- Email: [email protected]
[64] Platelet cGMP-binding phosphodiesterase
710
P H O S P H O D I E S T E R A S E ISOZYME M E T H O D S
[64]
We find that a TSK-250 column (0.75 × 60 cm) purchased from Bio-Rad has better resolution for PDE purification. The concentrated crude PDE (330/zl) is applied to a TSK-250 column (0.75 × 60 cm). The column has been equilibrated with buffer E. The chromatographic separation is carried out at a flow rate of 1 ml/min. The volume of each fraction is 0.5 ml/ tube. PDE activity is assayed as previously described.l~ A typical elution profile of the purified PDE is shown in Fig. 3. The purity of the pooled fractions (27 through 29) is examined by SDS-polyacrylamide slab gel electrophoresis and protein bands are stained with Coomassie blue (Fig. 4). The specific activity of the purified PDE is measured as 1100 mol of cGMP/mol of PDE/min, in the absence of any activating factors. When this assay is repeated in the presence of amphibian Ga/GTPTS [guanosine 5'-O-(3-thiotriphosphate)] it is found that the specific activity is then 11,000 mol of cGMP/mol of PDE/min, suggesting that the PDE purified by this methodology retains a significant amount of endogenous inhibitory activity presumably associated with the 13K subunit. Acknowledgment Los Alamos National Laboratory is operated by the University of California for the United States Department of Energy under contract W-7405-ENG-36. N W. J. Thompson and M. M. Appleman, Biochemistry 10, 311 (1971).
[64] P l a t e l e t c G M P - B i n d i n g P h o s p h o d i e s t e r a s e
By PAVEL HAMET and JOHANNE TREMBLAY Introduction It is generally accepted that at least a significant part of cGMP's function in the cell is expressed via its interaction with cGMP-dependent protein kinase, usually detected as a "binding" of the nucleotide. On the other hand, some tissues which appear to have reasonably well-modulated levels in relation to cell function do not present detectable cytosolic cGMP binding corresponding to cGMP-dependent protein kinase. Significant binding of a different type, one that is related to the activity of METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[64]
PLATELET cGMP-BINDINGPHOSPHODIESTERASE
711
cGMP-phosphodiesterase (PDE), has been observed in these tissues, which include platelets, the retina, and sea urchin sperm.l-3 At present, it is not known how this "significant binding" is implicated in the expression of cGMP's function. Goldberg e t al. 4 proposed that the function of cGMP may be expressed via its rapid and massive turnover leading to proton generation. This notion has been explored particularly in relation to sodium fluxes and the transmission of the light signal in the retina. Recently, interest in the regulation of cGMP hydrolysis has been renewed by the discovery of Fesenko e t al. 5 of a direct effect of cGMP on the regulation of the sodium channel in the retina. Although the direct action of cGMP on ionic fluxes has not yet been investigated in other systems, this notion needs to be examined in tissues such as platelets. Our earlier experiments on platelets and lungs 1,6 uncovered a cGMPbinding PDE distinct from other forms of cGMP-PDE such as calmodulinsensitive and cGMP-stimulated PDE. This protein was later independently noted by Francis e t al. 7 in the lung, and more recently by Coquil e t al. 8 in several other tissues. Similarly, at least as far as the potential "buffering capacity" of cGMP is concerned, an analogous type of PDE has been found in the retina, in which GMP-PDE also has a binding site for this nucleotide. 9 However, more recent studies have set apart retinal PDE as a distinctly regulated enzyme, immunologically and functionally different from the platelet enzyme, 1°,11and described in Chapter 63 in this volume. In the present chapter, we will deal specifically with cGMPbinding PDE from platelets. We will discuss the methods used for determination of the hydrolytic and binding activities of this enzyme, its partial purification, as well as modulation of both binding and enzymatic activities by agonists in v i t r o and e x v i v o . t p. Hamet and J.-F. Coquil, J. Cyclic Nucleotide Res. 4, 281 (1978). 2 j. S. Pober and M. W. Bitensky, Adv. Cyclic Nucleotide Res. 11, 265 (1979). 3 D. L. Garbers and G. S. Kopf, Adv. Cyclic Nucleotide Res. 13, 251(1980). 4 N. D. Goldberg, A. Ames III, J. E. Gander, and T. F. Walseth, J. Biol. Chem. 258, 9213 (1983). 5 E. E. Fesenko, S. S. Kolesnikov, and A. L. Lyubarsky, Nature (London) 313, 310 (1985). 6 J.-F. Coquil, D. J. Franks, J. N. Wells, M. Dupuis, and P. Hamet, Biochim. Biophys. Acta 631, 148 (1980). 7 S. H. Francis, T. M. Lincoln, and J. D. Corbin, J. Biol. Chem. 255, 620 (1980). 8 J.-F. Coquil, G. Brunelle, and J. Guedon, Biochem. Biophys. Res. Commun. 127, 226 (1985). 9 A. Yamazaki, I. Sen, and M. W. Bitensky, J. Biol. Chem. 255, 11619 (1980). 10 L. Stryer, Cold Spring Harbor Symp. Quant. Biol. 48, 841 (1983). 1~ R. L. Hurwitz, A. H. Bunt-Milam, M. L. Chang, and J. A. Beavo, J. Biol. Chem. 260, 568 (1985).
712
P H O S P H O D I E S T E R AISOZYME SE METHODS
[64]
Methods of Detection and Basic Characteristics of cGMP-Binding and PDE Activity
Standard Conditions for Measurement of cGMP Binding The standard conditions which we usually employ for cGMP-binding determinations are derived from those originally described by Gilman 12 and Lincoln et al. ~3The standard reaction mixture contains 30 mM Bicine (pH 9.0), 0.1 # M [3H]cGMP and 0.5 mg/ml bovine serum albumin in a final volume of 0.1 ml. Incubation is started with the addition of a cGMPbinding protein preparation containing 2 mM EDTA (the final concentration should be at least 0.4 mM) and 2 mM dithiothreitol (DTT). The duration of incubation is usually 90 min at 4°. At the end of the incubation period, 2 ml of a cold 20 mM potassium phosphate buffer (pH 7.0) is added, and the solution is poured onto Millipore or Gelman nitrocellulose filters (0.45 tzm), previously immersed in the same buffer, to separate free and bound [3H]cGMP. The tubes are rinsed with 4 ml of 20 mM potassium phosphate buffer, and the filters are washed with 30 ml of the same buffer. The filters are then dissolved in 2 ml of Cellosolve and 10 ml of scintillation liquid is added for the radioactivity determinations. Blank values are ascertained by performing the incubations without samples or by incubation with the binding protein preparation at time zero. Identical values (less than 50 cpm) are obtained with the two methods. The filters can also be washed with 20 m M Bicine buffer (pH 9.0), instead of potassium phosphate, and similar results are achieved. The data are expressed in picomoles [3H]cGMP bound per milligram protein or per milliliter of the binding protein preparation. An alternative procedure for the separation of bound and free cGMP molecules has been designed, using ammonium sulfate precipitation. At the conclusion of incubation, 1 ml of 70% ammonium sulfate is added to each tube. The contents of the tubes are then mixed, incubated for 20 min at 4°, and centrifuged at 3000 g for 15 rain. The supernatant is decanted, and a further 1 ml of ammonium sulfate is added to the precipitate with the procedure being repeated once more. Radioactivity in the dissolved pellet is counted after the addition of I ml of H20. Qualitative and quantitative correlation of the filtration and precipitation methods is excellent with r 2 = 0.94 (p <0.001) for 15 random, duplicate samples, the same degree of stimulation of activity by various agents, such as l-methyl-3-isobutylxanthine (MIX), is observed with the two methods. 6 However, due to greater 12 A. G. Gilman, Proc. Natl. Acad. Sci. U.S.A. 67, 305 (1970). 13 T. M. Lincoln, C. L. Hall, C. R. Park, and J. D. Corbin, Proc. Natl. Acad. Sci. U.S.A. 73, 2559 (1976).
[64]
PLATELETcGMP-BINDING PHOSPHODIESTERASE
713
variability, the samples separated by the precipitation methods are run in quadruplicate. Thus, for most purposes, the filtration procedure appears to be more suitable, yet the precipitation method can be used for the screening of binding in large-scale preparations, such as in eluates from column chromatography.
Standard Conditions for Measurement of cGMP-PDE This procedure is based of the method of Beavo et al. 14 and Wells et al. ~5 The assay mixture contains 60 mM TES, 3 mM MgCI2, 0.20 mg bovine serum albumin, 1/zM cGMP (unless otherwise stated), and 20-30 nCi of [3H]cGMP in a total volume of 200/xl. The reaction is started by the addition of 25/zl of enzyme preparation suitably diluted so that less than 20% of radioactive substrate is hydrolyzed when incubated at 30° for 30 min. The incubation is terminated by the addition of 25/zl of a reaction mixture containing 50 mM EDTA, 5 mM MIX, 15 mM cGMP, and 9 mM 5'-GMP. Twenty-five microliters of snake venom (Crotalus atrox, 2 mg/ ml) is then added, and the mixture incubated for a further 10 min at 30°. Incubation is stopped by the addition of 750 t~l of a solution containing 0.1 mM guanosine. The guanosine formed is separated from the nucleotides on QAE-Sephadex A-25 (formate form). ~5 In order to routinely verify both the 5'-nucleotidase step and the column recovery, we run the "100% control" of undiluted PDE samples to ensure the complete conversion of 5'-GMP to guanosine. The radiolabeled cyclic nucleotide should be kept at -70 ° to decrease 3H exchange w i t h H 2 0 . Periodically, the labeled [3H]cGMP stock is purified to reduce contamination by [3H]20, which is not separated at the last column step.
Stability o f ff H]cGMP in Binding Assay With the standard conditions employed for cGMP binding, the stability of the radiolabeled cGMP estimated by thin-layer chromatography on polyethyleneimine cellulose plates is over 99.4% of the original material added either in the absence or in the presence of MIX. 6 On the other hand, the stability of cGMP requires the presence of a chelator and incubation at lower temperatures. Thus, the conditions for the cGMP binding and PDE assays differ significantly with the presence or absence of free divalent cations as well as the incubation temperatures. The conditions used for studying cGMP binding could lead to underestimation of the 14 j. A. Beavo, J. G. Hardman, and E. W. Sutherland, J. Biol. Chem. 245, 5649 (1970). ~5j. N. Wells, C. E. Baird, Y. J. Wu, and J. G. Hardman, Biochim. Biophys. Acta 384, 430 (1975).
714
P H O S P H O D I E S T E R AISOZYME SE METHODS
[64]
cGMP-binding capacity which could exist in vivo and which could function as a "buffer" for available intracellular cGMP. Our attempts to measure the binding of cGMP in the presence of Mg z+, at 37°, suggest a much higher binding capacity, but the lability of the bound cGMP leads to unreliable results. Walseth et al.,16 using direct photoaffinity labeling to identify the cGMP-binding entity, verified the occurrence of cGMP binding and PDE activity in platelets and also confirmed the general characteristics discussed above. Effect o f p H and PDE Inhibitors cGMP binding to the 30,000 g supernatant from rat or human platelet homogenates is undetectable at a pH below 6.5. Binding can be observed at an alkaline pH, with maximum activity being obtained at pH 9.0. One of the most distinctive characteristics of this binding is its stimulation by MIX and other PDE inhibitors (discussed later in this chapter). Maximal stimulation by PDE inhibitors is seen at a physiological pH (up to 24-fold at pH 7.5), whereas a lower response is recorded at a pH where binding activity is the highest (8.4-fold at pH 9.5). Subcellular Distribution o f cGMP Binding In contrast to the MIX-stimulated cGMP binding noted in the retina, where the bulk of binding is associated with the membrane fraction, at least in isotonic media, 17,~8almost 100% of the cGMP binding in homogehates of rat platelets is recovered in the 12,000 g supernatant fraction. After centrifugation at 105,000 g for 60 min, 94% of binding remains in the supernatant fraction with little change in specific activity. We have not yet been able to find any condition (presence or absence of divalent cation, variation in the isotonicity of the buffer, presence of sucrose, or different types of homogenization procedure) which would suggest an attachment to the membrane fraction, at least in rat platelets. Tissue Distribution of cGMP Binding As we 1,6 and Francis et al. 7 have reported, tissues other than platelets frequently display two types of cGMP-binding activity, one (dealt with here) typically stimulated by MIX, and the other representing cGMP16 T. F. Walseth, P. S. T. Yuen, S. S. Panter, and N. D. Goldberg, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1854 (1985). ~7R. E. Kohnken, D. M. Eadie, A. Revzin, and D. G. McConnell, J. Biol. Chem. 256, 12502 (1981). 18 p. N. Tyminski and D. F. O'Brien, Biochemistry 23, 3986 0984).
[64]
PLATELET cGMP-BINDING PHOSPHODIESTERASE
715
dependent protein kinase. Our initial study 6 illustrated an analogous elution profile from DEAE-Sepharose for MIX-stimulated binding proteins (eluting at 0.12 M NaC1) in platelets, lung, and smooth muscle cells. In addition to that observed in the lungs (discussed by Francis and Corbin [65] in this volume), Coquil et al. 8 recently detected MIX-stimulated binding in the spleen, cerebellum, brain, liver, skeletal muscle, and kidney. In several of these tissues (spleen, lung, and brain), the binding was of the same magnitude as that of cGMP-dependent protein kinase, which elutes with higher concentrations of NaC1 (0.16 M). Purification of cGMP Binding and Association with cGMP-PDE It should be noted that complete purification of cGMP-binding PDE has not yet been reported.
DEAE-Sepharose Chromatography As mentioned earlier, cGMP-binding was eluted as a single peak by a NaC1 gradient (0.12 M) from DEAE-Sepharose which showed cGMPPDE activity. Kinetic and specificity studies were performed with this partially purified material. At equilibrium (300 rain), the Scatchard plot was linear, and using linear regression analysis, Kd values of 353 -+_60 nM and 13.4 -+ 1.5 n M were determined, respectively, in the absence and presence of 2 m M MIX. cGMP binding to this material was specific, since GTP, GDP, GMP, guanosine, adenosine, and AMP in a 1000-fold excess did not demonstrate any competition with cGMP. In the absence of MIX, a 150- to 500-fold excess of cAMP inhibited cGMP binding but in its presence, as a consequence of a higher affinity for cGMP, a 5000-fold excess of cAMP was required for competition at the cGMP-binding site.
Molecular Size The molecular size of the eazyme was established in concentrated material from DEAE-Sepharose by sucrose gradient centrifugation (19 hr at 39,000 rpm in an SW-41 rotor). Both cGMP-PDE and cGMP binding demonstrated the same mobility. The sedimentation coefficient was 6.44S. Assuming a partial specific volume of 0.769 cm3/g, the molecular weight (176,000) and frictional ratio (fifo 1.48) were calculated.
MIX-Affinity Chromatography This method of purification was designed to benefit from the presence of a MIX-binding site on the protein. The column was prepared by cou-
716
P H O S P H O D I E S T E R AISOZYME SE METHODS
[64]
piing 8-carboxyethyl-MIX in dimethylformamide and l-cyclohexyl-3-(2morpholinoethyl)-O-carbodiimide metho-p-toluene sulfonate to Affi-Gel 102 (40 ml packed gel made up to 100 ml in H20, mixed with 100 ml of 20 mM xanthine derivative). The reaction proceeded for 24 hr while the pH was maintained at 4.7 with 0.1 N HC1. 6 The resulting xanthine affinity gel contained about 10/zmol of xanthine/ml packed gel. The material from DEAE-Sepharose was applied to this column (0.9 x 12 cm) and washed with 50 ml of 20 m M sodium phosphate, 2 mM EDTA, and 2 mM DTT (pH 7.0). It was then eluted with a KCI gradient from 0 to 300 mM in the same buffer. The cGMP-PDE and cGMP-binding were once again coeluted, and a purification of about 500-fold was achieved for the two activities.
Gel Filtration The material from MIX-Affi-Gel was further purified by molecular sieving on Sephadex G-200 and Sepharose 6B. Both activities followed the same elution profile and, using the method of Siegel and Monty, 19the Stokes radius of cGMP-binding PDE was estimated to be 5.6 nm. Modulation of cGMP-Binding PDE Activity in Vitro
Effect of PDE lnhibitors We have already mentioned that while PDE activity is fully inhibited by the presence of MIX, the xanthine increases the binding affinity for cGMP. Other PDE inhibitors display similar effects, the most potent stimulator being dipyridamol, which elicits an 8-fold stimulation of cGMP binding at 50/zM when PDE activity is already fully suppressed. The capacity to inhibit PDE and to stimulate cGMP binding actually appears to be two relatively independent properties: the stimulatory effect of these compounds starts to be significant at a concentration at which PDE is inhibited more than 50-80% and still increases even when the PDE activity is no longer detectable. This finding stresses the possibility that the binding and hydrolysis sites for cGMP are two distinct entities, each associated with separate properties of the holoenzyme.6,z° On the other hand, it is evident that while performing experiments on the binding affinity for cGMP with PDE inhibitors, care should be taken to avoid any PDE activity in the binding assay, since this would falsely exaggerate the apparent binding increase due to substrate preservation. ~9 L. M. Siegel and K. J. Monty, Biochim. Biophys. Acta 112, 346 (1966). z0 p. Hamet, J.-F. Coquil, S. Bousseau-Lafortune, D. J. Franks, and J. Tremblay, Adv. Cyclic Nucleotide Protein Phosphorylation Res. 16, 119 (1984).
[64]
PLATELET cGMP-BIND1NGPHOSPHODIESTERASE
717
Effect of Chaotropic Salts and Temperature Chaotropic salts (NaCI, KC1, KBr, and KI) have a stimulatory influence of cGMP binding, but exert no effect on PDE. This stimulation is maximal with KBr and impacts only on "basal" activity, i.e., in the absence of MIX. Preincubation of partially purified preparations at progressively increasing temperatures also evokes a differential effect on binding and hydrolytic activity. Preincubation at 45 ° has a slightly stimulatory effect on binding (but the influence of MIX is abolished, the Kd being about 100 nM with or without MIX), while it destroys PDE activity .2o
Effect of Fatty Acids cGMP-binding PDE from the rat lung, which is similar to the platelet enzyme, is reportedly inhibited in vitro by unsaturated fatty acids, whereas saturated fatty acids are ineffective. 21 The inhibitory potency of unsaturated 18- and 20-carbon fatty acids increases with the number of double bonds in the hydrocarbon chain. However, the methyl esters of unsaturated fatty acids and saturated fatty acids of variable chain length have no appreciable effect on PDE. The inhibition of cGMP-binding PDE by up to 30 t~M arachidonic acid is reversible and appears to occur at only one inhibitory site. The inhibition by arachidonic acid is competitive with a Ki of 20/x M. In contrast, unsaturated fatty acids progressively stimulate cGMP binding with 50 to 75 ~ M registering a maximal effect. Higher concentrations result in a loss of activation. Methyl esters have only a slight effect on cGMP binding. 22 These studies suggest that the inhibitory action on PDE activity and the stimulation of cGMP binding by unsaturated fatty acids are directly linked. The difference in the K, and K~ of arachidonic acid, which also exists for other competitive PDE inhibitors (MIX, papaverine), can be explained by dissimilarities in the methods used for measuring PDE and cGMP binding (_+Mg2+) but as previously mentioned it also suggests that the binding site for cGMP is distinct from the catalytic site.
Effect of Hematin, 11202, Mn2+, and Oxygen We have recently observed 23 an apparent oxygen-dependent modulation of cGMP-binding PDE activity in rat platelets. Exposure of the enzyme to oxygen (at 4°) for up to 60 rain increases its activity up to 3-fold, ~.t J.-F. Coquil, Biochim. Biophys. Acta 743, 359 (1983). 22 J.-F. Coquil, Ph.D. thesis, presented to Universit6 de Paris-Sud, November 30, 1984. 23 p. Hamet and J. Tremblay, Can. Fed. Biol. Soc. Proc. 27, 106 (1984).
718
PHOSPHODIESTERASE ISOZYMEMETHODS
[64]
cGMP-PHOSPHODIESTERASE
~
5
J
21::.
~,
2 t
1
I
I
E
L
J
cGMP-BINDING 23
/
7
3
21
&
i 0
-MIX
I
I
I
I
I
I
fO
20
30
40
50
60
TIME (rain) FIG. 1. Effect of oxygenation on cGMP-binding PDE. Platelet supernatants were kept on ice for 30 min in an atmosphere of 100% Nz, which was then changed to 95% 02, and 5% CO2. Incubation was continued for the time periods indicated.
as compared to the enzyme kept under Nz in degassed buffer. This activation is DTT and EDTA independent. A similar increase is also observed for cGMP binding, both in the absence and in the presence of MIX (Fig. 1). Preincubation of platelet supernatant with HzOzinhibits PDE activity at concentrations above 1/zM, but DTT largely prevents this effect (Fig. 2). Oz-Dependent activation is further enhanced by the presence of Mn z+ with up to 10-fold increases being achieved with concentrations above 100 ~ M or even when diluted in the assay to trace amounts (Fig. 3). Unfortunately, the effect of Mn z+ on binding cannot be properly evaluated due to the stimulation of PDE activity. Hematin inhibits PDE activity at concentrations from 1 to 50/zM, but this effect is apparent only in the presence of DTT.24 24 j. Tremblay and P. Hamet, in "Platelets in Biology and Pathology: III" (J. L. Gordon and D. E. Maclntyre, eds.). Elsevier/North-Holland, Amsterdam, p. 433 (1987).
[64]
719
PLATELET ¢ G M P - B I N D I N G PHOSPHODIESTERASE 2.5 2.0
,.s
/•*DTT
t...)
ua
~
1.0
0.5 DTT 10 -2
I
I
t
I
i0 -I
10 0
101
10 2
H202 (#t~)
FIG. 2. Effect of H202 on c G M P - P D E . The platelet supernatant (protein concentration = 100 /xg/ml) was preincubated at 4 ° for 1 hr with various concentrations of H202 in the a b s e n c e or presence of 1 m M DTT. The supernatant was diluted 10 times before the cGMPP D E assay.
These recent data suggest a redox modulation of cGMP-PDE activity but further studies have to be performed with more purified material to exclude indirect interactions. Of significance is the observation that cGMP-PDE activity can be "restored" after the addition of Mn 2+ to the 2.0 02 Q
t~
1.5
N
N2 e
=
0.5
I //
0
I
I
0.1
I
MnCl 2 (raM)
FIG. 3. Effect of MnCI2 on c G M P - P D E . The platelet supernatant (protein concentration = 100/~g/ml) was preincubated for 1 hr with various concentrations of MnC12 u n d e r an N2 or 02 a t m o s p h e r e . T h e results are the m e a n of three experiments.
720
PHOSPHODIESTERASE ISOZYME METHODS
[64]
TABLE I RECOVERYOF cGMP-PDE ACTIVITYWITH MnClfl cGMP-PDE activity (nmol cGMP hydrolyzed/mg protein/rain) Enzyme frozen at -70 ° for 4 months Dialysis
Fresh enzyme
Before After
3.0 1.2
-MnC12 0.5 0.3
+MnCIe 6.5 6,0
" The protein fraction used was a pooled eluate (0.12 M NaCI) from DEAE-Sepharose. cGMP-PDE activity was measured before and after dialysis against 10 mM potassium phosphate buffer (pH 7.0) containing 0.5 mM EDTA and ! mM DTT. An aliquot was taken before dialysis, frozen immediately in liquid nitrogen and kept at -70 ° for 4 months. The cGMPPDE activity of this fraction was measured 4 months later, before and after dialysis, in the absence or presence of 100/zM MnCI2.
stored material, which otherwise appears to lose over 80% of its activity (Table I). M o d u l a t i o n of c G M P - B i n d i n g P D E e x V i v o B y e x v i v o m o d u l a t i o n , w e r e f e r to the p r e i n c u b a t i o n o f intact p l a t e l e t s with a g e n t s w h i c h , a f t e r p l a t e l e t w a s h i n g , results in a l t e r e d P D E and b i n d i n g a c t i v i t i e s . It is h y p o t h e s i z e d that this m o d u l a t o r y e f f e c t m a y be r e l e v a n t to the in v i v o situation.
Effect of MIX and PGE1 We have reported earlier that maximal stimulation of cGMP-PDE activity occurs after 30 sec of exposure of intact platelets to the combined presence of MIX and PGEI.25 A similar effect is observed for cGMP binding and PDE activity with MIX and forskolin. This preactivation is 2s p. Hamet, D. J. Franks, J. Tremblay, and J.-F. Coquil, Can. J. Biochem. Cell Biol. 61, 1158 (1983).
[64]
PLATELEXcGMP-BIND1NG PHOSPHODIESTERASE
721
relatively stable and persists even after DEAE-Sepharose or Bio-Sil TSK250 HPLC. 26 Possible M e c h a n i s m o f cGMP-Binding P D E Activation
In a recent study, we examined the possible mechanism of this ex vivo activation. 26 The stimulatory effect of forskolin on cGMP-PDE was correlated with the concomitant dissociation of cAMP-dependent protein kinase but was further enhanced by the presence of MIX without any additional dissociation of the protein kinase. The in vitro reconstitution of phosphorylating conditions (Mg 2+, ATP, catalytic subunit of cAMP-dependent protein kinase) imitated the stimulatory effect of forskolin and MIX added e x p i v o . 26 These observations led us to propose that cGMPbinding PDE can be modulated by cAMP-dependent phosphorylation. It is also conceivable that MIX increases PDE susceptibility to phosphorylation. It should be mentioned, however, that this activation can be achieved only with crude preparations, and not with phosphorylation of the partially purified material. Other than the possibility of indirect interactions, these findings suggest the presence of a regulatory subunit, rapidly lost during the initial purification steps and contributing to the difficulty encountered with the purification of this enzyme. The relevance of the ex vivo stimulation for the in vivo situation seems to be probable since Coquil (personal communication, 1986) has recently observed that exposure of platelets to MIX leads to an increased binding of endogenous cGMP to PDE as demonstrated by rapid filtration and ammonium sulfate precipitation after platelet homogenization. Conclusions One of the most intriguing aspects of cGMP-binding PDE is its possible involvement in the expression of cGMP's function, cGMP-dependent protein kinase has been convincingly demonstrated in membrane fractions of human and rat platelets, 27 but cGMP-binding PDE represents the major, if not the only, binding activity in platelet cytosol. Since the basal concentration of cGMP is around 4 pmol. mg protein ~ and the binding observed in supernatant is around 2-4 pmol. mg protein -l, it can potentially serve as a significant buffer for cGMP. Moreover, this binding activ26 j. Tremblay, B. Lachance, and P. Hamet, J. Cyclic Nucleotide Protein Phosphorylation Res. 10, 397 (1985). 27 R. Waldmann, S. Bauer, C. Gobel, F. Hofmann, K. H. Jakobs. and U. Walter, Eur. J. Biochem. 158, 203 (1986).
722
PHOSPHODIESTERASE
ISOZYME
METHODS
[65]
ity may be higher under in vivo conditions (higher temperature and the presence of cations) and may be stimulated by various agents and conditions described above. For these reasons, both binding and hydrolysis may be involved in the control of the physiological function of cGMP. Acknowledgments The authors acknowledge the help of Micheline Caron, Louise Chevrefil8, and Bruno Lachance in the preparation of this manuscript. This study was supported by a grant from MRC Group grant to the Multidisciplinary Group on Hypertension and FRSQ.
[65] P u r i f i c a t i o n o f c G M P - B i n d i n g P r o t e i n Phosphodiesterase from Rat Lung
By SHARRON H. FRANCIS and JACKIE D. CORBIN Rat lung tissue contains a cGMP phosphodiesterase which copurifies with a cGMP-binding activity and has thus been designated cGMP-binding protein phosphodiesterase. L: This cGMP-binding activity is clearly distinct from the cGMP-dependent protein kinase. The cGMP analog specificity at the binding site differs from the analog specificity of either the catalytic site of the associated cGMP phosphodiesterase or the cGMP kinase binding site.l The purification of this protein from rat lung is described below. Assays
cGMP Phosphodiesterase Assay The phosphodiesterase assay contains 50 mM Tris-HC1, pH 7.5, 10 mM magnesium chloride, 0.33 mg/ml metal-free bovine serum albumin, 10-20 tzM unlabeled cGMP, and 33 nM [3H]cGMP (-600,000 cpm) in a final volume of 150 /zl. 3 The assays are initiated by the addition of the enzyme diluted in 20 mM sodium phosphate buffer, pH 6.8, 2 mM EDTA, and 25 mM 2-mercaptoethanol. The assay mixture is incubated at 30° for 1 S. H. Francis, T. M. Lincoln, and J. D. Corbin, J. Biol. Chem. 255, 620 (1980). 2 p. Hamet and J. F. Coquil, J. Cyclic Nucleotide Res. 4, 281 (1978). 3 j. N. Wells, C. E. Baird, Y. J. Wu, and J. G. Hardman, Biochim. Biophys. Acta 384, 430 (1975).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
P H O S P H O D I E S T E R A S E ISOZYME M E T H O D S
[64]
We find that a TSK-250 column (0.75 × 60 cm) purchased from Bio-Rad has better resolution for PDE purification. The concentrated crude PDE (330/zl) is applied to a TSK-250 column (0.75 × 60 cm). The column has been equilibrated with buffer E. The chromatographic separation is carried out at a flow rate of 1 ml/min. The volume of each fraction is 0.5 ml/ tube. PDE activity is assayed as previously described.l~ A typical elution profile of the purified PDE is shown in Fig. 3. The purity of the pooled fractions (27 through 29) is examined by SDS-polyacrylamide slab gel electrophoresis and protein bands are stained with Coomassie blue (Fig. 4). The specific activity of the purified PDE is measured as 1100 mol of cGMP/mol of PDE/min, in the absence of any activating factors. When this assay is repeated in the presence of amphibian Ga/GTPTS [guanosine 5'-O-(3-thiotriphosphate)] it is found that the specific activity is then 11,000 mol of cGMP/mol of PDE/min, suggesting that the PDE purified by this methodology retains a significant amount of endogenous inhibitory activity presumably associated with the 13K subunit. Acknowledgment Los Alamos National Laboratory is operated by the University of California for the United States Department of Energy under contract W-7405-ENG-36. N W. J. Thompson and M. M. Appleman, Biochemistry 10, 311 (1971).
[64] P l a t e l e t c G M P - B i n d i n g P h o s p h o d i e s t e r a s e
By PAVEL HAMET and JOHANNE TREMBLAY Introduction It is generally accepted that at least a significant part of cGMP's function in the cell is expressed via its interaction with cGMP-dependent protein kinase, usually detected as a "binding" of the nucleotide. On the other hand, some tissues which appear to have reasonably well-modulated levels in relation to cell function do not present detectable cytosolic cGMP binding corresponding to cGMP-dependent protein kinase. Significant binding of a different type, one that is related to the activity of METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
[64]
PLATELET cGMP-BINDINGPHOSPHODIESTERASE
711
cGMP-phosphodiesterase (PDE), has been observed in these tissues, which include platelets, the retina, and sea urchin sperm.l-3 At present, it is not known how this "significant binding" is implicated in the expression of cGMP's function. Goldberg e t al. 4 proposed that the function of cGMP may be expressed via its rapid and massive turnover leading to proton generation. This notion has been explored particularly in relation to sodium fluxes and the transmission of the light signal in the retina. Recently, interest in the regulation of cGMP hydrolysis has been renewed by the discovery of Fesenko e t al. 5 of a direct effect of cGMP on the regulation of the sodium channel in the retina. Although the direct action of cGMP on ionic fluxes has not yet been investigated in other systems, this notion needs to be examined in tissues such as platelets. Our earlier experiments on platelets and lungs 1,6 uncovered a cGMPbinding PDE distinct from other forms of cGMP-PDE such as calmodulinsensitive and cGMP-stimulated PDE. This protein was later independently noted by Francis e t al. 7 in the lung, and more recently by Coquil e t al. 8 in several other tissues. Similarly, at least as far as the potential "buffering capacity" of cGMP is concerned, an analogous type of PDE has been found in the retina, in which GMP-PDE also has a binding site for this nucleotide. 9 However, more recent studies have set apart retinal PDE as a distinctly regulated enzyme, immunologically and functionally different from the platelet enzyme, 1°,11and described in Chapter 63 in this volume. In the present chapter, we will deal specifically with cGMPbinding PDE from platelets. We will discuss the methods used for determination of the hydrolytic and binding activities of this enzyme, its partial purification, as well as modulation of both binding and enzymatic activities by agonists in v i t r o and e x v i v o . t p. Hamet and J.-F. Coquil, J. Cyclic Nucleotide Res. 4, 281 (1978). 2 j. S. Pober and M. W. Bitensky, Adv. Cyclic Nucleotide Res. 11, 265 (1979). 3 D. L. Garbers and G. S. Kopf, Adv. Cyclic Nucleotide Res. 13, 251(1980). 4 N. D. Goldberg, A. Ames III, J. E. Gander, and T. F. Walseth, J. Biol. Chem. 258, 9213 (1983). 5 E. E. Fesenko, S. S. Kolesnikov, and A. L. Lyubarsky, Nature (London) 313, 310 (1985). 6 J.-F. Coquil, D. J. Franks, J. N. Wells, M. Dupuis, and P. Hamet, Biochim. Biophys. Acta 631, 148 (1980). 7 S. H. Francis, T. M. Lincoln, and J. D. Corbin, J. Biol. Chem. 255, 620 (1980). 8 J.-F. Coquil, G. Brunelle, and J. Guedon, Biochem. Biophys. Res. Commun. 127, 226 (1985). 9 A. Yamazaki, I. Sen, and M. W. Bitensky, J. Biol. Chem. 255, 11619 (1980). 10 L. Stryer, Cold Spring Harbor Symp. Quant. Biol. 48, 841 (1983). 1~ R. L. Hurwitz, A. H. Bunt-Milam, M. L. Chang, and J. A. Beavo, J. Biol. Chem. 260, 568 (1985).
712
P H O S P H O D I E S T E R AISOZYME SE METHODS
[64]
Methods of Detection and Basic Characteristics of cGMP-Binding and PDE Activity
Standard Conditions for Measurement of cGMP Binding The standard conditions which we usually employ for cGMP-binding determinations are derived from those originally described by Gilman 12 and Lincoln et al. ~3The standard reaction mixture contains 30 mM Bicine (pH 9.0), 0.1 # M [3H]cGMP and 0.5 mg/ml bovine serum albumin in a final volume of 0.1 ml. Incubation is started with the addition of a cGMPbinding protein preparation containing 2 mM EDTA (the final concentration should be at least 0.4 mM) and 2 mM dithiothreitol (DTT). The duration of incubation is usually 90 min at 4°. At the end of the incubation period, 2 ml of a cold 20 mM potassium phosphate buffer (pH 7.0) is added, and the solution is poured onto Millipore or Gelman nitrocellulose filters (0.45 tzm), previously immersed in the same buffer, to separate free and bound [3H]cGMP. The tubes are rinsed with 4 ml of 20 mM potassium phosphate buffer, and the filters are washed with 30 ml of the same buffer. The filters are then dissolved in 2 ml of Cellosolve and 10 ml of scintillation liquid is added for the radioactivity determinations. Blank values are ascertained by performing the incubations without samples or by incubation with the binding protein preparation at time zero. Identical values (less than 50 cpm) are obtained with the two methods. The filters can also be washed with 20 m M Bicine buffer (pH 9.0), instead of potassium phosphate, and similar results are achieved. The data are expressed in picomoles [3H]cGMP bound per milligram protein or per milliliter of the binding protein preparation. An alternative procedure for the separation of bound and free cGMP molecules has been designed, using ammonium sulfate precipitation. At the conclusion of incubation, 1 ml of 70% ammonium sulfate is added to each tube. The contents of the tubes are then mixed, incubated for 20 min at 4°, and centrifuged at 3000 g for 15 rain. The supernatant is decanted, and a further 1 ml of ammonium sulfate is added to the precipitate with the procedure being repeated once more. Radioactivity in the dissolved pellet is counted after the addition of I ml of H20. Qualitative and quantitative correlation of the filtration and precipitation methods is excellent with r 2 = 0.94 (p <0.001) for 15 random, duplicate samples, the same degree of stimulation of activity by various agents, such as l-methyl-3-isobutylxanthine (MIX), is observed with the two methods. 6 However, due to greater 12 A. G. Gilman, Proc. Natl. Acad. Sci. U.S.A. 67, 305 (1970). 13 T. M. Lincoln, C. L. Hall, C. R. Park, and J. D. Corbin, Proc. Natl. Acad. Sci. U.S.A. 73, 2559 (1976).
[64]
PLATELETcGMP-BINDING PHOSPHODIESTERASE
713
variability, the samples separated by the precipitation methods are run in quadruplicate. Thus, for most purposes, the filtration procedure appears to be more suitable, yet the precipitation method can be used for the screening of binding in large-scale preparations, such as in eluates from column chromatography.
Standard Conditions for Measurement of cGMP-PDE This procedure is based of the method of Beavo et al. 14 and Wells et al. ~5 The assay mixture contains 60 mM TES, 3 mM MgCI2, 0.20 mg bovine serum albumin, 1/zM cGMP (unless otherwise stated), and 20-30 nCi of [3H]cGMP in a total volume of 200/xl. The reaction is started by the addition of 25/zl of enzyme preparation suitably diluted so that less than 20% of radioactive substrate is hydrolyzed when incubated at 30° for 30 min. The incubation is terminated by the addition of 25/zl of a reaction mixture containing 50 mM EDTA, 5 mM MIX, 15 mM cGMP, and 9 mM 5'-GMP. Twenty-five microliters of snake venom (Crotalus atrox, 2 mg/ ml) is then added, and the mixture incubated for a further 10 min at 30°. Incubation is stopped by the addition of 750 t~l of a solution containing 0.1 mM guanosine. The guanosine formed is separated from the nucleotides on QAE-Sephadex A-25 (formate form). ~5 In order to routinely verify both the 5'-nucleotidase step and the column recovery, we run the "100% control" of undiluted PDE samples to ensure the complete conversion of 5'-GMP to guanosine. The radiolabeled cyclic nucleotide should be kept at -70 ° to decrease 3H exchange w i t h H 2 0 . Periodically, the labeled [3H]cGMP stock is purified to reduce contamination by [3H]20, which is not separated at the last column step.
Stability o f ff H]cGMP in Binding Assay With the standard conditions employed for cGMP binding, the stability of the radiolabeled cGMP estimated by thin-layer chromatography on polyethyleneimine cellulose plates is over 99.4% of the original material added either in the absence or in the presence of MIX. 6 On the other hand, the stability of cGMP requires the presence of a chelator and incubation at lower temperatures. Thus, the conditions for the cGMP binding and PDE assays differ significantly with the presence or absence of free divalent cations as well as the incubation temperatures. The conditions used for studying cGMP binding could lead to underestimation of the 14 j. A. Beavo, J. G. Hardman, and E. W. Sutherland, J. Biol. Chem. 245, 5649 (1970). ~5j. N. Wells, C. E. Baird, Y. J. Wu, and J. G. Hardman, Biochim. Biophys. Acta 384, 430 (1975).
714
P H O S P H O D I E S T E R AISOZYME SE METHODS
[64]
cGMP-binding capacity which could exist in vivo and which could function as a "buffer" for available intracellular cGMP. Our attempts to measure the binding of cGMP in the presence of Mg z+, at 37°, suggest a much higher binding capacity, but the lability of the bound cGMP leads to unreliable results. Walseth et al.,16 using direct photoaffinity labeling to identify the cGMP-binding entity, verified the occurrence of cGMP binding and PDE activity in platelets and also confirmed the general characteristics discussed above. Effect o f p H and PDE Inhibitors cGMP binding to the 30,000 g supernatant from rat or human platelet homogenates is undetectable at a pH below 6.5. Binding can be observed at an alkaline pH, with maximum activity being obtained at pH 9.0. One of the most distinctive characteristics of this binding is its stimulation by MIX and other PDE inhibitors (discussed later in this chapter). Maximal stimulation by PDE inhibitors is seen at a physiological pH (up to 24-fold at pH 7.5), whereas a lower response is recorded at a pH where binding activity is the highest (8.4-fold at pH 9.5). Subcellular Distribution o f cGMP Binding In contrast to the MIX-stimulated cGMP binding noted in the retina, where the bulk of binding is associated with the membrane fraction, at least in isotonic media, 17,~8almost 100% of the cGMP binding in homogehates of rat platelets is recovered in the 12,000 g supernatant fraction. After centrifugation at 105,000 g for 60 min, 94% of binding remains in the supernatant fraction with little change in specific activity. We have not yet been able to find any condition (presence or absence of divalent cation, variation in the isotonicity of the buffer, presence of sucrose, or different types of homogenization procedure) which would suggest an attachment to the membrane fraction, at least in rat platelets. Tissue Distribution of cGMP Binding As we 1,6 and Francis et al. 7 have reported, tissues other than platelets frequently display two types of cGMP-binding activity, one (dealt with here) typically stimulated by MIX, and the other representing cGMP16 T. F. Walseth, P. S. T. Yuen, S. S. Panter, and N. D. Goldberg, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1854 (1985). ~7R. E. Kohnken, D. M. Eadie, A. Revzin, and D. G. McConnell, J. Biol. Chem. 256, 12502 (1981). 18 p. N. Tyminski and D. F. O'Brien, Biochemistry 23, 3986 0984).
[64]
PLATELET cGMP-BINDING PHOSPHODIESTERASE
715
dependent protein kinase. Our initial study 6 illustrated an analogous elution profile from DEAE-Sepharose for MIX-stimulated binding proteins (eluting at 0.12 M NaC1) in platelets, lung, and smooth muscle cells. In addition to that observed in the lungs (discussed by Francis and Corbin [65] in this volume), Coquil et al. 8 recently detected MIX-stimulated binding in the spleen, cerebellum, brain, liver, skeletal muscle, and kidney. In several of these tissues (spleen, lung, and brain), the binding was of the same magnitude as that of cGMP-dependent protein kinase, which elutes with higher concentrations of NaC1 (0.16 M). Purification of cGMP Binding and Association with cGMP-PDE It should be noted that complete purification of cGMP-binding PDE has not yet been reported.
DEAE-Sepharose Chromatography As mentioned earlier, cGMP-binding was eluted as a single peak by a NaC1 gradient (0.12 M) from DEAE-Sepharose which showed cGMPPDE activity. Kinetic and specificity studies were performed with this partially purified material. At equilibrium (300 rain), the Scatchard plot was linear, and using linear regression analysis, Kd values of 353 -+_60 nM and 13.4 -+ 1.5 n M were determined, respectively, in the absence and presence of 2 m M MIX. cGMP binding to this material was specific, since GTP, GDP, GMP, guanosine, adenosine, and AMP in a 1000-fold excess did not demonstrate any competition with cGMP. In the absence of MIX, a 150- to 500-fold excess of cAMP inhibited cGMP binding but in its presence, as a consequence of a higher affinity for cGMP, a 5000-fold excess of cAMP was required for competition at the cGMP-binding site.
Molecular Size The molecular size of the eazyme was established in concentrated material from DEAE-Sepharose by sucrose gradient centrifugation (19 hr at 39,000 rpm in an SW-41 rotor). Both cGMP-PDE and cGMP binding demonstrated the same mobility. The sedimentation coefficient was 6.44S. Assuming a partial specific volume of 0.769 cm3/g, the molecular weight (176,000) and frictional ratio (fifo 1.48) were calculated.
MIX-Affinity Chromatography This method of purification was designed to benefit from the presence of a MIX-binding site on the protein. The column was prepared by cou-
716
P H O S P H O D I E S T E R AISOZYME SE METHODS
[64]
piing 8-carboxyethyl-MIX in dimethylformamide and l-cyclohexyl-3-(2morpholinoethyl)-O-carbodiimide metho-p-toluene sulfonate to Affi-Gel 102 (40 ml packed gel made up to 100 ml in H20, mixed with 100 ml of 20 mM xanthine derivative). The reaction proceeded for 24 hr while the pH was maintained at 4.7 with 0.1 N HC1. 6 The resulting xanthine affinity gel contained about 10/zmol of xanthine/ml packed gel. The material from DEAE-Sepharose was applied to this column (0.9 x 12 cm) and washed with 50 ml of 20 m M sodium phosphate, 2 mM EDTA, and 2 mM DTT (pH 7.0). It was then eluted with a KCI gradient from 0 to 300 mM in the same buffer. The cGMP-PDE and cGMP-binding were once again coeluted, and a purification of about 500-fold was achieved for the two activities.
Gel Filtration The material from MIX-Affi-Gel was further purified by molecular sieving on Sephadex G-200 and Sepharose 6B. Both activities followed the same elution profile and, using the method of Siegel and Monty, 19the Stokes radius of cGMP-binding PDE was estimated to be 5.6 nm. Modulation of cGMP-Binding PDE Activity in Vitro
Effect of PDE lnhibitors We have already mentioned that while PDE activity is fully inhibited by the presence of MIX, the xanthine increases the binding affinity for cGMP. Other PDE inhibitors display similar effects, the most potent stimulator being dipyridamol, which elicits an 8-fold stimulation of cGMP binding at 50/zM when PDE activity is already fully suppressed. The capacity to inhibit PDE and to stimulate cGMP binding actually appears to be two relatively independent properties: the stimulatory effect of these compounds starts to be significant at a concentration at which PDE is inhibited more than 50-80% and still increases even when the PDE activity is no longer detectable. This finding stresses the possibility that the binding and hydrolysis sites for cGMP are two distinct entities, each associated with separate properties of the holoenzyme.6,z° On the other hand, it is evident that while performing experiments on the binding affinity for cGMP with PDE inhibitors, care should be taken to avoid any PDE activity in the binding assay, since this would falsely exaggerate the apparent binding increase due to substrate preservation. ~9 L. M. Siegel and K. J. Monty, Biochim. Biophys. Acta 112, 346 (1966). z0 p. Hamet, J.-F. Coquil, S. Bousseau-Lafortune, D. J. Franks, and J. Tremblay, Adv. Cyclic Nucleotide Protein Phosphorylation Res. 16, 119 (1984).
[64]
PLATELET cGMP-BIND1NGPHOSPHODIESTERASE
717
Effect of Chaotropic Salts and Temperature Chaotropic salts (NaCI, KC1, KBr, and KI) have a stimulatory influence of cGMP binding, but exert no effect on PDE. This stimulation is maximal with KBr and impacts only on "basal" activity, i.e., in the absence of MIX. Preincubation of partially purified preparations at progressively increasing temperatures also evokes a differential effect on binding and hydrolytic activity. Preincubation at 45 ° has a slightly stimulatory effect on binding (but the influence of MIX is abolished, the Kd being about 100 nM with or without MIX), while it destroys PDE activity .2o
Effect of Fatty Acids cGMP-binding PDE from the rat lung, which is similar to the platelet enzyme, is reportedly inhibited in vitro by unsaturated fatty acids, whereas saturated fatty acids are ineffective. 21 The inhibitory potency of unsaturated 18- and 20-carbon fatty acids increases with the number of double bonds in the hydrocarbon chain. However, the methyl esters of unsaturated fatty acids and saturated fatty acids of variable chain length have no appreciable effect on PDE. The inhibition of cGMP-binding PDE by up to 30 t~M arachidonic acid is reversible and appears to occur at only one inhibitory site. The inhibition by arachidonic acid is competitive with a Ki of 20/x M. In contrast, unsaturated fatty acids progressively stimulate cGMP binding with 50 to 75 ~ M registering a maximal effect. Higher concentrations result in a loss of activation. Methyl esters have only a slight effect on cGMP binding. 22 These studies suggest that the inhibitory action on PDE activity and the stimulation of cGMP binding by unsaturated fatty acids are directly linked. The difference in the K, and K~ of arachidonic acid, which also exists for other competitive PDE inhibitors (MIX, papaverine), can be explained by dissimilarities in the methods used for measuring PDE and cGMP binding (_+Mg2+) but as previously mentioned it also suggests that the binding site for cGMP is distinct from the catalytic site.
Effect of Hematin, 11202, Mn2+, and Oxygen We have recently observed 23 an apparent oxygen-dependent modulation of cGMP-binding PDE activity in rat platelets. Exposure of the enzyme to oxygen (at 4°) for up to 60 rain increases its activity up to 3-fold, ~.t J.-F. Coquil, Biochim. Biophys. Acta 743, 359 (1983). 22 J.-F. Coquil, Ph.D. thesis, presented to Universit6 de Paris-Sud, November 30, 1984. 23 p. Hamet and J. Tremblay, Can. Fed. Biol. Soc. Proc. 27, 106 (1984).
718
PHOSPHODIESTERASE ISOZYMEMETHODS
[64]
cGMP-PHOSPHODIESTERASE
~
5
J
21::.
~,
2 t
1
I
I
E
L
J
cGMP-BINDING 23
/
7
3
21
&
i 0
-MIX
I
I
I
I
I
I
fO
20
30
40
50
60
TIME (rain) FIG. 1. Effect of oxygenation on cGMP-binding PDE. Platelet supernatants were kept on ice for 30 min in an atmosphere of 100% Nz, which was then changed to 95% 02, and 5% CO2. Incubation was continued for the time periods indicated.
as compared to the enzyme kept under Nz in degassed buffer. This activation is DTT and EDTA independent. A similar increase is also observed for cGMP binding, both in the absence and in the presence of MIX (Fig. 1). Preincubation of platelet supernatant with HzOzinhibits PDE activity at concentrations above 1/zM, but DTT largely prevents this effect (Fig. 2). Oz-Dependent activation is further enhanced by the presence of Mn z+ with up to 10-fold increases being achieved with concentrations above 100 ~ M or even when diluted in the assay to trace amounts (Fig. 3). Unfortunately, the effect of Mn z+ on binding cannot be properly evaluated due to the stimulation of PDE activity. Hematin inhibits PDE activity at concentrations from 1 to 50/zM, but this effect is apparent only in the presence of DTT.24 24 j. Tremblay and P. Hamet, in "Platelets in Biology and Pathology: III" (J. L. Gordon and D. E. Maclntyre, eds.). Elsevier/North-Holland, Amsterdam, p. 433 (1987).
[64]
719
PLATELET ¢ G M P - B I N D I N G PHOSPHODIESTERASE 2.5 2.0
,.s
/•*DTT
t...)
ua
~
1.0
0.5 DTT 10 -2
I
I
t
I
i0 -I
10 0
101
10 2
H202 (#t~)
FIG. 2. Effect of H202 on c G M P - P D E . The platelet supernatant (protein concentration = 100 /xg/ml) was preincubated at 4 ° for 1 hr with various concentrations of H202 in the a b s e n c e or presence of 1 m M DTT. The supernatant was diluted 10 times before the cGMPP D E assay.
These recent data suggest a redox modulation of cGMP-PDE activity but further studies have to be performed with more purified material to exclude indirect interactions. Of significance is the observation that cGMP-PDE activity can be "restored" after the addition of Mn 2+ to the 2.0 02 Q
t~
1.5
N
N2 e
=
0.5
I //
0
I
I
0.1
I
MnCl 2 (raM)
FIG. 3. Effect of MnCI2 on c G M P - P D E . The platelet supernatant (protein concentration = 100/~g/ml) was preincubated for 1 hr with various concentrations of MnC12 u n d e r an N2 or 02 a t m o s p h e r e . T h e results are the m e a n of three experiments.
720
PHOSPHODIESTERASE ISOZYME METHODS
[64]
TABLE I RECOVERYOF cGMP-PDE ACTIVITYWITH MnClfl cGMP-PDE activity (nmol cGMP hydrolyzed/mg protein/rain) Enzyme frozen at -70 ° for 4 months Dialysis
Fresh enzyme
Before After
3.0 1.2
-MnC12 0.5 0.3
+MnCIe 6.5 6,0
" The protein fraction used was a pooled eluate (0.12 M NaCI) from DEAE-Sepharose. cGMP-PDE activity was measured before and after dialysis against 10 mM potassium phosphate buffer (pH 7.0) containing 0.5 mM EDTA and ! mM DTT. An aliquot was taken before dialysis, frozen immediately in liquid nitrogen and kept at -70 ° for 4 months. The cGMPPDE activity of this fraction was measured 4 months later, before and after dialysis, in the absence or presence of 100/zM MnCI2.
stored material, which otherwise appears to lose over 80% of its activity (Table I). M o d u l a t i o n of c G M P - B i n d i n g P D E e x V i v o B y e x v i v o m o d u l a t i o n , w e r e f e r to the p r e i n c u b a t i o n o f intact p l a t e l e t s with a g e n t s w h i c h , a f t e r p l a t e l e t w a s h i n g , results in a l t e r e d P D E and b i n d i n g a c t i v i t i e s . It is h y p o t h e s i z e d that this m o d u l a t o r y e f f e c t m a y be r e l e v a n t to the in v i v o situation.
Effect of MIX and PGE1 We have reported earlier that maximal stimulation of cGMP-PDE activity occurs after 30 sec of exposure of intact platelets to the combined presence of MIX and PGEI.25 A similar effect is observed for cGMP binding and PDE activity with MIX and forskolin. This preactivation is 2s p. Hamet, D. J. Franks, J. Tremblay, and J.-F. Coquil, Can. J. Biochem. Cell Biol. 61, 1158 (1983).
[64]
PLATELEXcGMP-BIND1NG PHOSPHODIESTERASE
721
relatively stable and persists even after DEAE-Sepharose or Bio-Sil TSK250 HPLC. 26 Possible M e c h a n i s m o f cGMP-Binding P D E Activation
In a recent study, we examined the possible mechanism of this ex vivo activation. 26 The stimulatory effect of forskolin on cGMP-PDE was correlated with the concomitant dissociation of cAMP-dependent protein kinase but was further enhanced by the presence of MIX without any additional dissociation of the protein kinase. The in vitro reconstitution of phosphorylating conditions (Mg 2+, ATP, catalytic subunit of cAMP-dependent protein kinase) imitated the stimulatory effect of forskolin and MIX added e x p i v o . 26 These observations led us to propose that cGMPbinding PDE can be modulated by cAMP-dependent phosphorylation. It is also conceivable that MIX increases PDE susceptibility to phosphorylation. It should be mentioned, however, that this activation can be achieved only with crude preparations, and not with phosphorylation of the partially purified material. Other than the possibility of indirect interactions, these findings suggest the presence of a regulatory subunit, rapidly lost during the initial purification steps and contributing to the difficulty encountered with the purification of this enzyme. The relevance of the ex vivo stimulation for the in vivo situation seems to be probable since Coquil (personal communication, 1986) has recently observed that exposure of platelets to MIX leads to an increased binding of endogenous cGMP to PDE as demonstrated by rapid filtration and ammonium sulfate precipitation after platelet homogenization. Conclusions One of the most intriguing aspects of cGMP-binding PDE is its possible involvement in the expression of cGMP's function, cGMP-dependent protein kinase has been convincingly demonstrated in membrane fractions of human and rat platelets, 27 but cGMP-binding PDE represents the major, if not the only, binding activity in platelet cytosol. Since the basal concentration of cGMP is around 4 pmol. mg protein ~ and the binding observed in supernatant is around 2-4 pmol. mg protein -l, it can potentially serve as a significant buffer for cGMP. Moreover, this binding activ26 j. Tremblay, B. Lachance, and P. Hamet, J. Cyclic Nucleotide Protein Phosphorylation Res. 10, 397 (1985). 27 R. Waldmann, S. Bauer, C. Gobel, F. Hofmann, K. H. Jakobs. and U. Walter, Eur. J. Biochem. 158, 203 (1986).
722
PHOSPHODIESTERASE
ISOZYME
METHODS
[65]
ity may be higher under in vivo conditions (higher temperature and the presence of cations) and may be stimulated by various agents and conditions described above. For these reasons, both binding and hydrolysis may be involved in the control of the physiological function of cGMP. Acknowledgments The authors acknowledge the help of Micheline Caron, Louise Chevrefil8, and Bruno Lachance in the preparation of this manuscript. This study was supported by a grant from MRC Group grant to the Multidisciplinary Group on Hypertension and FRSQ.
[65] P u r i f i c a t i o n o f c G M P - B i n d i n g P r o t e i n Phosphodiesterase from Rat Lung
By SHARRON H. FRANCIS and JACKIE D. CORBIN Rat lung tissue contains a cGMP phosphodiesterase which copurifies with a cGMP-binding activity and has thus been designated cGMP-binding protein phosphodiesterase. L: This cGMP-binding activity is clearly distinct from the cGMP-dependent protein kinase. The cGMP analog specificity at the binding site differs from the analog specificity of either the catalytic site of the associated cGMP phosphodiesterase or the cGMP kinase binding site.l The purification of this protein from rat lung is described below. Assays
cGMP Phosphodiesterase Assay The phosphodiesterase assay contains 50 mM Tris-HC1, pH 7.5, 10 mM magnesium chloride, 0.33 mg/ml metal-free bovine serum albumin, 10-20 tzM unlabeled cGMP, and 33 nM [3H]cGMP (-600,000 cpm) in a final volume of 150 /zl. 3 The assays are initiated by the addition of the enzyme diluted in 20 mM sodium phosphate buffer, pH 6.8, 2 mM EDTA, and 25 mM 2-mercaptoethanol. The assay mixture is incubated at 30° for 1 S. H. Francis, T. M. Lincoln, and J. D. Corbin, J. Biol. Chem. 255, 620 (1980). 2 p. Hamet and J. F. Coquil, J. Cyclic Nucleotide Res. 4, 281 (1978). 3 j. N. Wells, C. E. Baird, Y. J. Wu, and J. G. Hardman, Biochim. Biophys. Acta 384, 430 (1975).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.