ANALYTICAL
BIOCHEMISTRY
72,380-388
(1976)
Simple Assay of Cyclic Nucleotide Phosphodiesterase Using 32Phosphorus Labeled Nucleotide; Application to Kidney Subcellular Fractions J. A. EISMAN’ AND T. J. MARTINS University of Melbourne
Department of Medicine, Austin Hospital, Heidelberg, Victoria, Australia
Received June 6, 1975; accepted October 24, 1975 A method is described for the estimation of cyclic nucleotide phosphodiesterase activity using ~*P~ospho~s-~abeIed cyclic nucleotides and separation of substrates from reaction products in a single-stage elution on dry neutral alumina columns. The method was used to study tiie subcellular distribution of cyclic AMP and cyclic GMP phosphodiesterase activity in rat renal cortex.
Intracellular hydrolysis of 3’5’-cyclic AMP and 3’5’-cyclic GMP by cyclic nucleotide phosphodiesteraseappearsto be the most important mechanismfor the disposal of cyclic nucleotidesfrom the cells in which they are generated(1,Z). The function and possible control of phosphodiesteraseactivity hasbeenstudiedextensivelysinceit wasfirst shown(3) to hydrolyse specifically the cyclic 3’:5’-monophosphatenucleotide to the 5’-monophosphate. Before radioisotopeassaysbecameavailable the enzyme activity was assayedat substrate concentrationstwo to three orders of magnitude higherthan physiologicallevels (4-8). Sensitivity at low substrateconcentrations has been obtained using PHI- and [14C]-labeled3’:5’-cyclic nucleotideas substrate,and various methodsof sep~ation of the labeled products. Barium sulfate precipitation of labeledS-AMP (9,lO) is unsatisfactory in studies of subcellular fractions containing 5’-nucleotidase activity, since adenosine,like its cyclic nucleotide, is not precipitatedby barium sulfate, Paper chromatographic separationis used (11,12),with conversion of generated5’-AMP to adenosineby addition of exogenous S-nucleotidase. The latter approach has also been used by Thompson and Appleman (13) in developing an ion exchangebinding system for phosphodiesteraseassay, which has been modified (14) to overcome problems of recovery of adenosineor guanosine.Filburn and Karn (15) 1 Present address: Department of Biochemistry, University of Wisconsin, Madison, Wis. 53706. * Present address: Department of Chemical Pathology, University of Sheffield Medical School, Sheffield, U.K. 380 Copytight Q 1976 by Academic Prerr. Inc. All right< of reproduction in any form I-ererved.
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
ASSAY
381
reported the use of alumina columns to separate [3H]- and [14C]-labeled cyclic AMP and cyclic GMP from their respective nucleotides, produced by the consecutive actions of phosphodiesterase and exogenous 5’nucleotidase. This method required a two-stage elution from alumina columns, and as with the chromatographic and ion exchange methods, the problem of 5’nucleotidase activity has to be overcome by addition of snake venom 5’-nucleotidase in excess. The present work describes a simple, reproducible cyclic nucleotide phosphodiesterase assay using [32P]-labeled cyclic AMP or cyclic GMP as substrate, and separation of labeled cyclic nucleotide from labeled product by a single-stage procedure. 5’-Nucleotidase activity can be disregarded, since both 5’-AMP and inorganic phosphate are virtually completely separated from cyclic AMP by the method, which is essentially that described by Ramachandran (16) for adenylate cyclase assay. The method has been applied to a study of the subcellular distribution of cyclic nucleotide phosphodiesterase in rat renal cortex. MATERIALS [3H]Cyclic AMP (11.0 Ci/mmol), [3H]cyclic GMP (6.0 Ci/mmol), [3H]5’AMP (21.0 Ci/mmol) and [3H]5’GMP (5.2 Ci/mmol) were obtained from The Radiochemical Centre, Amersham, Bucks., England. Carrierfree [32P]inorganic phosphate was obtained from the Australian Atomic Energy Commission, Lucas Heights, N.S.W. [32P]Cyclic AMP and [32P]cyclic GMP (2-8 Ci/mmol were synthesized in the laboratory of Dr. R. H. Symons, Department of Biochemistry, University of Adelaide, South Australia by his method (17). At times [32P]cyclic AMP (0.3 Ci/ mmol) was obtained from The Radiochemical Centre, Amersham. Unlabeled nucleosides and nucleotides were Sigma A grade, as was Trizma HCl and base. Bovine serum albumin fraction V was obtained from the Commonwealth Serum Laboratories, Parkville, Victoria, Australia. Neutral alumina was Merck grade 1 neutral aluminum oxide. All other reagents were analytical grade obtained from standard suppliers. METHODS Neutral Alumina
Columns
Glass columns with upper reservoirs were used to contain 0.9 x 6 cm columns of freshly poured dry neutral alumina, retained by glass wool plugs. Samples were applied to the dry neutral alumina and eluted directly into glass scintillation vials. The elution characteristics of inorganic phosphate and of various relevant nucleotides were studied. [3H]labeled cyclic AMP, cyclic GMP, 5’-AMP and 5’-GMP, and [32P]labeled inorganic phosphate were prepared at lop3 to lo+ M in 50 mM
382
EISMAN
AND
MARTIN
Tris HCl buffer (pH 7.4 at 37°C)containing 10mg% bovine serumalbumin, 5 mM MgC& and 5 mM MnCl,. Portions (0.4ml) were applied to columns andelutedwith 1- 10ml of 50mM Tris HCl (pH 7.0 at 25°C). Ten milliliters of Bray’s scintillant (18) were used to count [3H]-labeledsamples.Those containing [32P]as the only isotope were countedusing 1 ml of a Tritontoluene scintillant/4 ml of aqueoussample, with an efficiency of 90-95%. The scintillant consistedof 7.5 g/liter of 2,5-diphenyloxazoleand 250mg/ liter of 1,4-di(2-(5-phenyloxazolyl))benzene in Triton X-100:toluene (1:l). A Packard Tricarb liquid scintillation counter was used. Radioactivity recoveredwas expressedas a percentageof radioactivity applied. Phosphodiesterase
Activity
Incubations were carried out in a shakingwater bath at 37°C in Pyrex glasstest tubes (1 x 8 cm). Each 400 pliter incubation contained50 mM Tris HCl buffer (pH 7.4 at 37”C), 10 mg% bovine serum albumin and 5 mM MgCl,. MnCl, (5 mM) was present in the assaysof cyclic GMP phosphodiesteraseactivity. [32P]-Labeledcyclic nucleotide (5000dpm/ incubation) was added with unlabeled cyclic nucleotide to produce the desired final concentrations.Reactions were initiated by the addition of 5- 100pg of enzyme protein and were stoppedby immersion in a boiling water bath for 5 min. The incubationswere allowed to cool to room temperatureandthen wereappliedin toto to the dry neutralalumina columns. Each incubation mixture was allowed to enter the columns and then was eluted with 4 ml of 50 mM Tris HCl buffer (pH 7.4) directly into standard scintillation vials. Phosphodiesteraseactivity resulted in the loss of recovered radioactivity comparedto a control incubation, which was boiled as the enzyme was added. Activity was calculated from the percentageloss of radioactivity recoveredand was expressedas moles per minute. Tissue Fractionation
Sprague-Dawleyrats of either sex, weighing 100to 150g fed a standard diet ad lib, were usedfor all experiments.Animals were killed by stunning andcervical dislocation. The kidney cortices were separatedfrom medulla and homogenisedin a loose fitting Dounce homogeniser(Kontes GIass Co., New Jersey). The homogenatewas fractionated according to the method of Martin et al. (19) into 7OOg,SOOOg and 100,OOOg pellets and 100,OOOg supernatant. All sampleswere assayedimmediately for cyclic AMP and cyclic GMP phosphodiesteraseactivity using the assaymethod described. Stability of Particle-Associated
Activity
At times the twice washed pellets of the 7OOg,SOOOg and 100,OOOg centrifugation were subjectedto several strokesat top speedof a motor-
CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ASSAY
383
CAMP
ELUTION VOLUME
(ml)
FIG. 1. Elution of [3H]-labeled cyclic AMP, cyclic GMP, 5’.AMP and S’-GMP from columns of neutral alumina.
driven Teflon-glasshomogeniser.These homogenateswere centrifuged at 100,000gfor 45 min. The supernatantsand their appropriatehomogenateswere assayedfor phosph~iesterase activity immediately. Elution Characteristics of Alumina Columns Cyclic AMP and cyclic GMP were eluted from neutral ~umina by Tris HCl buffer whereasthe mononucleotidesand inorganic phosphate were retained(Fig. 1). Variations in the concentrationsof the nucleotides and phosphatefrom 10W3to 10W6~did not alter elution characteristics. E~u~onof inorganic phosphatewas less than OS%, and less than 3% of either mononucieotidewas elutedwith up to 10ml of the buffer. EIution of the cyclic nucleotides was not increasedsignificantly as the volume of eluting buffer was increasedabove 4 ml. The recovery of either cyclic nucleotidevaried from 70 to 95%between batchesof neutral alumina. The recovery deterioratedwith storageparticularly if the alumina was exposedto the atmospherefor any length of time, e.g., overnight. However, this deterioration was relatively slow and in any single experiment, the recovery was quite reproduciblewith a coefficient of variation of less than 1%. Deteriorated alumina could be restoredby heatingovernightin anovenattemperaturesgreaterthan 100°C.
384
EISMAN
AND MARTIN
FIG. 2. Time course of cyclic AMP hydrolysis by 100,OOOgsupematant phosphodiesterase activity from rat renal cortex. Cyclic AMP concentration was 2 x 10eB M. Phosphodiesterase
Assay
This technique of separating substrate from reaction products was appliedto cyclic AMP andcyclic GMP phosphodiesterase activity assays. All assayswere performed in triplicate with coefficients of variation of about 2.5% and any assay with coefficient of variation in excessof 5% was discarded.
FIG. 3. Lineweaver-Burk plot of hydrolysis of cyclic AMP by 100,OOOg supematant phosphodiesterase activity from rat renal cortex.
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
385
ASSAY
Using this method time course studies were performed using cyclic AMP and cyclic GMP as substrate. Cyclic AMP and cyclic GMP hydrolysis was found to be linear with respect to time over the range from 5 to 40% of substrate hydrolysis. An experiment illustrating this is shown in Fig. 2, in which cyclic AMP phosphodiesterase activity of rat kidney cortex cytosol was measured. Similarly linearity with respect to enzyme concentration was demonstrated over this range of 5-40% substrate hydrolysis. The assay has been used in detailed studies of the kinetics of cyclic AMP and cyclic GMP hydrolysis, and Fig. 3 shows a Lineweaver-Burke plot of results of cyclic AMP phosphodiesterase assay carried out on rat kidney cortex cytosol. Apparent K, values of 7.5 t 1.3 x IOF and 3.1 f 0.5 x 10e6 M (means -c SEM of 5 experiments) were obtained and the biphasic kinetics were similar to those found in brain (20), kidney (1 l), frog bladder (11) and liver (21). The value derived from the “high K,” component of the enzyme activity was not corrected for the contribution of the “low K,” activity to the velocity at the higher substrate concentrations. Kinetic studies of cyclic GMP phosphodiesterase revealed linear kinetics with apparent K, of 4.4 rt 1.5 x 10V5and 3.1 -I- 1.3 x 10T5 M for particle associated and cytosol fractions, respectively. These values are similar to those reported for rat brain by Thompson and Appleman (13) but are an order of magnitude greater than those reported for the same tissue by Brooker et at, (20) and for silkmoth fat body (15). Subcellular Distribution of Cyclic Nucleotide Phosphodiesterase and Instability of Particle-Associated Activity
Activity
The subcellular distribution of cyclic AMP phosphodiesterase activity is presented in Table I. The activity is expressed in nanomoles per minute per fraction derived from 3 pairs of renal cortices (about 3 g tissue), and also as a percentage of the total activity of the crude homogenate. Table 2 TABLE CYCLIC AMP PHOSPHODIESTERASEACTIVITY
I IN SUBCELLULAR
FRACTroNS
Activity Fraction
nmol/min/ fraction
% of homogenate
Crude homogenate 7OOg pellet-nuclei and plasma membranes 5,OOOgpellet-mitochond~ai l~,~ peiIet-microsomai 100,OOOgsupernatant-cytosol
56.5 15.7 1.1 0.4 28.2
100 27.8 1.9 0.6 49.9
Recovery
80.2
386
EISMAN
AND MARTIN TABLE
COMPARISON AND
OF SUBCELLULAR CYCLIC
GMP
2
DISTRIBUTION
OF CYCLIC
PHOSPHODIESTERASE
with substrates:
Activity
Cyclic AMP (n = 5)
Fraction Nuclei and plasma membranes Mitochondrial Microsomal Cytosol
AMP
ACTIVITIES
22.5 2.3 1.2 62.0
zi f * f
Cyclic GMP (n = 4)
3.5 0.3 0.3 9.5
20.1 7.4 3.3 67.4
+ 2 2 f
4.9 1.7 1.4 5.0
a Expressed as percentages of activity of crude homogenates. Means 2 SEM of several fractionations.
summarisesthe results of several such fractionations in which the subcellular distribution of both cyclic AMP and cyclic GMP phosphodiesteraseactivities was studied. The resultsindicate a closesimilarity between the distributions of the phosphodiesteraseactivities of the two cyclic nucleotides, and furthermore that at least a substantialproportion of eachenzymeactivity is associatedwith particulatecomponentsof cells. This activity probably derives from plasma membranefragments as, in TABLE EFFECT
OF VIGOROUS
3
HOMOGENISATION
PARTICLE-ASSOCIATED
ON “SOLUBILITY”
PHOSPHODIESTERASE
OF
ACTIVITY~
Activity (nmol/min/fraction) Fraction
Total
Supematant Cyclic AMP phosphodiesterase
Nuclei and plasma membranes Mitochondrial Microsomal
11.3 0.42 0.15
10.2 0.25 0.15
Cyclic GMP phosphodiesterase Nuclei and plasma membranes Mitochondrial Microsomal
5.0 1.35 0.69
(LAssays were carried out before and after motor-driven (see Methods).
5.7 1.14 1.03 Teflon-glass homogenisation
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
ASSAY
387
unpublished observations (J. A. Eisman and D. G. Legge), very low activity was found in nuclei isolated by the method of Chaveau et al. (23). Finally, as shown in Table 3, more drastic physical treatment of the twice washed particle fractions resulted in considerable release of activity into the 100,OOOg supernatant. DISCUSSION Separation of substrate from reaction products on the dry neutral alumina columns is rapid and requires only a single elution step. An assay consisting of 200 incubation tubes can be processed completely by one operator in less than 3 hr. Whatever the further metabolism of generated 5’-AMP in crude enzyme preparations, this method remains valid because of the negligible elution of inorganic phosphate from alumina under assay conditions. The fact that the assay measures um-eacted cyclic AMP present after incubation, rather than the product formed, might suggest a disadvantage in poor sensitivity at low rates of reaction. However, the coefficient of variation of triplicate assays was usually less than 2.5% and always less than 5%. This required that percentage hydrolysis be at least 5% to differentiate it reliably from zero hydrolysis, and the sensitivity and reproducibility under these conditions is satisfactory for any kinetic studies. In the example shown in Fig. 3, the results are similar to those reported for kidney (1 l), and were highly reproducible. The short half-life and expense of the [32P]-labeled substrates may be considered a disadvantage. However, the separation of the entire incubation medium on the alumina columns and the high counting efficiency with an inexpensive scintillant offset this. Less than 0.25 &i is required for a 100 tube assay, and a single batch of labeled substrate of relatively low initial specific activity (0.3 Cilmmole) can be used for at least 2 months. High specific activity material (4 Ciimmol) can be used for over 4 months. The simplicity of the pathways of [32P]-cyclic nucleotide hydrolysis make this assay pa~i~ularly suitable for the study of enzyme preparations as crude as those in subcellular fractions. The subcellular distribution of phosphodiesterase activity found in these experiments resembles that described by Campbell and Oliver (22), who used millimolar substrate concentrations. The parallelism in subcellular distribution of cyclic AMP and cyclic GMP phosphodiesterase activities has no immediate explanation, but we have found marked differences between the two enzyme activities in kinetic studies (unpublished data). The association of phosphodiesterase activity with plasma membrane fragments is supported by the findings of House et al. (24) with highly purified liver plasma membrane fragments. This association for cyclic AMP phosphodiesterase activity, at least, suggests a role in the regulation of cyclic AMP level at an intracellular site close to that of its hormonally
388
EISMAN
AND MARTIN
regulated production. This particle-associated phosphodiesterase activity is conveniently studied with the method described due to its tolerance of crude, nonhomogeneous enzyme preparations. Our finding that much of the particle-associated activity could be solubilised by further homogenisation suggests that the cytoplasmic activity may represent in part an artefact of the homogenisation procedure. Such a property should be borne in mind in analysing any studies of subcellular distribution of phosphodiesterase. This assay system for cyclic nucleotide phosphodiesterase is rapid and readily handles large numbers of individual assays, and as such has proved convenient for use in kinetic studies. The procedure is unaffected by a wide variety of incubation medium components, and the reaction is such that provision of exogenous 5’-nucleotidase is not necessary. These various factors make the assay useful for both purified and crude enzyme preparations, and particularly in comparing cyclic nucleotide phosphodiesterase activities in subcellular fractions. ACKNOWLEDGMENTS This work was supported by a grant from the National Health & Medical Research Council of Australia. J.A.E. was in receipt of a N.H. & M.R.C. Postgraduate Medical Research Scholarship. We are most grateful to Dr. R. H. Symons for his generous provision of facilities and of [32P]-cyclic nucleotides.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Blonde, L. W., Wehmann, R. E., and Steiner, A. L. (1974) J. Clin. Invest. 53, 163. Wehmann, R. E., Blonde, L. W., and Steiner, A. L. (1974) J. Clin. Invest. 53, 173. Butcher, R. W., and Sutherland, E. W. (1962) J. Biol. Chem. 237, 1244. Sutherland, E. W., and Rail, T. W. (1958) J. Biol. Chem. 232, 1077. Drummond, G. I., and Perrott-Yee, S. (1961) J. Biol. Chem. 236, 1126. Cheung, W. Y. (1%6) Biochem. Biophys. Res. Commun. 23, 214. Cheung, W. Y. (1%9) Anal. Biochem. 28, 182. DouSa, T., and Rychhk, I. (1970) Biochim. Biophys. Acta 204, 10. Chase, L. R., Fedak, S. A., and Aurbach, G. D. (1969) Endocrinology 84, 761. Marcus, R., and Aurbach, G. D. (1971) Biochim. Biophys. Acta 242, 410. Jard, S., and Bernard, M. (1970) Biochem. Biophys. Res. Commun. 41,781. Lagarde, A., and Colobert, L. (1972) Biochim. Biophys. Acta 276,444. Thompson, W. J., and Appleman, M. M. (1971) Biochemistry 10, 311. Boudreau, R. J., and Drummond, G. I. (1975)Anal. Biochem. 63, 388. Filburn, C. R., and Kam, J. (1973) Anal. Biochem. 52, 505. Ramachandran, J. (1971) Anal. Biochem. 43,226. Symons, R. H. (1973) Biochim. Biophys. Acta 320, 535. Bray, G. A. (l%O)Anal. Biochem. 1, 279. Martin, T. J., Melick, R. A., and deluise, M. (1%9) Biochem, J 111, 509. Brooker, G., Thomas, L. J., Jr., and Appleman, M. M. (1968) Biochem. J. 7, 4177. Clark, J. F., Morris, H. P., and Weber, G. (1973) Concev Res. 33, 356. Campbell, M. T., and Oliver, I. T. (1972) Eur. J. Biochem. 28, 30. Chaveau, J., Moult, Y., and Rouiller, G. (1956) Exp. Cell Res. 11, 317. House, P. D. R., Poulis, P., and Weidemann, M. J. (1972) Eur. J. Biochem. 24, 429.
BIOCHEMISTRY
72,380-388
(1976)
Simple Assay of Cyclic Nucleotide Phosphodiesterase Using 32Phosphorus Labeled Nucleotide; Application to Kidney Subcellular Fractions J. A. EISMAN’ AND T. J. MARTINS University of Melbourne
Department of Medicine, Austin Hospital, Heidelberg, Victoria, Australia
Received June 6, 1975; accepted October 24, 1975 A method is described for the estimation of cyclic nucleotide phosphodiesterase activity using ~*P~ospho~s-~abeIed cyclic nucleotides and separation of substrates from reaction products in a single-stage elution on dry neutral alumina columns. The method was used to study tiie subcellular distribution of cyclic AMP and cyclic GMP phosphodiesterase activity in rat renal cortex.
Intracellular hydrolysis of 3’5’-cyclic AMP and 3’5’-cyclic GMP by cyclic nucleotide phosphodiesteraseappearsto be the most important mechanismfor the disposal of cyclic nucleotidesfrom the cells in which they are generated(1,Z). The function and possible control of phosphodiesteraseactivity hasbeenstudiedextensivelysinceit wasfirst shown(3) to hydrolyse specifically the cyclic 3’:5’-monophosphatenucleotide to the 5’-monophosphate. Before radioisotopeassaysbecameavailable the enzyme activity was assayedat substrate concentrationstwo to three orders of magnitude higherthan physiologicallevels (4-8). Sensitivity at low substrateconcentrations has been obtained using PHI- and [14C]-labeled3’:5’-cyclic nucleotideas substrate,and various methodsof sep~ation of the labeled products. Barium sulfate precipitation of labeledS-AMP (9,lO) is unsatisfactory in studies of subcellular fractions containing 5’-nucleotidase activity, since adenosine,like its cyclic nucleotide, is not precipitatedby barium sulfate, Paper chromatographic separationis used (11,12),with conversion of generated5’-AMP to adenosineby addition of exogenous S-nucleotidase. The latter approach has also been used by Thompson and Appleman (13) in developing an ion exchangebinding system for phosphodiesteraseassay, which has been modified (14) to overcome problems of recovery of adenosineor guanosine.Filburn and Karn (15) 1 Present address: Department of Biochemistry, University of Wisconsin, Madison, Wis. 53706. * Present address: Department of Chemical Pathology, University of Sheffield Medical School, Sheffield, U.K. 380 Copytight Q 1976 by Academic Prerr. Inc. All right< of reproduction in any form I-ererved.
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
ASSAY
381
reported the use of alumina columns to separate [3H]- and [14C]-labeled cyclic AMP and cyclic GMP from their respective nucleotides, produced by the consecutive actions of phosphodiesterase and exogenous 5’nucleotidase. This method required a two-stage elution from alumina columns, and as with the chromatographic and ion exchange methods, the problem of 5’nucleotidase activity has to be overcome by addition of snake venom 5’-nucleotidase in excess. The present work describes a simple, reproducible cyclic nucleotide phosphodiesterase assay using [32P]-labeled cyclic AMP or cyclic GMP as substrate, and separation of labeled cyclic nucleotide from labeled product by a single-stage procedure. 5’-Nucleotidase activity can be disregarded, since both 5’-AMP and inorganic phosphate are virtually completely separated from cyclic AMP by the method, which is essentially that described by Ramachandran (16) for adenylate cyclase assay. The method has been applied to a study of the subcellular distribution of cyclic nucleotide phosphodiesterase in rat renal cortex. MATERIALS [3H]Cyclic AMP (11.0 Ci/mmol), [3H]cyclic GMP (6.0 Ci/mmol), [3H]5’AMP (21.0 Ci/mmol) and [3H]5’GMP (5.2 Ci/mmol) were obtained from The Radiochemical Centre, Amersham, Bucks., England. Carrierfree [32P]inorganic phosphate was obtained from the Australian Atomic Energy Commission, Lucas Heights, N.S.W. [32P]Cyclic AMP and [32P]cyclic GMP (2-8 Ci/mmol were synthesized in the laboratory of Dr. R. H. Symons, Department of Biochemistry, University of Adelaide, South Australia by his method (17). At times [32P]cyclic AMP (0.3 Ci/ mmol) was obtained from The Radiochemical Centre, Amersham. Unlabeled nucleosides and nucleotides were Sigma A grade, as was Trizma HCl and base. Bovine serum albumin fraction V was obtained from the Commonwealth Serum Laboratories, Parkville, Victoria, Australia. Neutral alumina was Merck grade 1 neutral aluminum oxide. All other reagents were analytical grade obtained from standard suppliers. METHODS Neutral Alumina
Columns
Glass columns with upper reservoirs were used to contain 0.9 x 6 cm columns of freshly poured dry neutral alumina, retained by glass wool plugs. Samples were applied to the dry neutral alumina and eluted directly into glass scintillation vials. The elution characteristics of inorganic phosphate and of various relevant nucleotides were studied. [3H]labeled cyclic AMP, cyclic GMP, 5’-AMP and 5’-GMP, and [32P]labeled inorganic phosphate were prepared at lop3 to lo+ M in 50 mM
382
EISMAN
AND
MARTIN
Tris HCl buffer (pH 7.4 at 37°C)containing 10mg% bovine serumalbumin, 5 mM MgC& and 5 mM MnCl,. Portions (0.4ml) were applied to columns andelutedwith 1- 10ml of 50mM Tris HCl (pH 7.0 at 25°C). Ten milliliters of Bray’s scintillant (18) were used to count [3H]-labeledsamples.Those containing [32P]as the only isotope were countedusing 1 ml of a Tritontoluene scintillant/4 ml of aqueoussample, with an efficiency of 90-95%. The scintillant consistedof 7.5 g/liter of 2,5-diphenyloxazoleand 250mg/ liter of 1,4-di(2-(5-phenyloxazolyl))benzene in Triton X-100:toluene (1:l). A Packard Tricarb liquid scintillation counter was used. Radioactivity recoveredwas expressedas a percentageof radioactivity applied. Phosphodiesterase
Activity
Incubations were carried out in a shakingwater bath at 37°C in Pyrex glasstest tubes (1 x 8 cm). Each 400 pliter incubation contained50 mM Tris HCl buffer (pH 7.4 at 37”C), 10 mg% bovine serum albumin and 5 mM MgCl,. MnCl, (5 mM) was present in the assaysof cyclic GMP phosphodiesteraseactivity. [32P]-Labeledcyclic nucleotide (5000dpm/ incubation) was added with unlabeled cyclic nucleotide to produce the desired final concentrations.Reactions were initiated by the addition of 5- 100pg of enzyme protein and were stoppedby immersion in a boiling water bath for 5 min. The incubationswere allowed to cool to room temperatureandthen wereappliedin toto to the dry neutralalumina columns. Each incubation mixture was allowed to enter the columns and then was eluted with 4 ml of 50 mM Tris HCl buffer (pH 7.4) directly into standard scintillation vials. Phosphodiesteraseactivity resulted in the loss of recovered radioactivity comparedto a control incubation, which was boiled as the enzyme was added. Activity was calculated from the percentageloss of radioactivity recoveredand was expressedas moles per minute. Tissue Fractionation
Sprague-Dawleyrats of either sex, weighing 100to 150g fed a standard diet ad lib, were usedfor all experiments.Animals were killed by stunning andcervical dislocation. The kidney cortices were separatedfrom medulla and homogenisedin a loose fitting Dounce homogeniser(Kontes GIass Co., New Jersey). The homogenatewas fractionated according to the method of Martin et al. (19) into 7OOg,SOOOg and 100,OOOg pellets and 100,OOOg supernatant. All sampleswere assayedimmediately for cyclic AMP and cyclic GMP phosphodiesteraseactivity using the assaymethod described. Stability of Particle-Associated
Activity
At times the twice washed pellets of the 7OOg,SOOOg and 100,OOOg centrifugation were subjectedto several strokesat top speedof a motor-
CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ASSAY
383
CAMP
ELUTION VOLUME
(ml)
FIG. 1. Elution of [3H]-labeled cyclic AMP, cyclic GMP, 5’.AMP and S’-GMP from columns of neutral alumina.
driven Teflon-glasshomogeniser.These homogenateswere centrifuged at 100,000gfor 45 min. The supernatantsand their appropriatehomogenateswere assayedfor phosph~iesterase activity immediately. Elution Characteristics of Alumina Columns Cyclic AMP and cyclic GMP were eluted from neutral ~umina by Tris HCl buffer whereasthe mononucleotidesand inorganic phosphate were retained(Fig. 1). Variations in the concentrationsof the nucleotides and phosphatefrom 10W3to 10W6~did not alter elution characteristics. E~u~onof inorganic phosphatewas less than OS%, and less than 3% of either mononucieotidewas elutedwith up to 10ml of the buffer. EIution of the cyclic nucleotides was not increasedsignificantly as the volume of eluting buffer was increasedabove 4 ml. The recovery of either cyclic nucleotidevaried from 70 to 95%between batchesof neutral alumina. The recovery deterioratedwith storageparticularly if the alumina was exposedto the atmospherefor any length of time, e.g., overnight. However, this deterioration was relatively slow and in any single experiment, the recovery was quite reproduciblewith a coefficient of variation of less than 1%. Deteriorated alumina could be restoredby heatingovernightin anovenattemperaturesgreaterthan 100°C.
384
EISMAN
AND MARTIN
FIG. 2. Time course of cyclic AMP hydrolysis by 100,OOOgsupematant phosphodiesterase activity from rat renal cortex. Cyclic AMP concentration was 2 x 10eB M. Phosphodiesterase
Assay
This technique of separating substrate from reaction products was appliedto cyclic AMP andcyclic GMP phosphodiesterase activity assays. All assayswere performed in triplicate with coefficients of variation of about 2.5% and any assay with coefficient of variation in excessof 5% was discarded.
FIG. 3. Lineweaver-Burk plot of hydrolysis of cyclic AMP by 100,OOOg supematant phosphodiesterase activity from rat renal cortex.
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
385
ASSAY
Using this method time course studies were performed using cyclic AMP and cyclic GMP as substrate. Cyclic AMP and cyclic GMP hydrolysis was found to be linear with respect to time over the range from 5 to 40% of substrate hydrolysis. An experiment illustrating this is shown in Fig. 2, in which cyclic AMP phosphodiesterase activity of rat kidney cortex cytosol was measured. Similarly linearity with respect to enzyme concentration was demonstrated over this range of 5-40% substrate hydrolysis. The assay has been used in detailed studies of the kinetics of cyclic AMP and cyclic GMP hydrolysis, and Fig. 3 shows a Lineweaver-Burke plot of results of cyclic AMP phosphodiesterase assay carried out on rat kidney cortex cytosol. Apparent K, values of 7.5 t 1.3 x IOF and 3.1 f 0.5 x 10e6 M (means -c SEM of 5 experiments) were obtained and the biphasic kinetics were similar to those found in brain (20), kidney (1 l), frog bladder (11) and liver (21). The value derived from the “high K,” component of the enzyme activity was not corrected for the contribution of the “low K,” activity to the velocity at the higher substrate concentrations. Kinetic studies of cyclic GMP phosphodiesterase revealed linear kinetics with apparent K, of 4.4 rt 1.5 x 10V5and 3.1 -I- 1.3 x 10T5 M for particle associated and cytosol fractions, respectively. These values are similar to those reported for rat brain by Thompson and Appleman (13) but are an order of magnitude greater than those reported for the same tissue by Brooker et at, (20) and for silkmoth fat body (15). Subcellular Distribution of Cyclic Nucleotide Phosphodiesterase and Instability of Particle-Associated Activity
Activity
The subcellular distribution of cyclic AMP phosphodiesterase activity is presented in Table I. The activity is expressed in nanomoles per minute per fraction derived from 3 pairs of renal cortices (about 3 g tissue), and also as a percentage of the total activity of the crude homogenate. Table 2 TABLE CYCLIC AMP PHOSPHODIESTERASEACTIVITY
I IN SUBCELLULAR
FRACTroNS
Activity Fraction
nmol/min/ fraction
% of homogenate
Crude homogenate 7OOg pellet-nuclei and plasma membranes 5,OOOgpellet-mitochond~ai l~,~ peiIet-microsomai 100,OOOgsupernatant-cytosol
56.5 15.7 1.1 0.4 28.2
100 27.8 1.9 0.6 49.9
Recovery
80.2
386
EISMAN
AND MARTIN TABLE
COMPARISON AND
OF SUBCELLULAR CYCLIC
GMP
2
DISTRIBUTION
OF CYCLIC
PHOSPHODIESTERASE
with substrates:
Activity
Cyclic AMP (n = 5)
Fraction Nuclei and plasma membranes Mitochondrial Microsomal Cytosol
AMP
ACTIVITIES
22.5 2.3 1.2 62.0
zi f * f
Cyclic GMP (n = 4)
3.5 0.3 0.3 9.5
20.1 7.4 3.3 67.4
+ 2 2 f
4.9 1.7 1.4 5.0
a Expressed as percentages of activity of crude homogenates. Means 2 SEM of several fractionations.
summarisesthe results of several such fractionations in which the subcellular distribution of both cyclic AMP and cyclic GMP phosphodiesteraseactivities was studied. The resultsindicate a closesimilarity between the distributions of the phosphodiesteraseactivities of the two cyclic nucleotides, and furthermore that at least a substantialproportion of eachenzymeactivity is associatedwith particulatecomponentsof cells. This activity probably derives from plasma membranefragments as, in TABLE EFFECT
OF VIGOROUS
3
HOMOGENISATION
PARTICLE-ASSOCIATED
ON “SOLUBILITY”
PHOSPHODIESTERASE
OF
ACTIVITY~
Activity (nmol/min/fraction) Fraction
Total
Supematant Cyclic AMP phosphodiesterase
Nuclei and plasma membranes Mitochondrial Microsomal
11.3 0.42 0.15
10.2 0.25 0.15
Cyclic GMP phosphodiesterase Nuclei and plasma membranes Mitochondrial Microsomal
5.0 1.35 0.69
(LAssays were carried out before and after motor-driven (see Methods).
5.7 1.14 1.03 Teflon-glass homogenisation
CYCLIC
NUCLEOTIDE
PHOSPHODIESTERASE
ASSAY
387
unpublished observations (J. A. Eisman and D. G. Legge), very low activity was found in nuclei isolated by the method of Chaveau et al. (23). Finally, as shown in Table 3, more drastic physical treatment of the twice washed particle fractions resulted in considerable release of activity into the 100,OOOg supernatant. DISCUSSION Separation of substrate from reaction products on the dry neutral alumina columns is rapid and requires only a single elution step. An assay consisting of 200 incubation tubes can be processed completely by one operator in less than 3 hr. Whatever the further metabolism of generated 5’-AMP in crude enzyme preparations, this method remains valid because of the negligible elution of inorganic phosphate from alumina under assay conditions. The fact that the assay measures um-eacted cyclic AMP present after incubation, rather than the product formed, might suggest a disadvantage in poor sensitivity at low rates of reaction. However, the coefficient of variation of triplicate assays was usually less than 2.5% and always less than 5%. This required that percentage hydrolysis be at least 5% to differentiate it reliably from zero hydrolysis, and the sensitivity and reproducibility under these conditions is satisfactory for any kinetic studies. In the example shown in Fig. 3, the results are similar to those reported for kidney (1 l), and were highly reproducible. The short half-life and expense of the [32P]-labeled substrates may be considered a disadvantage. However, the separation of the entire incubation medium on the alumina columns and the high counting efficiency with an inexpensive scintillant offset this. Less than 0.25 &i is required for a 100 tube assay, and a single batch of labeled substrate of relatively low initial specific activity (0.3 Cilmmole) can be used for at least 2 months. High specific activity material (4 Ciimmol) can be used for over 4 months. The simplicity of the pathways of [32P]-cyclic nucleotide hydrolysis make this assay pa~i~ularly suitable for the study of enzyme preparations as crude as those in subcellular fractions. The subcellular distribution of phosphodiesterase activity found in these experiments resembles that described by Campbell and Oliver (22), who used millimolar substrate concentrations. The parallelism in subcellular distribution of cyclic AMP and cyclic GMP phosphodiesterase activities has no immediate explanation, but we have found marked differences between the two enzyme activities in kinetic studies (unpublished data). The association of phosphodiesterase activity with plasma membrane fragments is supported by the findings of House et al. (24) with highly purified liver plasma membrane fragments. This association for cyclic AMP phosphodiesterase activity, at least, suggests a role in the regulation of cyclic AMP level at an intracellular site close to that of its hormonally
388
EISMAN
AND MARTIN
regulated production. This particle-associated phosphodiesterase activity is conveniently studied with the method described due to its tolerance of crude, nonhomogeneous enzyme preparations. Our finding that much of the particle-associated activity could be solubilised by further homogenisation suggests that the cytoplasmic activity may represent in part an artefact of the homogenisation procedure. Such a property should be borne in mind in analysing any studies of subcellular distribution of phosphodiesterase. This assay system for cyclic nucleotide phosphodiesterase is rapid and readily handles large numbers of individual assays, and as such has proved convenient for use in kinetic studies. The procedure is unaffected by a wide variety of incubation medium components, and the reaction is such that provision of exogenous 5’-nucleotidase is not necessary. These various factors make the assay useful for both purified and crude enzyme preparations, and particularly in comparing cyclic nucleotide phosphodiesterase activities in subcellular fractions. ACKNOWLEDGMENTS This work was supported by a grant from the National Health & Medical Research Council of Australia. J.A.E. was in receipt of a N.H. & M.R.C. Postgraduate Medical Research Scholarship. We are most grateful to Dr. R. H. Symons for his generous provision of facilities and of [32P]-cyclic nucleotides.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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