mean value of five sampies. Brackets indicate the standard error.

of Rat ~er~~~~7?~. The ~hos~~~lodiesteras~ actj~~ty of the supernatant fraction of rat cerebral ~lo~nog~x~ates was 52 nmoles of cyclic GMP hydrolyzed per mg protein per minute in the absence of added cations (Fig. 6). This activity is completely abolished by the addition of 100 pv

Ac~i~~~~

,I6 ,. .' - 0.1 ..4.-

0i

-_.--~. 7157

31

62 5 @A cyclic

dl ,. -

125 3: 5’ GMP

62,,$3 ---____

64

05 250

FIG.7. Cyclic GMP phosphodiesterase activity as a function oi substrate concentration. The soluble s~~perna~an~ fraction of rat cerebral ho~logenate (equivalent to 0.55 pg of protein) was assayed for cyclic GMP phosphodiesterase activity in the presence of varying concentrations of cyclic GMP as described in the section on Methods. Each point is the mean of sis determinations. Brackets indicate the standard error,

394

FlXM3L

AND

WEISS

ethylenediamine tetraacetic acid (EDTA) suggcating that divalent cations are required for activity. The inhibitory effect of EDT.4 could be partially reversed by t.he addition of as little as 40 alar. ,11g2+, and optimum activity was reached when 0.2 rnM Mgz+ \vas added. A similar Mg’+ dependence for cyclic GMP phospIlodiester~se has previously been reported (25). Several other workers have found that cyclic AMP phosphodiesterase requires a higher conce~tratjon of Mg2+ for optimal activity (2526). In contrast to the effects of Mg’+, calcium ions, at concentrations of 0.5 rnM and above, inhibited cyclic GMP phosphodie&erase nct,ivity, a phenomenon previously observed with cyclic AMP, phosphodi~?sterase (26,271. CycEic GMP Phosphodiesterase Activity as a Fmction of Substrate Concentration. Figure 7 shows the effect of increasing concentrations of cyclic GMP on the cyclic GMP phosphodiesterase activity of a soluble supernatant fraction of rat cerebra1 homogenates. In this experiment signi~~a~~t enzyme activity Teas obtained with as little as 2 f.tM cyclic GMP. A I~ine~~ea~rer-Burk plot (28) of these data indicates an apparent Michaelis constant I++,, of 10 FM. This value is similar to that obtained by other laboratories for the cyclic GMP phosphociiesterase of rat liver (ZS), and rat brain (7). By increasing t,he sensitivity of the assay, lower concentrations of substrate ran be used, and ~,herefore enzymes wit& much IOU-cr IL’s can be studied. Compa?Tison Between Cyebic AMP and Cyclic G%P Phosphodiesterases of Several Tissues of the Rat. The cyclic AMP and cyclic GMP phosphodiesternse activities of several rat tissues are shona jn Table 1. There is a general parallel between the activity of cyclic AXP phosphodiesterase and cyclic GXP p~losp~~o~~iesterase in the tissues studied. For example, cere~)runl has the hig~le~t activity of both cyclic AMP and cyclic GMP p~~osphodiesterase! while whole blood and skeletal muscle have t~he lowest activity of both phosphodiestprases. The data shown in the table seem to indicate that t,here is a greater activity of cyclic GMP phosphod~esterase than cyclic AMP phosphodie&erase in each of the tissues studied. However, this is probably due to the dif!ferenee in the Km values of Lhe t,wo enzymes. The IC, for cyclic GMP phosphodiesterase is about’ 10 ,LI.M,and t,hc K, for the predominant form of cyclic AMP phosphodiest#ernse is 100 PM (30). Therefore, the results in Table 1, which were obt,ained using 200 p.~ cyclic nucleotide for both enzymes, compares the activity of cyclic GMP phosphod~esterase using a sllbstrate concentratjon 20 times its -Fir, value wit,h the activity of cyclic AMP phosphodiesterase using a substrate concentration only twice its Km value. Thus, one might expect that. higher maximal values of cyclic AMP phosphodiesterase activity could be obtained if higher

CYCLIC

GMP

PHOSPHODIESTERASE

TABLE 1 The Activity of Cyclic AMP and Cyclic GMP in the Supernatant Fraction of Homogenates Cyclic Tissue Cerebrum Adrenal Liver Cerebellum Kidney Lung Skeletal muscle Whole blood

GMP phosphodiest,erase (nmoles/mg prot/min) 93 23 19 19 18 9.5 1.7 0.39

+_ 13 It 4 t 3 +_ 6 F 5 + 3.4 + 0.4 + 0.17

395

ASSAY

Phosphodiesterases of Rat Tissuesa

Cyclic

AMP phosphodiesterase (nmoles/mg prot/min) 49 + 6 2.3 F 0.6 1.3

*

0.4

4.9 4.5 2.1 .68 .063

f + k + +

1.5 0.9 0.6 0.4 .024

a Tissues from 200 g male rats were prepared &s described in the section on methods. Each tissue was analyzed for cyclic GMP phosphodiesterase and cyclic AMP phosphodie&erase using 200 PM cyclic nucleotide as substrate. Incubations were conducted for 60 min. at 37°C. Enzyme activities are given as nmoles cyclic nucleotide hydrolyzed per mg protein per min. Each number represents the mean value + SE for at least four animals.

of cyclic AMP were used. In fact, when a substrate concentration of 1 mM is used, the activity of cyclic AMP phosphodiesterase in these tissues is 24-fold greater than the activities determined here cm. Cyclic AMP and Cyclic GMP Phosphodiesterase Activities of Purified Fractions of Rat Caudate Nucleus. The assay described in this report is particularly suited for determining cyclic GMP phosphodiesterase activity in purified fractions of tissue which may contain small quantities of the enzyme. Previous studies (10) have shown that the soluble supernatant fraction of rat, cerebellum cont.ains six forms of cyclic AMP phosphodie&erase (designated I to VI, according to the order in which they emerged from a gel electrophoresis column). Using similar techniques we fractionated the supernatant portion of a homogenate of rat caudate nuclei and measured the cyclic AYIP phosphodiesterase and cyclic GMP phosphodiesterase in these fractions (Fig. 8). Two peaks of cyclic AMP phosphodiesterase and a single peak of cyclic GMP phosphodiesterase were found in this tissue. A minor peak of cyclic AMP phosphodiesterase occurred in fractions 22-28, and a major peak of cyclic AMP phosphodie&erase was detected in fractions 35-55. This major peak has properties similar to those of the peak III fraction which had been previously isolated from rat, brain (10). The peak of cyclic GMP phosphodiesterase from the caudate nucleus is coincident with the major cyclic AMP phosphodiesterase peak. Other studies of partially purified preparations have concentrations

396

FEXTEL

AND

WEISS

also indicated that there is a coincidence between tllc major peaks of the two cyclic nucleotide phosphodiestcrases (7,19,1. Possible Source of Ever. There are several possible sources of error which must be taken into consideration when using this assay. First, since the assay is so sensitive to YGMP, any contaminant of 5’-GMP, eit,hcr in the cyclic GMP or the guanylate kinase, will cause an increase in the reaction blank. There are several published methods for separating 5’GM’ from cyclic GMP. We have used alumina adsorption (31)) followed by further purification on Dowex 50 cation exchange resin. The commercial guany~ate kinase prep~~rat~on has a slight conta~ni~~~ltion of 5’GMP which me were unable t80 remove by dialysis. This does not affect the present assay, but if the assay were to be used for the determination of cyclic GMP, greater sensitivity would be required, and therefore an

140 J

.i IO

20

30

40

FRACTION

50

60

70

a0

NUMBER

FIG. 8. Cyclic AMP and cyclic GMP phosphodiesterase activities of purified fractions of rat caudak nucleus. Rat caudate nuclei from four rats were homogenized, sonicated, and centrifuged as described in the test. One milliliter of the supernatant fraction was placed on a preparative polpacrylamide gel clectrophoresis column, and the electrophoresis was conducted as described previously (10). Fractions (2 ml each) were collected and analyzed for phosphodiesterase activity using 500 pM cyclic AMP and 200 /tM cyclic GMP. ‘Each point represents the mean of two determinations. The results are expressed as pmoles of cyclic nucleotide hydrolyzed per ml of fraction per min.

CYCLlC

GMP

PHOSPHODIESTI?RASE

397

ASSAY

enzyme preparat,ion with less 5’-GXIP contamination might be necessary. A seponij factor \yhich must be considcrcd is the presence of 5'-llUChtidase in the tissue to be studid. Since the assay depends on a direct correlation betr\*eeii cyclic ( :-1IP liytlrolpzed an11 5’-GNP formed, ally loss of j’-C;_\lP in the :w:~y q&cm ~0~111lead to calculated results which arc loxcr than actual ~a!ucs. This problem is especially acute when working with l~hosplioclic~st,~~rnsc preparations which are essentially mlpurified, such a:: l)articulate fractions. 111 most instances, howver, it is possible to comlxnsntc for 5’-nurlcoti~lasc contamination by dctcrmining the effect of nuclcotidaw ill the tissue on the 5’4;hIP standard curve, and using this standard curve for c:alculat~iiig the true pEiosl~liotliesterase activity. Thus, n-e have mcas~~rcd cyclic (+,11P phosphocliestcrase activity in the 100,OOOg Ixllet of rat cerebrum, and found an activity of approximately 90 nmolcs cyclic C;JLP l~ydrol~zed,‘n~g llrotcin, a value compnrnblc to that found for the soluble frnct,ion. Fwfher dpplicnfions of this Assn!/. Although this assay was designed to mcasurc cyclic (:JlP pliosl~liodicstcr:ise, thcl general procedures described in this rcljort, witJ\ certain moclificationu, can be used to mcnsure Y-GNP, qclic G?\lP, an11 guanylate kinnw. In fact, in a recent, paper, Schultz et nl. (32) utilize cyclic GRIP l~hos~~l~odiesteratse and guanylate ltinasc as l)nrt of their lworcclurc for mc:Wukg cyclic GNP.

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TKIKEII.

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(in

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AND

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