- Email: [email protected]
[62] Isolation and comparison of bovine heart cGMP-inhibited and cGMP-stimulated phosphodiesterases
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
685
corticoids appear to exert effects via the low-Km cAMP isoenzyrne. 9 Similarly, FSH can induce a state of refractoriness in the Sertoli cell by enhancing the activity of the cAMP-dependent isoform. 1° Thus, the link between calcium, calmodulin, and cGMP hydrolysis and the involvement of this system in hormone action requires clarification. Before this can be achieved more research in the role of cGMP in hormone action is necessary. Ion-exchange chromatography of tissue cytosols combined with the use of calmodulin and micromolar concentrations of substrate provide a convenient means of mapping the various l o w - K i n isoforms of cyclic nucleotide phosphodiesterase in tissue cytosols. However, to exclude the possibility that these isoforms are not simply degradation products and therefore artifacts, differences in kinetics, thermal stability, molecular mass, and the responses to calmodulin and drugs of these isolated enzymes should be sought. 9 W. J. Thompson and S. J. Strada, Recept. Horm. Action 3, 553. ~0 M. Conti, M. V. Toscano, L. Petrelli, R. Geremia, and M. Stephanini, Endocrinology 113, 1845 (1983).
[62] I s o l a t i o n a n d C o m p a r i s o n o f B o v i n e H e a r t cGMP-Inhibited and cGMP-Stimulated Phosphodiesterases
By S. A. HARRISON, N. BEIER, T. J. MARTINS, and J. A. BEAVO Introduction Most tissues and cells contain mixtures of cyclic nucleotide phosphodiesterases that have very different physical and kinetic characteristics. 1-3 One approach to understanding these complex systems is to isolate and to study pure preparations of individual phosphodiesterases from a single cell or tissue type. This allows one to compare the physical and chemical characteristics of the enzymes, and to develop specific probes for studying the relative contributions of individual phosphodiesterases in J. N. Wells and J. G. Hardman, Adv. Cyclic Nucleotide Res. 8, 119 (1977). 2 M. Vaughan, M. A. Danello, V. C. Mangeniello, and G. J. Strewler, Adv. Cyc. Nuc. Res. 14, 263 (1981). 3 j. A. Beavo, R. S. Hansen, S. A. Harrison, R. L. Hurwitz, T. J. Martins, and M. C. Mumby, Mol. Cell. Endocrinol. 28, 387 (1982).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
686
P H O S P H O D I E S T E R AISOZYME SE METHODS
[62]
crude tissue preparations. These probes also enable one to examine the locations and functions of the enzymes in vivo. Three major cyclic nucleotide phosphodiesterases have been identified and purified from bovine cardiac muscle. 4-8 This chapter summarizes our recent studies with two phosphodiesterase isozymes, the cGMP-stimulated and cGMP-inhibited phosphodiesterases, which we isolate from bovine cardiac muscle. Three different methods for producing stable, homogeneous preparations of these trace regulatory enzymes are described and a detailed comparison of the enzymes is presented. Figure 1 provides diagrammatic representations of the purification procedures described below. Procedure A enables one to purify both cGMP-stimulated and cGMP-inhibited phosphodiesterases from bovine cardiac muscle by taking advantage of the enzymes' differential binding to affinity resins. Procedure B utilizes isozyme-specific monoclonal antibodies to rapidly isolate cardiac muscle phosphodiesterase-antibody complexes for use in studies requiring preparations of catalytically active, nonproteolyzed enzymes. Procedure C represents a modification of the antibody isolation procedure B. This method results in homogeneous preparations of denatured but nonproteolyzed phosphodiesterases for use in a variety of protein structural studies. Note that although procedures B and C are described for purification of cGMP-inhibited phosphodiesterase, they can also be used to isolate cGMP-stimulated and CaZ+/calmodulin activated phosphodiesterases.
Reagents The various buffers used in the assay and isolation procedures are listed below. Buffer A: 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.5, 2.5 mM ethylene glycol bis(fl-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA), 37.5 mM MgCI2, 0.5 mg/ml bovine serum albumin (BSA) Buffer B: 100 mM Tris-HCl, pH 8.0 (at room temperature) and 15 mM 2-mercaptoethanol Buffer C: 40 mM Tris-HCl, pH 7.8 (at room temperature), 15 mM 2mercaptoethanol, 2 mM ethylenediaminetetraacetic acid (EDTA), and 100 mM NaC1 4 D. C. LaPorte, W. A. Toscano, and D. R. Storm, Biochemistry 18, 2820 (1979). 5 R. K. Sharma, T. H. Wang, E. Wirch, and J. H. Wang, J. Biol. Chem. 255, 5916 (1980). 6 R. S. Hansen and J. A. Beavo, Proc. Natl. Acad. Sci. U.S.A. 79, 2788 (1982). 7 T. J. Martins, M. C. Mumby, and J. A. Beavo, J. Biol. Chem. 25, 1973 (1982). 8 S. A. Harrison, D. H. Reifsnyder, B. Gallis, G. G. Cadd, and J. A. Beavo, Mol. Pharmacol. 29, 506-514 (1986).
A
B
BovineCardiac Muscle Homogenize Spin r 1 Pellet Hypotonic extract
BovineCardiac Muscle ~ omogenize Spin i pellet
~ DEAE-Cellulose
Immunoadsorbspin
DEAE pool
~
Supematant
Antibodypellets
cAMP Sepharose ---- cGMP Sepharose
Bound fraction
~
I
Hypotonic extract
CN-Sepharoses-unbound fraction
cGMPelution
cGMP-stimulatedPDE
CGI-PDE CGS-PDE Ca2+/Cam-PDE
l
Bluedextran-Sepharose cAMP elution
Blue dextran-Sepharosepool
PhosphodiesterascActivity and InhibitorAssays
~ Anion-exchangeHPLC cGMP-inhibitedPDE C BovineCardiacMuscle Homogenize Spin r
Pellet
I
Hypotonic extract Add antibody
~
~ DEAE-Cellulose DEAE pool Protein A-Sepharose Protein A-Sepharose pooled fractions SDS-PAGE Gel slice Electroelution cGMP-inhibitedPDE protein FIG. 1. Diagram of cardiac muscle phosphodiesterase isolation procedures. Three procedures are described for isolating cGMP-stimulated and cGMP-inhibited phosphodiesterases from bovine cardiac muscle. (A) The first procedure utilizes high-affinity binding of the phosphodiesterase isozymes to ligands coupled to solid supports, and subsequent elutions with cyclic nucleotides. (B) The second procedure utilizes monoclonal antibodies directed against the phosphodiesterase of interest. Phosphodiesterases are bound to solid-phase antibody reagents and isolated from crude enzyme preparations. These antibodies do not inhibit phosphodiesterase activity and the antibody-phosphodiesterase complex can be used to characterize catalytic properties of the bound enzyme. (C) A modification of the latter procedure results in purification of nonproteolyzed, denatured cGMP-inhibited phosphodiesterase protein for use in determinations of amino acid composition and amino acid sequence.
688
PHOSPHODIESTERASE ISOZYME METHODS
[62]
Buffer D: 20 mM MOPS, pH 7.5, 15 mM 2-mercaptoethanol, 0.1 mM EDTA, and 200 mM NaCI Buffer-E: 40 mM Tris-HCl, pH 7.5 at room temperature, 15 mM 2mercaptoethanol, and 10 mM MgCI2 Buffer F: 5 mM Tris-HC1, pH 7.5 at room temperature, and 250 mM NaC1 Buffer G: 10 mM phosphate, pH 7.4, 150 mM NaCl, and 0.5 mg/ml BSA Buffer H: l0 mM Tris-HCl, pH 8.0 at room temperature, 15 mM 2mercaptoethanol, and 300 mM NaC1 Phosphodiesterase Activity Assay Most of the phosphodiesterase assays described in the literature can be used to measure phosphodiesterase activity during the isolation procedures presented in this chapter. We use a previously described method, 9 with minor modifications. Enzyme samples (0-50 tzl) are diluted with distilled water to a final volume of 0.05 m] and preincubated with 100/zl of buffer A at 30° for 2 rain. The reaction is initiated by addition of 100/zl [3H]cAMP, diluted to a desired final concentration with unlabeled cAMP. The reactions are terminated by boiling 1 rain. After treatment with 25/zg of Crotalus atrox venom for 3 min at room temperature, the sample is diluted to 0.5 ml with water and applied to 0.8 m] DEAE-Sephadex A-25 columns equilibrated in 20 mM Tris-HCl buffer, pH 7.5. Liquid scintillation vials are then placed under the columns and [3H]adenosine is separated from unreacted cAMP by eluting the resin three times with 1-ml aliquots of 20 mM Tris-HC1 buffer, pH 7.5. cAMP hydrolysis should be less than 20% during the time course of the assays. [3H]cAMP is routinely repurified by chromatography on DEAE-Sephadex A-25, and assay blanks contain less than 2% of the total 3H in an assay. If cGMP stimulation or inhibition of cAMP hydrolysis is to be measured, low concentrations of cAMP (0.1-1.0/zM) must be used, and these assays should be initiated with enzyme, rather than with substrate. This modification allows one to add cGMP to the assay without that modulator being hydrolyzed during the preincubation step. Phosphodiesterase assays on resuspended immune pellets are performed as described above. Solid-phase antibody reagents are prepared using heat-inactivated, formalin-fixed cells of the Cowan I strain of Staphylococcus aureus (Staph A) and rabbit anti-mouse IgG antiserum (RAM). Resuspended Staph A-RAM-primary antibody-phosphodiesterase pellets are used as enzyme samples, and Staph A-RAM control pellets are used as enzyme blanks. [3H]cGMP 9 j. A. Beavo, J. G. H a r d m a n , and E. W. Sutherland, J. Biol. Chem. 245, 5649 (1970).
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
689
hydrolysis is assayed using the same procedure described for measuring cAMP hydrolysis. Preparation of Affinity Resins N6-H2N(CH2)2-cAMP-Sepharose and C S - H 2 N ( C H 2 ) 2 - N H - c A M P Sepharose can be prepared by coupling the cAMP derivatives to cyanogen bromide-activated Sepharose as described in detail previously, t° or the resins can be purchased from P. L. Biochemicals or Pharmacia. The cGMP-Sepharose resin is prepared by coupling cGMP to epoxyactivated Sepharose 4B as described in detail previously7 and summarized below. Sepharose resin is swollen in distilled water and 60 ml of resin is activated during an 8-hr, room-temperature incubation with 60 ml of 0.5 M NaOH and 2 mg/ml sodium borohydride, and 60 ml of 1,4-butanediol diglycidyl ether. The resin is washed with 5 to 10 vol of cold distilled water and then incubated with 60 ml of coupling buffer containing 20 mM sodium borate, pH 10, 200 mM NaCI, and 20 mM cGMP for 36 hr at room temperature. It is finally blocked overnight at 4° by incubation with nucleotide-free coupling buffer containing 1 M ethanolamine (with pH readjusted to 11). Blue dextran-Sepharose resin is produced by coupling 3.2 g of Pharmacia blue dextran 2000 to 135 ml of swollen Pharmacia cyanogen bromide-activated Sepharose 4B, according to the manufacturer's instructions supplied with the resin. A blue dextran-agarose resin is available commercially from Sigma; however, that resin has not yet been tested in our laboratory. Protein A-Sepharose can be purchased from Sigma or Pharmacia. Several of the antibody affinity resins described below will be available in the near future from Chemicon International, Inc. Identification of Cardiac cGMP-Stimulated and cGMP-Inhibited Phosphodiesterases While the cardiac Ca2+/calmodulin-activated phosphodiesterase is easily identified, cGMP-stimulated and cGMP-inhibited phosphodiesterases are more difficult to identify and study in bovine cardiac muscle extracts. When these extracts are assayed for phosphodiesterase activity at 1/xM cAMP, in the presence or absence of Ca2+/calmodulin or cGMP, some Ca2+/calmodulin activation is detected, but no effect of cGMP is observed (Fig. 2). The contributions of each enzyme can only be seen ~0W. L. Dills, Jr., J. A. Beavo, P. J. Bechtel, K. R. Myers, L. J. Sakai, and E. (3. Krebs, Biochemistry 15, 3724 (1976).
690
PHOSPHODIESTERASE ISOZYME METHODS
[62]
.6 i O
Ca 2+
EGTA
EGTA
+
+
CAM
cGMP
FIG. 2. Cardiac muscle extract phosphodiesterase activities measured at 1 /xM cAMP. The soluble fraction of a hypotonic extract from bovine cardiac muscle was assayed for phosphodiesterase activity in the presence of maximal concentrations of effector(s). Hydrolysis of 1/xM c A M P was measured in the presence of Ca 2÷ and calmodulin, EGTA, or E G T A and 1/xM c G M P . Although some Ca2÷/calmodulin activation was evident, no cGMP stimulation was observed in the muscle extract.
after specific antibody immunoadsorption analysis is performed (Fig. 3). This approach utilizes isozyme-specific monoclonal antibodies to remove and identify individual phosphodiesterase isozymes from cardiac extracts. The antibodies used in Fig. 3, ACAP-1 (anti-CaZ+/calmodulin-acti vated phosphodiesterase antibody) and CGS-2 (anti-cGMP-stimulated phosphodiesterase antibody), have been described in detail previously. 6,~ In contrast to Fig. 2, Fig. 3 clearly demonstrates the presence of both Ca2+/calmodulin-activated and cGMP-stimulated phosphodiesterases on the antibody pellets, and shows the relative contributions of each enzyme in the presence of regulatory molecules. Each enzyme makes a significant H M. C. M u m b y , T. J. Martins, M. L. Chang, and J. A. Beavo, J. Biol. Chem. 257, 13283 (1982).
[62]
BOVINE HEART c G M P PHOSPHODIESTERASES
4
691
S = 10"6M cAMP
E
._~ ~"E3
ACAP Pellet
CGS-2 Pellet
ACAP + CGS-2 Supernatant
E e= .~_ 2 r~
~'1
l/
EGTA
Ca 2+ +
--
+
cGMP
--
+
cGMP
CAM FIG. 3. Immunoadsorption analysis of cardiac muscle extract phosphodiesterase activities measured at 1 /~M cAMP. Solid-phase monoclonal antibodies were used to remove CaZ+/calmodulin-activated and cGMP-stimulated phosphodiesterases from cardiac muscle extracts. Antibody pellets and supernatants were separated by centrifugation and assayed for cAMP hydrolysis in the presence of Ca 2. and calmodulin, EGTA, or EGTA and 1 p,M cGMP, In contrast to the experiment performed without antibody fractionation (Fig. 2), this figure demonstrates the presence of the cGMP-stimulated phosphodiesterase, and it shows the relative contributions of the isozymes in the presence of the effectors. Notice that a cGMP-inhibited phosphodiesterase activity was revealed when the masking isozyme was removed.
contribution to total substrate hydrolysis at 1 ~M cAMP in the presence of the appropriate activator. (CaZ+/calmodulin-activated phosphodiesterase is activated when bound to ACAP-1; Hansen and Beavo, unpublished observations). In addition, a cGMP-inhibited phosphodiesterase activity is revealed in the antibody-treated supernatant after removal of the masking cGMP-stimulated enzyme. Chromatography of phosphodiesterase activities on DEAE cellulose is routinely reported as a method of isolating individual isozymes for characterization of physical and catalytic properties and for determination of pharmacological profiles. A typical elution profile of the bovine cardiac muscle phosphodiesterase activities is shown in Fig. 4. Two major peaks are observed, the first of which is activated by Ca 2+/calmodulin. As in the extract, no cGMP-sensitive phosphodiesterase activity is detected in the fractions after chromatography on DEAE-cellulose. However, immunoadsorption analysis using a monoclonal antibody directed against the cGMP-stimulated phosphodiesterase reveals the presence of both cGMPstimulated and cGMP-inhibited phosphodiesterase activities in the elution
692
PHOSPHODIESTERASE [SOZYME METHODS l
~
20
i
i
[62]
I
S : 10"6M cAMP
E .-~ ~'~ 16 "~
[] -"
I
-- Ca + CAM [] EGTA 2+ '= cGMP
"~=12 r~
o
10
20
30 40 50 Number FIG. 4. DEAE chromatographyof cardiac muscle phosphodiesterase activities measured at 1/zM cAMP. Heart extract was adsorbed to DEAE cellulose and eluted with a linear NaCl gradient. Fractions collected after anion-exchange chromatographywere assayed for cAMP hydrolysis in the presence of Ca2÷ and calmodulin, EGTA, or EGTA and I /zM cGMP. Notice that only two peaks of activity were measured. CaZ÷/calmodulin-activatedphosphodiesterase was detected; however, neither the cGMP-stimulated phosphodiesterase nor the cGMP-inhibited phosphodiesterase was detected in these fractions. Fraction
profile. These data are shown in Fig. 5. The upper panel identifies a large peak of cGMP-inhibited phosphodiesterase activity which is only detected after removal of the cGMP-stimulated form. The lower panel demonstrates that the cGMP-stimulated enzyme elutes with, and masks the presence of the cGMP-inhibited activity, even after chromatography on the DEAE-resin. These data clearly illustrate the need for characterizing the physical and kinetic properties of highly purified phosphodiesterases, rather than crude preparations which may contain more than one isozyme. Procedure A: Affinity Purification of Cardiac cGMP-Stimulated and cGMP-Inhibited Phosphodiesterases The extraction and DEAE-cellulose chromatography procedures described below can be scaled up or down to accommodate 0.01-25 kg of heart muscle. A large-scale (20-kg) preparation is described below. Obtain bovine cardiac muscle flesh from an abattoir and keep the tissue on ice
[62]
BOVINE HEART c G M P PHOSPHODIESTERASES I
t
I
I
ACAP-1 + CGS-2 Supematant
A/
I
S=
10-6
693
M cAMP
12
8
E4 E
E
0
L.
~3
0
10
20
30
40
50
Fraction Number FIG. 5. DEAE chromatography and immunoadsorption analysis of cardiac phosphodiesterase activity measured at 1 #M cAMP. Heart extract was adsorbed to DEAE-cellulose and eluted with a linear NaCI gradient. Fractions collected after anion-exchange chromatography were incubated with solid-phase monoclonal antibody reagent, centrifuged to separate pellets and supernatants, and assayed for cAMP hydrolysis in the presence of Ca 2÷ and calmodulin, EGTA, or EGTA and 1 /zM cGMP. (A) A large peak of cGMP-inhibited phosphodiesterase activity was detected after removal of the Ca2+/calmodulin-activated and cGMP-stimulated phosphodiesterases by monoclonal antibody reagents. (B) The cGMPstimulated phosphodiesterase was detected on the CGS-2 pellet. These data confirm the presence of a cGMP-inhibited phosphodiesterase in bovine heart muscle. This isozyme elutes with heart cGMP-stimulated phosphodiesterase, and each isozyme masks the others presence in crude enzyme preparations.
while transporting it to the laboratory. All subsequent operations are performed at 4°. Trim and slice approximately 20 kg of ventricular mtiscle, pass the tissue through a coarse disk of a meat grinder, and then homogenize the ground tissue in 2.5 vol (w/v) of buffer B in a Waring blender for 15 sec at high speed. Add crystalline PMSF to a concentration of 0.2 mM during the homogenization. Centrifuge the homogenate at 4000 g for 20 min and filter the resulting supernatant through glass wool and cheesecloth. Adjust the supernatant to pH 7.8 and add crystalline PMSF
694
PHOSPHODIESTERASE ISOZYME METHODS
[62]
to a final concentration of 0.4 mM. Batch adsorb the filtered supernatant onto 10 liters of DEAE-cellulose for 30 min, stirring with a paddle attached to a large drill motor. Wash the resin batchwise with 8-10 vol of buffer C, make a slurry of the resin in buffer C, pour into a large column (e.g., a Glenco column with a 15.4-cm diameter), elute with a linear 20liter NaC1 gradient (0.1-0.7 M), and collect fractions after the conductivity begins to increase in the eluate. This procedure allows the removal of CaZ+/calmodulin-activated phosphodiesterase during the wash step. Assay the fractions for phosphodiesterase activity using either 1 mM cGMP or 1 /xM cAMP. The 1 mM cGMP assay detects primarily cGMP-stimulated phosphodiesterase because the cGMP-inhibited phosphodiesterase hydrolyzes cGMP very poorly. The 1 tzM cAMP assay measures primarily cGMP-inhibited phosphodiesterase in the fractions, since cGMP-stimulated phosphodiesterase is relatively inactive under these conditions (Figs. 3 and 5). cGMP-Stimulated Phosphodiesterase Purification. As demonstrated in Fig. 5, the fractions pooled following anion-exchange chromatography contain both the cGMP-stimulated and cGMP-inhibited phosphodiesterases. Add benzamidine to the pooled fractions to a final concentration of 15 mM. Then apply the sample by gravity flow to two columns connected in series, in which the first column contains 20 ml of NHzN(CH2)2-cAMP-Sepharose and the second contains 60 ml of cGMPSepharose. Both resins should be equilibrated with buffer D prior to sample application. This step allows the removal and purification of cGMP-stimulated phosphodiesterase which binds specifically to the cGMP resin. The cAMP resin is mainly used to remove the type II regulatory subunit of cyclic nucleotide-dependent protein kinase (RII), to prevent that protein from competing with cGMP-stimulated phosphodiesterase for binding to the cGMP resin. Separate the two columns and wash the cGMP resin first with 250 ml of buffer D, and then with 250 ml of buffer D containing a final NaC1 concentration of 0.5 M, and finally wash again with 100 ml of buffer D. Warm the resin to room temperature and elute the cGMP-stimulated phosphodiesterase with buffer D containing 10 mM cGMP at a slow flow rate (10-20 ml/hr). Eluted fractions are collected in an adjacent cold box at 4° and assayed for phosphodiesterase activity and protein. Note that aliquots of each fraction should be incubated with 10 mM MgCI2 for 1 hr on ice, prior to the assay for phosphodiesterase activity. Any contaminating RII can be removed by adding the following steps. The phosphodiesterase-containing pooled fractions are incubated with 10 mM MgC12 for 1 hr on ice to allow complete hydrolysis of the cGMP. The completeness of the hydrolysis can be checked by thin-layer chromatog-
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
695
raphy of small aliquots from this incubation on polyethyleneimine-impregnated plastic strips using n-propanol: ammonium hydroxide:water (6:3:1). The phosphodiesterase samples are then passed through C 8H2N(CH2)2-NH-cAMP-Sepharose resin (0.9 x 8 cm) which was previously equilibrated with buffer D. The unbound cGMP-stimulated phosphodiesterase is essentially free of contaminating RII. This procedure routinely provides stable preparations of phosphodiesterase that are greater than 95% homogeneous, based on analysis by SDS-PAGE. The enzyme can be stored in this solution at 4° or in 20% glycerol at -20 °. Freezing or lyophilizing the native enzyme is not recommended. The enzyme is purified 16,400-fold by this procedure, with a yield of 19% and a specific activity of approximately 105 U/min. mg for cAMP or cGMP. 7 cGMP-Inhibited Phosphodiesterase Purification. cGMP-inhibited phosphodiesterase is prepared from the activity which flows through the cyclic nucleotide resins. Add MgCI2 to the flow-through fraction to a final concentration of 10 mM, and dilute the pool to a conductivity of 7.0 mmho (at 4°) with buffer E. The pH of the diluted sample should be between 7.8 and 8.0. Equilibrate 120 ml of blue dextran-Sepharose in buffer E and batch adsorb the sample to this resin overnight using a slowly turning paddle attached to a drill motor (do not use a magnetic stirring bar because it fractures the gel beads and results in very slow flow rates). Then wash the resin with 2 liters of the dilution buffer in a sintered glass funnel. Add enough wash buffer to make a slurry and pour the washed resin into a 4.5 x 7.5 cm column. Elute the phosphodiesterase activity from the resin with a 600-ml linear gradient of cAMP (0-8 mM) in buffer E. The ratio of resin volume to column height appears to be a critical variable. For example, poor chromatography is obtained with a column 2.5 x 24 cm, possibly because the cAMP used to elute the enzyme is hydrolyzed by the adsorbed phosphodiesterase during elution from the longer column. This chromatography step provides an additional 100 to 200-fold purification of the cGMP-inhibited phosphodiesterase activity. Pool the blue dextranSepharose fractions containing phosphodiesterase activity and adsorb the activity to an HLPC anion-exchange resin (TSK-DEAE-5-PW) using a large sample injection loop (e.g., Pharmacia Superloop) or multiple injections with a smaller sample injection loop. This work can be done at room temperature, but the fractions are collected at 4° in an adjacent cold box. Column life is prolonged by centrifuging the sample for 10 min in a Beckman airfuge at full speed prior to the HPLC step. The sample is loaded onto the column and then the column is washed in buffer F, and eluted with buffer F containing additional NaC1 added to a final concentration of 0.33 M NaC1. A second peak containing a smaller amount of phosphodiesterase activity is eluted with 0.5 M NaC1. The concentration of NaC1
696
PHOSPHODIESTERASE ISOZYME METHODS
[62]
required to elute the cGMP-inhibited phosphodiesterase may vary with different TSK-DEAE columns. Therefore, one should perform a pilot elution with an NaC1 gradient to determine the actual concentration of NaCI necessary to elute the phosphodiesterase. The enzyme is purified 16,000-fold with a yield of approximately 5% and a specific activity of 6/zmol of cAMP hydrolyzed/min • mg. 8 This procedure produces a highly purified phosphodiesterase; however, the enzyme preparations usually contain a mixture of several proteolytically related polypeptides ranging in molecular weight from 80,000 to 60,000. 8 We have not yet obtained a stable, homogeneous preparation of the enzyme, using this procedure. However, we have utilized the HPLC-purified phosphodiesterase as antigen for the production of polyclonal antisera and monoclonal antibodies. This preparation of phosphodiesterase appears to be very antigenic, since all of the mice injected with this protein developed isozyme-specific antisera. These antibodies have in turn proved to be very useful as specific probes for identifying cGMPinhibited phosphodiesterase protein and for isolating a stable enzyme, with an apparent subunit molecular weight of 110,000. 8 Procedure B: Rapid Isolation of Catalytic Amounts of Bovine Cardiac Muscle Phosphodiesterases on Solid-Phase Monoclonal Antibodies Procedures B and C are useful for rapidly isolating cGMP-inhibited, cGMP-stimulated, and Ca2+-calmodulin-activated phosphodiesterases. Since the solid-phase antibody procedures are the only methods available at this time for isolating nondegraded cGMP-inhibited phosphodiesterase, we have used that enzyme as an example for the description of these methods. Catalytic amounts of nondegraded cGMP-inhibited phosphodiesterase can be bound to the solid-phase monoclonal antibody reagent and the complex isolated for in vitro studies of phosphodiesterase kinetics, regulation, and pharmacology. 8 Polyclonal mouse antisera and hybridoma cell lines producing monoclonal antibodies directed against the cGMP-inhibited phosphodiesterase can be produced by established procedures, 8,12using the cGMP-inhibited phosphodiesterase preparation described in procedure A. Solid-phase antibody reagents are prepared using Staph A and RAM. The use of solidphase reagents allows isolation of a specific phosphodiesterase by precipitation of phosphodiesterase-antibody complexes. None of the antibodies used inhibits enzyme activity, and the phosphodiesterase is readily assayed on the resuspended immune pellet. In general, Staph A - R A M is t2 S. F a z e k a s de St.Groth and D. Scheidegger, J. lmmunol. Methods 35, 1 (1980).
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
697
prepared as follows: wash 0.5 ml of 10% Staph A twice with buffer G, resuspend in 0.5 ml of buffer G containing 150/zg of rabbit anti-mouse IgG antibodies, and incubate for 1 hr. Then wash the solid phase reagent two or three items with buffer G, This reagent is then resuspended in buffer G containing approximately 50/xg of anti-phosphodiesterase antibodies (for example CGI-5, an anti-cGMP-inhibited phosphodiesterase monoclonal antibody), incubated for 1 hr, and the washes are repeated. All steps in the procedure are performed on ice or in a cold room at 4°. Note that when CGI-5 antibody reagents are used, 0.1% Tween-20 is routinely used to replace BSA in buffer G. The detergent may interfere, however, with ACAP-1- and CGS-2-phosphodiesterase interactions. Incubate 1.5 ml of the bovine heart extract or DEAE-pooled fractions described above for 1.5 hr with either Staph A-RAM-primary antibody or Staph A-RAM control reagent, centrifuge, and then wash the solid-phase reagent once with 20 mM Tris-HCl buffer, pH 7.8 (at 4°), containing 0.5 M NaCI and twice with Tris-HC1 buffer minus the NaC1. Resuspend immune and control pellets in Tris-HC1 buffer using an appropriate dilution and assay for phosphodiesterase activity. Immune pellets with bound phosphodiesterase-antibody complexes can also be resuspended in 20 mM Tris-HC1 buffer containing 50% glycerol, pH 7.8, and stored for several months at - 2 0 °. This approach can also be used to isolate cardiac Ca2+/calmodulinactivated and cGMP-stimulated phosphodiesterases using ACAP-1 and CGS-2 monoclonal antibodies, as demonstrated in Figs. 3 and 5. Procedure C: Purification of Large Quantities of Phosphodiesterase Protein Using Solid-Phase Monoclonal Antibodies A modification of the monoclonal antibody purification procedure can be used to isolate homogeneous preparations of nonproteolyzed, but denatured, cGMP-inhibited phosphodiesterase protein in sufficient quantities for use in a variety of structural studies. All steps are performed on ice or at 4°. Prepare an extract from 2 kg of bovine cardiac muscle as described above. Add ascitic fluid containing 5 mg of CGI-5 to the extract, incubate for 1 hr, and then batch adsorb the extract onto 1 liter of DEAEcellulose for 30 min. Wash the resin with 10 liters of buffer C in a 2-liter sintered glass funnel. Elute the proteins from the resin stepwise with buffer C containing a final concentration of 300 mM NaCI, collect 250 to 300-ml fractions, and assay the fractions for conductivity and phosphodiesterase activity (measured at 1 tzM cAMP). Note that the conductivity of the first two fractions should be approximately 4.5 mmho and the conductivity of the third fraction should increase to approximately 7.07.5 mmho. The phosphodiesterase activity will elute in the third DEAE-
698
P H O S P H O D I E S T E R AISOZYME SE METHODS
[62]
cellulose fraction. Next, check the pH of this fraction (the pH should be 8.2-8.5) and apply by gravity flow onto 5 ml of protein A-Sepharose resin which has been previously equilibrated in buffer H at 4°. This resin will bind the CGI-5 monoclonal antibody : cGMP-inhibited phosphodiesterase complex. Wash the resin extensively, first with I0 vol of buffer H and then with 10 vol of buffer H containing no NaC|, and elute with 0.1 N acetic acid. Collect 2-ml fractions and measure eluted protein by A280. In our laboratory, the identity of the eluted protein is confirmed at this point in the procedure by protein immunoblot analysis and this polypeptide is the only Coomassie staining band in the region of 110 kDa on the SDS gels. Other polypeptides in this sample include IgG heavy and light chains and polypeptides with molecular weights of approximately 200,000-220,000 and 45,000 (these polypeptides comigrate with reduced fibronectin and with actin; however, their identities have not yet been determined). Phosphodiesterase-containing fractions are then lyophilized and stored prior to electrophoresis by SDS-PAGE. Lyophilized sample is resuspended in Laemmli sample buffer ~3 and polypeptides are separated electrophoretically on 10% polyacrylamide gels. The 110-kDa cGMP-inhibited phosphodiesterase protein is visualized by Coomassie staining and is then excised from the gel and electrophoretically transferred from the gel slices by the method of Hunkapillar et al.14 Dry the recovered protein by vacuum rotary evaporation. Next, wash the pellet with distilled and deionized water and dry the protein again by vacuum rotary evaporation. Repeat the washing and drying steps two more times. Residual Coomassie dye can be removed as follows: resuspend the dried protein in neat methanol for 1 hr at 4°, centrifuge the suspension for 15 rain at full speed in an Eppendorf microfuge, removed and discard the supernatant fraction. Then wash with distilled water and dry by evaporation three times, as described above. This protein preparation is suitable for studying the protein chemistry of cyclic nucleotide phosphodiesterases, as demonstrated below. Comparison of cGMP-Inhibited and cGMP-Stimulated Phosphodiesterases Table I summarizes the amino acid compositions that have been determined for cGMP-inhibited and cGMP-stimulated phosphodiesterases using enzymes isolated by the electroelution procedure. The composition of 13 U. K. Laemmli, Nature (London) 227, 680 (1970). ~4M. W. Hunkapillar, E. Lujan, F. Ostrander, and L. E. Hood, this series, Vol. 91, p. 227.
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
699
TABLE I AMINO ACID COMPOSITIONS OF cGMP-STIMULATED AND cGMP-INHIBITED PHOSPHODIESTERASES~
Amino acids Asx b Thr Ser Glx' Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Trp
Affinity-purified cGMP-stimulated pbosphodiesterase (mol%) 10.4 4.1 5.4 13.1 5.9 5.6 9.0 NI a 6.4 2.6 4.6 13. l 2.9 4.2 5.1 3.0 4.4 NI
Electroeluted cGMP-stimulated phosphodiesterase (mol%) 10.9 3.8 5.7 13.9 4.0 8.0 7.9 ND ~ 6.4 2.4 4.9 13.2 2.5 3.8 5.3 2.8 4.4 ND
Electroeluted cGMP-inhibited phosphodiesterase (mol%) 10.1 4.5 5.6 13.0 7.7 6.0 7.9 ND 6.5 1.8 5.6 10.6 2.2 3.9 6.0 3.0 5.6 ND
Determined by the method of B. A. Bidlingmeyer, S. A. Cohen, and T. L. Tarvin, J. Chromatogr. 336, 93 (1984). h Combined asparagine and aspartic acid. ' Combined glutamine and glutamic acid. ,/NI, not included in the calculations of mol% for purposes of this comparison. '~ ND, not determined.
the electroeluted cGMP-stimulated phosphodiesterase was compared with the composition determined for cGMP-stimulated phosphodiesterase isolated by procedure A, as described above. The comparison between these two methods enabled us to use the cGMP-stimulated enzyme as a positive control for the cGMP-inhibited phosphodiesterase protein, purified by procedure C. The two phosphodiesterase isozymes have nearly identical amino acid compositions, which is not surprising considering the similar sizes and functions of the two proteins. It will be very interesting to compare the amino acid sequences of these two cGMP-sensitive phosphodiesterase isozymes. Table II presents a variety of physical and catalytic properties of the bovine heart cGMP-inhibited and cGMP-stimulated
700
[62]
PHOSPHODIESTERASE ISOZYME METHODS T A B L E 11 PROPERTIES OF cGMP-STIMULATED AND cGMP-INHIBITED PHOSPHODIESTERASES
Properties
cGMP-stimulated phosphodiesterase
cGMP-inhibited phosphodiesterase
Monomer MW Subunit structure
Dimer
K m cAMP Km cGMP Vmax cAMP b Vmax cGMP"
30 k~M 10 ~ M 105 U/mg 105 U/mg
110,00 Dimer? 0.15 # M 0.06/~M ~ 6.0 U/mg 0.6 U/mg J
Modulator Phosphorylation e
cGMP (stimulation) No
cGMP (inhibition) Yes
105,000
Inhibitor Ki (kLM)
lnhibitors Milrinone Fenoximone IBMX
Papaverine RO 20-1724
cGMP-stimulated
cGMP-inhibited
PDE
PDE
180 430 14 4.5 300
0.26 1.5 1.3 0.66 62
" Ki of cGMP as a competitive inhibitor of cAMP hydrolysis. h U = ~mol cAMP hydrolyzed/min at 30 °. ' U = p.mol cGMP hydrolyzed/min at 30 °. a Estimated for pure enzyme from Vmaxratios of cAMP/cGMP hydrolysis by preparations of enzyme-antibody complex. e I n vitro phosphorylation by cAMP-dependent protein kinase.
phosphodiesterases. These two enzymes represent potential control points, at which cGMP could regulate cAMP metabolism. Notice that although the two isozymes coelute from DEAE-cellulose and are similar in size and sensitivity to cGMP, they exhibit very different catalytic properties, regulation by cGMP, and affinities for inhibitors. The differential sensitivities to inhibitors is particularly striking for the new cardiotonic agents, milrinone and fenoximone. Highly selective inhibition of cardiac cGMP-inhibited phosphodiesterase, relative to the other heart isozymes, is a possible mechanism of action for these drugs. 8 In addition, cGMPinhibited phosphodiesterase is phosphorylated in vitro by cAMP-dependent protein kinase under conditions which do not result in phosphorylation of cGMP-stimulated phosphodiesterase.
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
701
General Comments Three isolation procedures are described in this chapter. The first procedure results in purification of cGMP-stimulated phosphodiesterase by affinity chromatography using cGMP-Sepharose and purification of cGMP-inhibited phosphodiesterase by pseudoaffinity chromatography on blue dextran-Sepharose. The second approach utilizes specific monoclonal antibodies for isolation of antibody-phosphodiesterase complexes. This immunological method is demonstrated by the rapid isolation of cGMP-inhibited phosphodiesterase, an enzyme which is very sensitive to proteolytic degradation. The third method combines specific binding of the phosphodiesterase to a monoclonal antibody, adsorption of that complex to an antibody-binding resin, and electroelution of the phosphodiesterase protein from a gel slice after SDS-PAGE. This method results in purification of sufficient quantities of nonproteolyzed, denatured phosphodiesterase for structural studies. These approaches allow one to study the physical and catalytic characteristics of the phosphodiesterase isozymes. It is our experience that when EDTA is present in the cardiac muscle homogenization buffer, as originally reported by Martins el al., 7 the cGMP-inhibited phosphodiesterase activity is significantly decreased by an as yet unknown mechanism. This chelator-dependent decrease in cGMP-inhibited phosphodiesterase activity effectively unmasks the cGMP-stimulated phosphodiesterase in bovine heart extracts (Harrison and Beavo, unpublished observations). The large-scale ligand affinity purification procedure described in this chapter is highly satisfactory for purification of large quantities of cGMP-stimulated phosphodiesterase. Note, however, that solutions containing greater than 0.5 mg/ml of the purified cGMP-stimulated phosphodiesterase protein have a tendency to aggregate. One must also take care not to saturate the cAMP resin and contaminate the phosphodiesterase preparation with RII. Enzyme purified by this method has been useful for characterization of physical and catalytic properties 7 and is now proving useful for determining the amino acid sequence of the cGMP-stimulated phosphodiesterase. Problems still exist with pseudoaffinity purification of the cGMP-inhibited phosphodiesterase. The procedure produces predominantly an 80kDa polypeptide which slowly degrades to 67- and 60-kDa polypeptides. 8 Some improvement in the stability of the blue dextran-Sepharose-purifled protein can be obtained by adjusting buffer E to include 10 tzM leupeptin, 10 p.M pepstatin A, 2 mM o-phenanthroline, 2 mM EGTA, and 3 mM EDTA. HPLC-purified cGMP-inhibited phosphodiesterase contained several enzyme fragments which are eluted from the TSK-DEAE
702
PHOSPHODIESTERASE ISOZYME METHODS
[63]
resin by 0.33 M NaCI. Recently we have found that the 110-kDa enzyme is eluted by a higher salt concentration. Use of the protease inhibitors allows us to obtain a greater proportion of the enzyme in the second HPLC peak containing the 110-kDa form; however, as stated above, we have not yet obtained a stable, homogeneous preparation of the enzyme by this procedure, cGMP-inhibited phosphodiesterase purification using monoclonal antibodies does allow us to identify a high-molecular-weight form, characterize catalytic properties, detect phosphorylation by cAMPdependent protein kinase, and identify several highly selective inhibitors. 8,~5 The large-scale version of this method should also be useful for producing enzyme for protein structural studies, and it should be useful in the development of probes for studying the molecular biology of the enzyme. Acknowledgements The authors would like to thank Dr. H. Charbonneau for his helpful discussions and for his expert assistance with our determinations of the phosphodiesterase amino acid compositions. This work was supported by National Institutes of Health Grant AM 21723. 15 S. A. Harrison, M. L. Chang, and J. A. Bevo, Circulation (Suppl. I11), 73, 109-116 March (1986).
[63] P u r i f i c a t i o n o f R o d O u t e r S e g m e n t G T P - B i n d i n g Protein Subunits and cGMP Phosphodiesterase by Single-Step Column Chromatography
By AKIO YAMAZAKI, MASAHIROTATSUMI, and MARK W. BITENSKY Introduction There is a well-established homology between the hormone-sensitive adenylate cyclase and the light-activated cGMP phosphodiesterase (PDE) in rod outer segments (ROS).~ The adenylate cyclase system is composed of three principal segments2: A receptor moiety for stereospecific binding of hormones or neurotransmitter molecules, the GTP-binding compoA. Yamazaki, K. R. Halliday, J. S. George, S. Nagao, C.-H. Kuo, K. S. Ailsworth, and M. W. Bitensky, Adu. Cyclic Nucleotide Protein Phosphorylation Res. 19, 113 (1985). 2 M. Rodbell, Nature (London) 284, 17 (1980).
METHODS IN ENZYMOLOGY,VOL. 159
Copyright © 1988by Academic Press, Inc. All rights of reproductionin any form reserved.
BOVINE HEARTcGMP PHOSPHODIESTERASES
685
corticoids appear to exert effects via the low-Km cAMP isoenzyrne. 9 Similarly, FSH can induce a state of refractoriness in the Sertoli cell by enhancing the activity of the cAMP-dependent isoform. 1° Thus, the link between calcium, calmodulin, and cGMP hydrolysis and the involvement of this system in hormone action requires clarification. Before this can be achieved more research in the role of cGMP in hormone action is necessary. Ion-exchange chromatography of tissue cytosols combined with the use of calmodulin and micromolar concentrations of substrate provide a convenient means of mapping the various l o w - K i n isoforms of cyclic nucleotide phosphodiesterase in tissue cytosols. However, to exclude the possibility that these isoforms are not simply degradation products and therefore artifacts, differences in kinetics, thermal stability, molecular mass, and the responses to calmodulin and drugs of these isolated enzymes should be sought. 9 W. J. Thompson and S. J. Strada, Recept. Horm. Action 3, 553. ~0 M. Conti, M. V. Toscano, L. Petrelli, R. Geremia, and M. Stephanini, Endocrinology 113, 1845 (1983).
[62] I s o l a t i o n a n d C o m p a r i s o n o f B o v i n e H e a r t cGMP-Inhibited and cGMP-Stimulated Phosphodiesterases
By S. A. HARRISON, N. BEIER, T. J. MARTINS, and J. A. BEAVO Introduction Most tissues and cells contain mixtures of cyclic nucleotide phosphodiesterases that have very different physical and kinetic characteristics. 1-3 One approach to understanding these complex systems is to isolate and to study pure preparations of individual phosphodiesterases from a single cell or tissue type. This allows one to compare the physical and chemical characteristics of the enzymes, and to develop specific probes for studying the relative contributions of individual phosphodiesterases in J. N. Wells and J. G. Hardman, Adv. Cyclic Nucleotide Res. 8, 119 (1977). 2 M. Vaughan, M. A. Danello, V. C. Mangeniello, and G. J. Strewler, Adv. Cyc. Nuc. Res. 14, 263 (1981). 3 j. A. Beavo, R. S. Hansen, S. A. Harrison, R. L. Hurwitz, T. J. Martins, and M. C. Mumby, Mol. Cell. Endocrinol. 28, 387 (1982).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
686
P H O S P H O D I E S T E R AISOZYME SE METHODS
[62]
crude tissue preparations. These probes also enable one to examine the locations and functions of the enzymes in vivo. Three major cyclic nucleotide phosphodiesterases have been identified and purified from bovine cardiac muscle. 4-8 This chapter summarizes our recent studies with two phosphodiesterase isozymes, the cGMP-stimulated and cGMP-inhibited phosphodiesterases, which we isolate from bovine cardiac muscle. Three different methods for producing stable, homogeneous preparations of these trace regulatory enzymes are described and a detailed comparison of the enzymes is presented. Figure 1 provides diagrammatic representations of the purification procedures described below. Procedure A enables one to purify both cGMP-stimulated and cGMP-inhibited phosphodiesterases from bovine cardiac muscle by taking advantage of the enzymes' differential binding to affinity resins. Procedure B utilizes isozyme-specific monoclonal antibodies to rapidly isolate cardiac muscle phosphodiesterase-antibody complexes for use in studies requiring preparations of catalytically active, nonproteolyzed enzymes. Procedure C represents a modification of the antibody isolation procedure B. This method results in homogeneous preparations of denatured but nonproteolyzed phosphodiesterases for use in a variety of protein structural studies. Note that although procedures B and C are described for purification of cGMP-inhibited phosphodiesterase, they can also be used to isolate cGMP-stimulated and CaZ+/calmodulin activated phosphodiesterases.
Reagents The various buffers used in the assay and isolation procedures are listed below. Buffer A: 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.5, 2.5 mM ethylene glycol bis(fl-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA), 37.5 mM MgCI2, 0.5 mg/ml bovine serum albumin (BSA) Buffer B: 100 mM Tris-HCl, pH 8.0 (at room temperature) and 15 mM 2-mercaptoethanol Buffer C: 40 mM Tris-HCl, pH 7.8 (at room temperature), 15 mM 2mercaptoethanol, 2 mM ethylenediaminetetraacetic acid (EDTA), and 100 mM NaC1 4 D. C. LaPorte, W. A. Toscano, and D. R. Storm, Biochemistry 18, 2820 (1979). 5 R. K. Sharma, T. H. Wang, E. Wirch, and J. H. Wang, J. Biol. Chem. 255, 5916 (1980). 6 R. S. Hansen and J. A. Beavo, Proc. Natl. Acad. Sci. U.S.A. 79, 2788 (1982). 7 T. J. Martins, M. C. Mumby, and J. A. Beavo, J. Biol. Chem. 25, 1973 (1982). 8 S. A. Harrison, D. H. Reifsnyder, B. Gallis, G. G. Cadd, and J. A. Beavo, Mol. Pharmacol. 29, 506-514 (1986).
A
B
BovineCardiac Muscle Homogenize Spin r 1 Pellet Hypotonic extract
BovineCardiac Muscle ~ omogenize Spin i pellet
~ DEAE-Cellulose
Immunoadsorbspin
DEAE pool
~
Supematant
Antibodypellets
cAMP Sepharose ---- cGMP Sepharose
Bound fraction
~
I
Hypotonic extract
CN-Sepharoses-unbound fraction
cGMPelution
cGMP-stimulatedPDE
CGI-PDE CGS-PDE Ca2+/Cam-PDE
l
Bluedextran-Sepharose cAMP elution
Blue dextran-Sepharosepool
PhosphodiesterascActivity and InhibitorAssays
~ Anion-exchangeHPLC cGMP-inhibitedPDE C BovineCardiacMuscle Homogenize Spin r
Pellet
I
Hypotonic extract Add antibody
~
~ DEAE-Cellulose DEAE pool Protein A-Sepharose Protein A-Sepharose pooled fractions SDS-PAGE Gel slice Electroelution cGMP-inhibitedPDE protein FIG. 1. Diagram of cardiac muscle phosphodiesterase isolation procedures. Three procedures are described for isolating cGMP-stimulated and cGMP-inhibited phosphodiesterases from bovine cardiac muscle. (A) The first procedure utilizes high-affinity binding of the phosphodiesterase isozymes to ligands coupled to solid supports, and subsequent elutions with cyclic nucleotides. (B) The second procedure utilizes monoclonal antibodies directed against the phosphodiesterase of interest. Phosphodiesterases are bound to solid-phase antibody reagents and isolated from crude enzyme preparations. These antibodies do not inhibit phosphodiesterase activity and the antibody-phosphodiesterase complex can be used to characterize catalytic properties of the bound enzyme. (C) A modification of the latter procedure results in purification of nonproteolyzed, denatured cGMP-inhibited phosphodiesterase protein for use in determinations of amino acid composition and amino acid sequence.
688
PHOSPHODIESTERASE ISOZYME METHODS
[62]
Buffer D: 20 mM MOPS, pH 7.5, 15 mM 2-mercaptoethanol, 0.1 mM EDTA, and 200 mM NaCI Buffer-E: 40 mM Tris-HCl, pH 7.5 at room temperature, 15 mM 2mercaptoethanol, and 10 mM MgCI2 Buffer F: 5 mM Tris-HC1, pH 7.5 at room temperature, and 250 mM NaC1 Buffer G: 10 mM phosphate, pH 7.4, 150 mM NaCl, and 0.5 mg/ml BSA Buffer H: l0 mM Tris-HCl, pH 8.0 at room temperature, 15 mM 2mercaptoethanol, and 300 mM NaC1 Phosphodiesterase Activity Assay Most of the phosphodiesterase assays described in the literature can be used to measure phosphodiesterase activity during the isolation procedures presented in this chapter. We use a previously described method, 9 with minor modifications. Enzyme samples (0-50 tzl) are diluted with distilled water to a final volume of 0.05 m] and preincubated with 100/zl of buffer A at 30° for 2 rain. The reaction is initiated by addition of 100/zl [3H]cAMP, diluted to a desired final concentration with unlabeled cAMP. The reactions are terminated by boiling 1 rain. After treatment with 25/zg of Crotalus atrox venom for 3 min at room temperature, the sample is diluted to 0.5 ml with water and applied to 0.8 m] DEAE-Sephadex A-25 columns equilibrated in 20 mM Tris-HCl buffer, pH 7.5. Liquid scintillation vials are then placed under the columns and [3H]adenosine is separated from unreacted cAMP by eluting the resin three times with 1-ml aliquots of 20 mM Tris-HC1 buffer, pH 7.5. cAMP hydrolysis should be less than 20% during the time course of the assays. [3H]cAMP is routinely repurified by chromatography on DEAE-Sephadex A-25, and assay blanks contain less than 2% of the total 3H in an assay. If cGMP stimulation or inhibition of cAMP hydrolysis is to be measured, low concentrations of cAMP (0.1-1.0/zM) must be used, and these assays should be initiated with enzyme, rather than with substrate. This modification allows one to add cGMP to the assay without that modulator being hydrolyzed during the preincubation step. Phosphodiesterase assays on resuspended immune pellets are performed as described above. Solid-phase antibody reagents are prepared using heat-inactivated, formalin-fixed cells of the Cowan I strain of Staphylococcus aureus (Staph A) and rabbit anti-mouse IgG antiserum (RAM). Resuspended Staph A-RAM-primary antibody-phosphodiesterase pellets are used as enzyme samples, and Staph A-RAM control pellets are used as enzyme blanks. [3H]cGMP 9 j. A. Beavo, J. G. H a r d m a n , and E. W. Sutherland, J. Biol. Chem. 245, 5649 (1970).
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
689
hydrolysis is assayed using the same procedure described for measuring cAMP hydrolysis. Preparation of Affinity Resins N6-H2N(CH2)2-cAMP-Sepharose and C S - H 2 N ( C H 2 ) 2 - N H - c A M P Sepharose can be prepared by coupling the cAMP derivatives to cyanogen bromide-activated Sepharose as described in detail previously, t° or the resins can be purchased from P. L. Biochemicals or Pharmacia. The cGMP-Sepharose resin is prepared by coupling cGMP to epoxyactivated Sepharose 4B as described in detail previously7 and summarized below. Sepharose resin is swollen in distilled water and 60 ml of resin is activated during an 8-hr, room-temperature incubation with 60 ml of 0.5 M NaOH and 2 mg/ml sodium borohydride, and 60 ml of 1,4-butanediol diglycidyl ether. The resin is washed with 5 to 10 vol of cold distilled water and then incubated with 60 ml of coupling buffer containing 20 mM sodium borate, pH 10, 200 mM NaCI, and 20 mM cGMP for 36 hr at room temperature. It is finally blocked overnight at 4° by incubation with nucleotide-free coupling buffer containing 1 M ethanolamine (with pH readjusted to 11). Blue dextran-Sepharose resin is produced by coupling 3.2 g of Pharmacia blue dextran 2000 to 135 ml of swollen Pharmacia cyanogen bromide-activated Sepharose 4B, according to the manufacturer's instructions supplied with the resin. A blue dextran-agarose resin is available commercially from Sigma; however, that resin has not yet been tested in our laboratory. Protein A-Sepharose can be purchased from Sigma or Pharmacia. Several of the antibody affinity resins described below will be available in the near future from Chemicon International, Inc. Identification of Cardiac cGMP-Stimulated and cGMP-Inhibited Phosphodiesterases While the cardiac Ca2+/calmodulin-activated phosphodiesterase is easily identified, cGMP-stimulated and cGMP-inhibited phosphodiesterases are more difficult to identify and study in bovine cardiac muscle extracts. When these extracts are assayed for phosphodiesterase activity at 1/xM cAMP, in the presence or absence of Ca2+/calmodulin or cGMP, some Ca2+/calmodulin activation is detected, but no effect of cGMP is observed (Fig. 2). The contributions of each enzyme can only be seen ~0W. L. Dills, Jr., J. A. Beavo, P. J. Bechtel, K. R. Myers, L. J. Sakai, and E. (3. Krebs, Biochemistry 15, 3724 (1976).
690
PHOSPHODIESTERASE ISOZYME METHODS
[62]
.6 i O
Ca 2+
EGTA
EGTA
+
+
CAM
cGMP
FIG. 2. Cardiac muscle extract phosphodiesterase activities measured at 1 /xM cAMP. The soluble fraction of a hypotonic extract from bovine cardiac muscle was assayed for phosphodiesterase activity in the presence of maximal concentrations of effector(s). Hydrolysis of 1/xM c A M P was measured in the presence of Ca 2÷ and calmodulin, EGTA, or E G T A and 1/xM c G M P . Although some Ca2÷/calmodulin activation was evident, no cGMP stimulation was observed in the muscle extract.
after specific antibody immunoadsorption analysis is performed (Fig. 3). This approach utilizes isozyme-specific monoclonal antibodies to remove and identify individual phosphodiesterase isozymes from cardiac extracts. The antibodies used in Fig. 3, ACAP-1 (anti-CaZ+/calmodulin-acti vated phosphodiesterase antibody) and CGS-2 (anti-cGMP-stimulated phosphodiesterase antibody), have been described in detail previously. 6,~ In contrast to Fig. 2, Fig. 3 clearly demonstrates the presence of both Ca2+/calmodulin-activated and cGMP-stimulated phosphodiesterases on the antibody pellets, and shows the relative contributions of each enzyme in the presence of regulatory molecules. Each enzyme makes a significant H M. C. M u m b y , T. J. Martins, M. L. Chang, and J. A. Beavo, J. Biol. Chem. 257, 13283 (1982).
[62]
BOVINE HEART c G M P PHOSPHODIESTERASES
4
691
S = 10"6M cAMP
E
._~ ~"E3
ACAP Pellet
CGS-2 Pellet
ACAP + CGS-2 Supernatant
E e= .~_ 2 r~
~'1
l/
EGTA
Ca 2+ +
--
+
cGMP
--
+
cGMP
CAM FIG. 3. Immunoadsorption analysis of cardiac muscle extract phosphodiesterase activities measured at 1 /~M cAMP. Solid-phase monoclonal antibodies were used to remove CaZ+/calmodulin-activated and cGMP-stimulated phosphodiesterases from cardiac muscle extracts. Antibody pellets and supernatants were separated by centrifugation and assayed for cAMP hydrolysis in the presence of Ca 2. and calmodulin, EGTA, or EGTA and 1 p,M cGMP, In contrast to the experiment performed without antibody fractionation (Fig. 2), this figure demonstrates the presence of the cGMP-stimulated phosphodiesterase, and it shows the relative contributions of the isozymes in the presence of the effectors. Notice that a cGMP-inhibited phosphodiesterase activity was revealed when the masking isozyme was removed.
contribution to total substrate hydrolysis at 1 ~M cAMP in the presence of the appropriate activator. (CaZ+/calmodulin-activated phosphodiesterase is activated when bound to ACAP-1; Hansen and Beavo, unpublished observations). In addition, a cGMP-inhibited phosphodiesterase activity is revealed in the antibody-treated supernatant after removal of the masking cGMP-stimulated enzyme. Chromatography of phosphodiesterase activities on DEAE cellulose is routinely reported as a method of isolating individual isozymes for characterization of physical and catalytic properties and for determination of pharmacological profiles. A typical elution profile of the bovine cardiac muscle phosphodiesterase activities is shown in Fig. 4. Two major peaks are observed, the first of which is activated by Ca 2+/calmodulin. As in the extract, no cGMP-sensitive phosphodiesterase activity is detected in the fractions after chromatography on DEAE-cellulose. However, immunoadsorption analysis using a monoclonal antibody directed against the cGMP-stimulated phosphodiesterase reveals the presence of both cGMPstimulated and cGMP-inhibited phosphodiesterase activities in the elution
692
PHOSPHODIESTERASE [SOZYME METHODS l
~
20
i
i
[62]
I
S : 10"6M cAMP
E .-~ ~'~ 16 "~
[] -"
I
-- Ca + CAM [] EGTA 2+ '= cGMP
"~=12 r~
o
10
20
30 40 50 Number FIG. 4. DEAE chromatographyof cardiac muscle phosphodiesterase activities measured at 1/zM cAMP. Heart extract was adsorbed to DEAE cellulose and eluted with a linear NaCl gradient. Fractions collected after anion-exchange chromatographywere assayed for cAMP hydrolysis in the presence of Ca2÷ and calmodulin, EGTA, or EGTA and I /zM cGMP. Notice that only two peaks of activity were measured. CaZ÷/calmodulin-activatedphosphodiesterase was detected; however, neither the cGMP-stimulated phosphodiesterase nor the cGMP-inhibited phosphodiesterase was detected in these fractions. Fraction
profile. These data are shown in Fig. 5. The upper panel identifies a large peak of cGMP-inhibited phosphodiesterase activity which is only detected after removal of the cGMP-stimulated form. The lower panel demonstrates that the cGMP-stimulated enzyme elutes with, and masks the presence of the cGMP-inhibited activity, even after chromatography on the DEAE-resin. These data clearly illustrate the need for characterizing the physical and kinetic properties of highly purified phosphodiesterases, rather than crude preparations which may contain more than one isozyme. Procedure A: Affinity Purification of Cardiac cGMP-Stimulated and cGMP-Inhibited Phosphodiesterases The extraction and DEAE-cellulose chromatography procedures described below can be scaled up or down to accommodate 0.01-25 kg of heart muscle. A large-scale (20-kg) preparation is described below. Obtain bovine cardiac muscle flesh from an abattoir and keep the tissue on ice
[62]
BOVINE HEART c G M P PHOSPHODIESTERASES I
t
I
I
ACAP-1 + CGS-2 Supematant
A/
I
S=
10-6
693
M cAMP
12
8
E4 E
E
0
L.
~3
0
10
20
30
40
50
Fraction Number FIG. 5. DEAE chromatography and immunoadsorption analysis of cardiac phosphodiesterase activity measured at 1 #M cAMP. Heart extract was adsorbed to DEAE-cellulose and eluted with a linear NaCI gradient. Fractions collected after anion-exchange chromatography were incubated with solid-phase monoclonal antibody reagent, centrifuged to separate pellets and supernatants, and assayed for cAMP hydrolysis in the presence of Ca 2÷ and calmodulin, EGTA, or EGTA and 1 /zM cGMP. (A) A large peak of cGMP-inhibited phosphodiesterase activity was detected after removal of the Ca2+/calmodulin-activated and cGMP-stimulated phosphodiesterases by monoclonal antibody reagents. (B) The cGMPstimulated phosphodiesterase was detected on the CGS-2 pellet. These data confirm the presence of a cGMP-inhibited phosphodiesterase in bovine heart muscle. This isozyme elutes with heart cGMP-stimulated phosphodiesterase, and each isozyme masks the others presence in crude enzyme preparations.
while transporting it to the laboratory. All subsequent operations are performed at 4°. Trim and slice approximately 20 kg of ventricular mtiscle, pass the tissue through a coarse disk of a meat grinder, and then homogenize the ground tissue in 2.5 vol (w/v) of buffer B in a Waring blender for 15 sec at high speed. Add crystalline PMSF to a concentration of 0.2 mM during the homogenization. Centrifuge the homogenate at 4000 g for 20 min and filter the resulting supernatant through glass wool and cheesecloth. Adjust the supernatant to pH 7.8 and add crystalline PMSF
694
PHOSPHODIESTERASE ISOZYME METHODS
[62]
to a final concentration of 0.4 mM. Batch adsorb the filtered supernatant onto 10 liters of DEAE-cellulose for 30 min, stirring with a paddle attached to a large drill motor. Wash the resin batchwise with 8-10 vol of buffer C, make a slurry of the resin in buffer C, pour into a large column (e.g., a Glenco column with a 15.4-cm diameter), elute with a linear 20liter NaC1 gradient (0.1-0.7 M), and collect fractions after the conductivity begins to increase in the eluate. This procedure allows the removal of CaZ+/calmodulin-activated phosphodiesterase during the wash step. Assay the fractions for phosphodiesterase activity using either 1 mM cGMP or 1 /xM cAMP. The 1 mM cGMP assay detects primarily cGMP-stimulated phosphodiesterase because the cGMP-inhibited phosphodiesterase hydrolyzes cGMP very poorly. The 1 tzM cAMP assay measures primarily cGMP-inhibited phosphodiesterase in the fractions, since cGMP-stimulated phosphodiesterase is relatively inactive under these conditions (Figs. 3 and 5). cGMP-Stimulated Phosphodiesterase Purification. As demonstrated in Fig. 5, the fractions pooled following anion-exchange chromatography contain both the cGMP-stimulated and cGMP-inhibited phosphodiesterases. Add benzamidine to the pooled fractions to a final concentration of 15 mM. Then apply the sample by gravity flow to two columns connected in series, in which the first column contains 20 ml of NHzN(CH2)2-cAMP-Sepharose and the second contains 60 ml of cGMPSepharose. Both resins should be equilibrated with buffer D prior to sample application. This step allows the removal and purification of cGMP-stimulated phosphodiesterase which binds specifically to the cGMP resin. The cAMP resin is mainly used to remove the type II regulatory subunit of cyclic nucleotide-dependent protein kinase (RII), to prevent that protein from competing with cGMP-stimulated phosphodiesterase for binding to the cGMP resin. Separate the two columns and wash the cGMP resin first with 250 ml of buffer D, and then with 250 ml of buffer D containing a final NaC1 concentration of 0.5 M, and finally wash again with 100 ml of buffer D. Warm the resin to room temperature and elute the cGMP-stimulated phosphodiesterase with buffer D containing 10 mM cGMP at a slow flow rate (10-20 ml/hr). Eluted fractions are collected in an adjacent cold box at 4° and assayed for phosphodiesterase activity and protein. Note that aliquots of each fraction should be incubated with 10 mM MgCI2 for 1 hr on ice, prior to the assay for phosphodiesterase activity. Any contaminating RII can be removed by adding the following steps. The phosphodiesterase-containing pooled fractions are incubated with 10 mM MgC12 for 1 hr on ice to allow complete hydrolysis of the cGMP. The completeness of the hydrolysis can be checked by thin-layer chromatog-
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
695
raphy of small aliquots from this incubation on polyethyleneimine-impregnated plastic strips using n-propanol: ammonium hydroxide:water (6:3:1). The phosphodiesterase samples are then passed through C 8H2N(CH2)2-NH-cAMP-Sepharose resin (0.9 x 8 cm) which was previously equilibrated with buffer D. The unbound cGMP-stimulated phosphodiesterase is essentially free of contaminating RII. This procedure routinely provides stable preparations of phosphodiesterase that are greater than 95% homogeneous, based on analysis by SDS-PAGE. The enzyme can be stored in this solution at 4° or in 20% glycerol at -20 °. Freezing or lyophilizing the native enzyme is not recommended. The enzyme is purified 16,400-fold by this procedure, with a yield of 19% and a specific activity of approximately 105 U/min. mg for cAMP or cGMP. 7 cGMP-Inhibited Phosphodiesterase Purification. cGMP-inhibited phosphodiesterase is prepared from the activity which flows through the cyclic nucleotide resins. Add MgCI2 to the flow-through fraction to a final concentration of 10 mM, and dilute the pool to a conductivity of 7.0 mmho (at 4°) with buffer E. The pH of the diluted sample should be between 7.8 and 8.0. Equilibrate 120 ml of blue dextran-Sepharose in buffer E and batch adsorb the sample to this resin overnight using a slowly turning paddle attached to a drill motor (do not use a magnetic stirring bar because it fractures the gel beads and results in very slow flow rates). Then wash the resin with 2 liters of the dilution buffer in a sintered glass funnel. Add enough wash buffer to make a slurry and pour the washed resin into a 4.5 x 7.5 cm column. Elute the phosphodiesterase activity from the resin with a 600-ml linear gradient of cAMP (0-8 mM) in buffer E. The ratio of resin volume to column height appears to be a critical variable. For example, poor chromatography is obtained with a column 2.5 x 24 cm, possibly because the cAMP used to elute the enzyme is hydrolyzed by the adsorbed phosphodiesterase during elution from the longer column. This chromatography step provides an additional 100 to 200-fold purification of the cGMP-inhibited phosphodiesterase activity. Pool the blue dextranSepharose fractions containing phosphodiesterase activity and adsorb the activity to an HLPC anion-exchange resin (TSK-DEAE-5-PW) using a large sample injection loop (e.g., Pharmacia Superloop) or multiple injections with a smaller sample injection loop. This work can be done at room temperature, but the fractions are collected at 4° in an adjacent cold box. Column life is prolonged by centrifuging the sample for 10 min in a Beckman airfuge at full speed prior to the HPLC step. The sample is loaded onto the column and then the column is washed in buffer F, and eluted with buffer F containing additional NaC1 added to a final concentration of 0.33 M NaC1. A second peak containing a smaller amount of phosphodiesterase activity is eluted with 0.5 M NaC1. The concentration of NaC1
696
PHOSPHODIESTERASE ISOZYME METHODS
[62]
required to elute the cGMP-inhibited phosphodiesterase may vary with different TSK-DEAE columns. Therefore, one should perform a pilot elution with an NaC1 gradient to determine the actual concentration of NaCI necessary to elute the phosphodiesterase. The enzyme is purified 16,000-fold with a yield of approximately 5% and a specific activity of 6/zmol of cAMP hydrolyzed/min • mg. 8 This procedure produces a highly purified phosphodiesterase; however, the enzyme preparations usually contain a mixture of several proteolytically related polypeptides ranging in molecular weight from 80,000 to 60,000. 8 We have not yet obtained a stable, homogeneous preparation of the enzyme, using this procedure. However, we have utilized the HPLC-purified phosphodiesterase as antigen for the production of polyclonal antisera and monoclonal antibodies. This preparation of phosphodiesterase appears to be very antigenic, since all of the mice injected with this protein developed isozyme-specific antisera. These antibodies have in turn proved to be very useful as specific probes for identifying cGMPinhibited phosphodiesterase protein and for isolating a stable enzyme, with an apparent subunit molecular weight of 110,000. 8 Procedure B: Rapid Isolation of Catalytic Amounts of Bovine Cardiac Muscle Phosphodiesterases on Solid-Phase Monoclonal Antibodies Procedures B and C are useful for rapidly isolating cGMP-inhibited, cGMP-stimulated, and Ca2+-calmodulin-activated phosphodiesterases. Since the solid-phase antibody procedures are the only methods available at this time for isolating nondegraded cGMP-inhibited phosphodiesterase, we have used that enzyme as an example for the description of these methods. Catalytic amounts of nondegraded cGMP-inhibited phosphodiesterase can be bound to the solid-phase monoclonal antibody reagent and the complex isolated for in vitro studies of phosphodiesterase kinetics, regulation, and pharmacology. 8 Polyclonal mouse antisera and hybridoma cell lines producing monoclonal antibodies directed against the cGMP-inhibited phosphodiesterase can be produced by established procedures, 8,12using the cGMP-inhibited phosphodiesterase preparation described in procedure A. Solid-phase antibody reagents are prepared using Staph A and RAM. The use of solidphase reagents allows isolation of a specific phosphodiesterase by precipitation of phosphodiesterase-antibody complexes. None of the antibodies used inhibits enzyme activity, and the phosphodiesterase is readily assayed on the resuspended immune pellet. In general, Staph A - R A M is t2 S. F a z e k a s de St.Groth and D. Scheidegger, J. lmmunol. Methods 35, 1 (1980).
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
697
prepared as follows: wash 0.5 ml of 10% Staph A twice with buffer G, resuspend in 0.5 ml of buffer G containing 150/zg of rabbit anti-mouse IgG antibodies, and incubate for 1 hr. Then wash the solid phase reagent two or three items with buffer G, This reagent is then resuspended in buffer G containing approximately 50/xg of anti-phosphodiesterase antibodies (for example CGI-5, an anti-cGMP-inhibited phosphodiesterase monoclonal antibody), incubated for 1 hr, and the washes are repeated. All steps in the procedure are performed on ice or in a cold room at 4°. Note that when CGI-5 antibody reagents are used, 0.1% Tween-20 is routinely used to replace BSA in buffer G. The detergent may interfere, however, with ACAP-1- and CGS-2-phosphodiesterase interactions. Incubate 1.5 ml of the bovine heart extract or DEAE-pooled fractions described above for 1.5 hr with either Staph A-RAM-primary antibody or Staph A-RAM control reagent, centrifuge, and then wash the solid-phase reagent once with 20 mM Tris-HCl buffer, pH 7.8 (at 4°), containing 0.5 M NaCI and twice with Tris-HC1 buffer minus the NaC1. Resuspend immune and control pellets in Tris-HC1 buffer using an appropriate dilution and assay for phosphodiesterase activity. Immune pellets with bound phosphodiesterase-antibody complexes can also be resuspended in 20 mM Tris-HC1 buffer containing 50% glycerol, pH 7.8, and stored for several months at - 2 0 °. This approach can also be used to isolate cardiac Ca2+/calmodulinactivated and cGMP-stimulated phosphodiesterases using ACAP-1 and CGS-2 monoclonal antibodies, as demonstrated in Figs. 3 and 5. Procedure C: Purification of Large Quantities of Phosphodiesterase Protein Using Solid-Phase Monoclonal Antibodies A modification of the monoclonal antibody purification procedure can be used to isolate homogeneous preparations of nonproteolyzed, but denatured, cGMP-inhibited phosphodiesterase protein in sufficient quantities for use in a variety of structural studies. All steps are performed on ice or at 4°. Prepare an extract from 2 kg of bovine cardiac muscle as described above. Add ascitic fluid containing 5 mg of CGI-5 to the extract, incubate for 1 hr, and then batch adsorb the extract onto 1 liter of DEAEcellulose for 30 min. Wash the resin with 10 liters of buffer C in a 2-liter sintered glass funnel. Elute the proteins from the resin stepwise with buffer C containing a final concentration of 300 mM NaCI, collect 250 to 300-ml fractions, and assay the fractions for conductivity and phosphodiesterase activity (measured at 1 tzM cAMP). Note that the conductivity of the first two fractions should be approximately 4.5 mmho and the conductivity of the third fraction should increase to approximately 7.07.5 mmho. The phosphodiesterase activity will elute in the third DEAE-
698
P H O S P H O D I E S T E R AISOZYME SE METHODS
[62]
cellulose fraction. Next, check the pH of this fraction (the pH should be 8.2-8.5) and apply by gravity flow onto 5 ml of protein A-Sepharose resin which has been previously equilibrated in buffer H at 4°. This resin will bind the CGI-5 monoclonal antibody : cGMP-inhibited phosphodiesterase complex. Wash the resin extensively, first with I0 vol of buffer H and then with 10 vol of buffer H containing no NaC|, and elute with 0.1 N acetic acid. Collect 2-ml fractions and measure eluted protein by A280. In our laboratory, the identity of the eluted protein is confirmed at this point in the procedure by protein immunoblot analysis and this polypeptide is the only Coomassie staining band in the region of 110 kDa on the SDS gels. Other polypeptides in this sample include IgG heavy and light chains and polypeptides with molecular weights of approximately 200,000-220,000 and 45,000 (these polypeptides comigrate with reduced fibronectin and with actin; however, their identities have not yet been determined). Phosphodiesterase-containing fractions are then lyophilized and stored prior to electrophoresis by SDS-PAGE. Lyophilized sample is resuspended in Laemmli sample buffer ~3 and polypeptides are separated electrophoretically on 10% polyacrylamide gels. The 110-kDa cGMP-inhibited phosphodiesterase protein is visualized by Coomassie staining and is then excised from the gel and electrophoretically transferred from the gel slices by the method of Hunkapillar et al.14 Dry the recovered protein by vacuum rotary evaporation. Next, wash the pellet with distilled and deionized water and dry the protein again by vacuum rotary evaporation. Repeat the washing and drying steps two more times. Residual Coomassie dye can be removed as follows: resuspend the dried protein in neat methanol for 1 hr at 4°, centrifuge the suspension for 15 rain at full speed in an Eppendorf microfuge, removed and discard the supernatant fraction. Then wash with distilled water and dry by evaporation three times, as described above. This protein preparation is suitable for studying the protein chemistry of cyclic nucleotide phosphodiesterases, as demonstrated below. Comparison of cGMP-Inhibited and cGMP-Stimulated Phosphodiesterases Table I summarizes the amino acid compositions that have been determined for cGMP-inhibited and cGMP-stimulated phosphodiesterases using enzymes isolated by the electroelution procedure. The composition of 13 U. K. Laemmli, Nature (London) 227, 680 (1970). ~4M. W. Hunkapillar, E. Lujan, F. Ostrander, and L. E. Hood, this series, Vol. 91, p. 227.
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
699
TABLE I AMINO ACID COMPOSITIONS OF cGMP-STIMULATED AND cGMP-INHIBITED PHOSPHODIESTERASES~
Amino acids Asx b Thr Ser Glx' Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Trp
Affinity-purified cGMP-stimulated pbosphodiesterase (mol%) 10.4 4.1 5.4 13.1 5.9 5.6 9.0 NI a 6.4 2.6 4.6 13. l 2.9 4.2 5.1 3.0 4.4 NI
Electroeluted cGMP-stimulated phosphodiesterase (mol%) 10.9 3.8 5.7 13.9 4.0 8.0 7.9 ND ~ 6.4 2.4 4.9 13.2 2.5 3.8 5.3 2.8 4.4 ND
Electroeluted cGMP-inhibited phosphodiesterase (mol%) 10.1 4.5 5.6 13.0 7.7 6.0 7.9 ND 6.5 1.8 5.6 10.6 2.2 3.9 6.0 3.0 5.6 ND
Determined by the method of B. A. Bidlingmeyer, S. A. Cohen, and T. L. Tarvin, J. Chromatogr. 336, 93 (1984). h Combined asparagine and aspartic acid. ' Combined glutamine and glutamic acid. ,/NI, not included in the calculations of mol% for purposes of this comparison. '~ ND, not determined.
the electroeluted cGMP-stimulated phosphodiesterase was compared with the composition determined for cGMP-stimulated phosphodiesterase isolated by procedure A, as described above. The comparison between these two methods enabled us to use the cGMP-stimulated enzyme as a positive control for the cGMP-inhibited phosphodiesterase protein, purified by procedure C. The two phosphodiesterase isozymes have nearly identical amino acid compositions, which is not surprising considering the similar sizes and functions of the two proteins. It will be very interesting to compare the amino acid sequences of these two cGMP-sensitive phosphodiesterase isozymes. Table II presents a variety of physical and catalytic properties of the bovine heart cGMP-inhibited and cGMP-stimulated
700
[62]
PHOSPHODIESTERASE ISOZYME METHODS T A B L E 11 PROPERTIES OF cGMP-STIMULATED AND cGMP-INHIBITED PHOSPHODIESTERASES
Properties
cGMP-stimulated phosphodiesterase
cGMP-inhibited phosphodiesterase
Monomer MW Subunit structure
Dimer
K m cAMP Km cGMP Vmax cAMP b Vmax cGMP"
30 k~M 10 ~ M 105 U/mg 105 U/mg
110,00 Dimer? 0.15 # M 0.06/~M ~ 6.0 U/mg 0.6 U/mg J
Modulator Phosphorylation e
cGMP (stimulation) No
cGMP (inhibition) Yes
105,000
Inhibitor Ki (kLM)
lnhibitors Milrinone Fenoximone IBMX
Papaverine RO 20-1724
cGMP-stimulated
cGMP-inhibited
PDE
PDE
180 430 14 4.5 300
0.26 1.5 1.3 0.66 62
" Ki of cGMP as a competitive inhibitor of cAMP hydrolysis. h U = ~mol cAMP hydrolyzed/min at 30 °. ' U = p.mol cGMP hydrolyzed/min at 30 °. a Estimated for pure enzyme from Vmaxratios of cAMP/cGMP hydrolysis by preparations of enzyme-antibody complex. e I n vitro phosphorylation by cAMP-dependent protein kinase.
phosphodiesterases. These two enzymes represent potential control points, at which cGMP could regulate cAMP metabolism. Notice that although the two isozymes coelute from DEAE-cellulose and are similar in size and sensitivity to cGMP, they exhibit very different catalytic properties, regulation by cGMP, and affinities for inhibitors. The differential sensitivities to inhibitors is particularly striking for the new cardiotonic agents, milrinone and fenoximone. Highly selective inhibition of cardiac cGMP-inhibited phosphodiesterase, relative to the other heart isozymes, is a possible mechanism of action for these drugs. 8 In addition, cGMPinhibited phosphodiesterase is phosphorylated in vitro by cAMP-dependent protein kinase under conditions which do not result in phosphorylation of cGMP-stimulated phosphodiesterase.
[62]
BOVINE HEARTcGMP PHOSPHODIESTERASES
701
General Comments Three isolation procedures are described in this chapter. The first procedure results in purification of cGMP-stimulated phosphodiesterase by affinity chromatography using cGMP-Sepharose and purification of cGMP-inhibited phosphodiesterase by pseudoaffinity chromatography on blue dextran-Sepharose. The second approach utilizes specific monoclonal antibodies for isolation of antibody-phosphodiesterase complexes. This immunological method is demonstrated by the rapid isolation of cGMP-inhibited phosphodiesterase, an enzyme which is very sensitive to proteolytic degradation. The third method combines specific binding of the phosphodiesterase to a monoclonal antibody, adsorption of that complex to an antibody-binding resin, and electroelution of the phosphodiesterase protein from a gel slice after SDS-PAGE. This method results in purification of sufficient quantities of nonproteolyzed, denatured phosphodiesterase for structural studies. These approaches allow one to study the physical and catalytic characteristics of the phosphodiesterase isozymes. It is our experience that when EDTA is present in the cardiac muscle homogenization buffer, as originally reported by Martins el al., 7 the cGMP-inhibited phosphodiesterase activity is significantly decreased by an as yet unknown mechanism. This chelator-dependent decrease in cGMP-inhibited phosphodiesterase activity effectively unmasks the cGMP-stimulated phosphodiesterase in bovine heart extracts (Harrison and Beavo, unpublished observations). The large-scale ligand affinity purification procedure described in this chapter is highly satisfactory for purification of large quantities of cGMP-stimulated phosphodiesterase. Note, however, that solutions containing greater than 0.5 mg/ml of the purified cGMP-stimulated phosphodiesterase protein have a tendency to aggregate. One must also take care not to saturate the cAMP resin and contaminate the phosphodiesterase preparation with RII. Enzyme purified by this method has been useful for characterization of physical and catalytic properties 7 and is now proving useful for determining the amino acid sequence of the cGMP-stimulated phosphodiesterase. Problems still exist with pseudoaffinity purification of the cGMP-inhibited phosphodiesterase. The procedure produces predominantly an 80kDa polypeptide which slowly degrades to 67- and 60-kDa polypeptides. 8 Some improvement in the stability of the blue dextran-Sepharose-purifled protein can be obtained by adjusting buffer E to include 10 tzM leupeptin, 10 p.M pepstatin A, 2 mM o-phenanthroline, 2 mM EGTA, and 3 mM EDTA. HPLC-purified cGMP-inhibited phosphodiesterase contained several enzyme fragments which are eluted from the TSK-DEAE
702
PHOSPHODIESTERASE ISOZYME METHODS
[63]
resin by 0.33 M NaCI. Recently we have found that the 110-kDa enzyme is eluted by a higher salt concentration. Use of the protease inhibitors allows us to obtain a greater proportion of the enzyme in the second HPLC peak containing the 110-kDa form; however, as stated above, we have not yet obtained a stable, homogeneous preparation of the enzyme by this procedure, cGMP-inhibited phosphodiesterase purification using monoclonal antibodies does allow us to identify a high-molecular-weight form, characterize catalytic properties, detect phosphorylation by cAMPdependent protein kinase, and identify several highly selective inhibitors. 8,~5 The large-scale version of this method should also be useful for producing enzyme for protein structural studies, and it should be useful in the development of probes for studying the molecular biology of the enzyme. Acknowledgements The authors would like to thank Dr. H. Charbonneau for his helpful discussions and for his expert assistance with our determinations of the phosphodiesterase amino acid compositions. This work was supported by National Institutes of Health Grant AM 21723. 15 S. A. Harrison, M. L. Chang, and J. A. Bevo, Circulation (Suppl. I11), 73, 109-116 March (1986).
[63] P u r i f i c a t i o n o f R o d O u t e r S e g m e n t G T P - B i n d i n g Protein Subunits and cGMP Phosphodiesterase by Single-Step Column Chromatography
By AKIO YAMAZAKI, MASAHIROTATSUMI, and MARK W. BITENSKY Introduction There is a well-established homology between the hormone-sensitive adenylate cyclase and the light-activated cGMP phosphodiesterase (PDE) in rod outer segments (ROS).~ The adenylate cyclase system is composed of three principal segments2: A receptor moiety for stereospecific binding of hormones or neurotransmitter molecules, the GTP-binding compoA. Yamazaki, K. R. Halliday, J. S. George, S. Nagao, C.-H. Kuo, K. S. Ailsworth, and M. W. Bitensky, Adu. Cyclic Nucleotide Protein Phosphorylation Res. 19, 113 (1985). 2 M. Rodbell, Nature (London) 284, 17 (1980).
METHODS IN ENZYMOLOGY,VOL. 159
Copyright © 1988by Academic Press, Inc. All rights of reproductionin any form reserved.