- Email: [email protected]
[69] Isolation and characterization of insulin-stimulated, high-affinity cAMP phosphodiesterases from rat liver
[69]
HIGH-AFFINITY LIVER cAMP PHOSPHODIESTERASE
751
presence of 1 m M EDTA. 7 The Stokes radius of the solubilized basal e n z y m e is 8.7 nm, 7 that of the solubilized plus-insulin enzyme is 9.4 nm, 7 and that of the catalytic domain is 4.6-5.4 nm. 9 Acknowledgments The methods presented above have been tested and improved over a period of several years in collaboration with F. W. Robinson, J. A. Sarver, Dr. R. H. Pointer, Dr. P. M. de Buschiazzo, Dr, H. Makino, J. E. Jordan, and Dr. M. Ueda. Our original studies on this subject were supported by NIH Grant 5R01 AM 06725.
[69] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f I n s u l i n - S t i m u l a t e d , High-Affinity cAMP
Phosphodiesterases
from Rat Liver
By MILES D. HOUSLAY, NIGEL J. PYNE, and MICHAEL E. COOPER Introduction The ability of glucagon to elevate intracellular cAMP concentrations in hepatocytes is antagonized by insulin.l This effect is achieved through inhibition of adenylate cyclasC and activation of specific cAMP phosphodiesterases.l,3,4 The activation of two specific cAMP phosphodiesterases can readily be assessed using a rapid Percoll gradient fractionation procedure to resolve the various membrane fractions. 1 Both of these enzymes express a high affinity and specificity for cAMP. One of them is a peripheral protein found associated exclusively with the plasma membrane 5 and is activated by its phosphorylation, which is triggered by insulin. 6 The other is associated with an as yet undefined intracellular dense-vesicle fraction.~ The mechanism of its activation has yet to be defined but may involve m e m b r a n e processing/translocation reactions. 7 The dense-vesicle e n z y m e can also be activated by glucagon through a process which is distinct from that whereby insulin elicits its activation. 1,7 i C. M. Heyworth, A. V. Wallace, and M. D. Houslay, Biochem. J. 214, 99 (1983). 2 C. M. Heyworth and M. D. Houslay, Biochem. J. 214, 547 (1983). 3 M. D. Houslay, A. V. Wallace, S. R. Wilson, R. J. Marchmont, and C. M. Heyworth, Horm. Cell Regul. 7, 105 (1983). 4 M. D. Houslay, Mol. Aspects Cell. Regul. 4, 279 (1985). 5 M. D. Houslay and R. J. Marchmont, Biochem. J. 198, 703 (1981). 6 R. J. Marchmont and M. D. Houslay, Biochem. J. 195, 653 (1981). 7 S. R. Wilson, A. V. Wallace, and M. D. Houslay, Biochem. J. 216, 245 (1983).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
752
PHOSPHODIESTERASE ISOZYME METHODS
[69]
Reagents All buffers were prepared in glass-distilled water which had been deionized and were then kept at 0-4 °. Adult Sprague-Dawley rats, 200-300 g weight Bicarbonate buffer: 1 mM potassium bicarbonate, pH 7.4 Buffer A: 0.5 M NaCI, 20 mM Tris-HC1 buffer, final pH 7.4, at 4° Buffer B: 0.075 M NaC1, 5 m M MgCl2, 2 mM 2-mercaptoethanol, 20 m M Tris-HC1, final pH 7.4. Also a solution of this buffer but with 0.4 M NaC1, final pH 7.4 Buffer C: As buffer B but with 0.2 M NaCI, final pH 7.4 Buffer D: As buffer B but with 1.0 M NaCI, final pH 7.4 Buffer E: 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris-HC1 buffer, final pH 7.4 at 4° Buffer F: 1 m M EDTA, 10 mM Tris-HCl buffer, final pH 7.4 Buffer G: 1 m M MgCI2, 10 mM Tris-HCl buffer, final pH 7.4 Buffer H: 1 m M MgC12, 0.15 M NaCl, 20% ethylene glycol, 10 mM Tris-HC1 buffer, final pH 7.4 Coupling buffer: 0.1 M NaHCO3 + 0.5 M NaCl, final pH 9.8 0.5 M NaC1, 0.! M sodium acetate, final pH 4.0 Whatman DEAE-cellulose Butyl-agarose Aminopentyl-agarose Affi-Gel blue Sephadex G-25 ECTEOLA-cellulose 6-Chloroguanine Ethylenediamine Ethanolamine CNBr-activated Sepharose 4B PMSF Dowex 1 (200-400 mesh) Ophiophagus hannah venom [3H]cAMP [3H]Adenosine A s s a y of c A M P P h o s p h o d i e s t e r a s e c A M P p h o s p h o d i e s t e r a s e ( P D E ) a c t i v i t y in p u r i f i e d / p a r t i a l l y purified p r e p a r a t i o n s w a s m e a s u r e d b y a m o d i f i c a t i o n 8 o f the t w o - s t e p p r o c e d u r e o f T h o m p s o n a n d A p p l e m a n . 9 W h e n P D E a c t i v i t y w a s m e a s u r e d in ho8 R. J. Marchmont and M. D. Houslay, Biochem. J. 187, 381 (1980). 9 W. J. Thompson and M. M. Appleman, Biochemistry 10, 311 (1981).
[69]
HIGH-AFFINITY LIVERcAMP PHOSPHODIESTERASE
753
mogenates, in order to compensate for any underestimation of activity owing to adenosine deaminase ~° activity being present, the Dowex resin was prepared fresh as a l : 1 : 1 (v/v/v) slurry of resin : H20 : ethanol and used thus. ~1Routinely a [cAMP] of 1.0/zM was employed for the peripheral PDE and 0.1/zM for the dense-vesicIe PDE, both with an [Mg2~] of 5 mM. It was checked that cAMP degradation was linear with respect to time and initial rates of degradation were assessed. Activity is expressed in international units (i.u.) when 1 U is the production of 1/zmol product/ min/mg protein. A 100-/zl reaction mixture, in Eppendorf (I .8-ml) tubes, contained final concentrations of 5 m M MgCI2, [3H]cAMP ( - 100,000 cpm), 20 mM TrisHCI, final pH 7.4, and an appropriate amount of enzyme. During kinetic analysis [cAMP] range from 4 x 10 -7 to 1! X 10-3 M. Reaction tubes were vortexed and incubated at 30° for an appropriate time interval, usually 15 min (linearity should be ascertained under all conditions for initial rate analysis). Termination of assays was achieved by removal of tubes to a boiling H20 bath for 2 rain. The tubes were then cooled to 4° before the addition of 25 p~g of Ophiophagus hannah venom (l mg/ml). This was then incubated for 10 min at 30° in order to allow for the conversion of 5'AMP to adenosine; 0.4 ml of resin slurry (Dowex 1; 200-400 mesh; C1form as resin : H20, ! : 2 (v/v) mixture) was then added to the tubes. They were then vortexed several times over a 15-rain period before being centrifuged at 14,000 gay for 4 rain in order to sediment the resin thoroughly. A 150-/.d aliquot of the supernatant was taken for liquid scintillation counting of [3H]adenosine. The resin will bind some adenosine, often as much as 50%, and this can be evaluated by control experiments by assessing the binding of [3H]adenosine over a range of concentrations. In all cases blanks were performed with no enzyme added and these counts subtracted from test experiments. The value of the blank can be affected by changes in buffer constituents (e.g., NaC[). This needs careful attention, especially during purification. Also high substrate [cAMP] may affect [3H]adenosine binding, which again can be tested for. Product inhibition studies, using 5'-AMP, require careful evaluation of the completion of conversion of 5'-AMP to adenosine when treating with the snake venom. Purification of the Peripheral cAMP Phosphodiesterase from Rat Liver Plasma Membranes The method described below uses a crude membrane fraction for the extraction of this enzyme. This obviates the need to purify liver plasma ~0 W. J. Rutten, B. M. Schoot, and J. S. H. Dupont, Biochim. Biophys. Acta 315, 378 (1983). H j. L o n d e s b o r o u g h , Anal. Biochem. 71, 623 (1976).
754
PHOSPHODIESTERASE ISOZYME METHODS
[69]
membranes for the starting material, as has been used for an alternative purification strategy described elsewhere by us.12 In that procedure the plasma membrane PDE was eluted from Affi-Gel blue by high salt only. However, batches used over the last 2 years demand the addition of cAMP + MgCI2 to elicit its elution. Step 1. Using a glass homogenizer and rotating Teflon pestle, six freshly isolated rat livers were homogenized in ice-cold bicarbonate buffer [3 : 1 (v/v), liver : buffer) using six to eight additional strokes at 400 rpm after dispersion. This was centrifuged for 10 min at 1500 gay, the resultant supernatant discarded, and the pellet resuspended in ice-cold bicarbonate buffer [1 : 3 (v/v), pellet : buffer]. This was recentrifuged at 10,000 gay for I0 min and the resuspension and centrifugation process repeated once more to yield a washed pellet fraction. Step 2. The washed pellet fraction was resuspended in 120-150 ml of ice-cold buffer A and left at 4° for 1 hr before being centrifuged at 48,000 gay for 30 min at 4°. The supernatant was then dialyzed overnight at 4 ° against two changes of buffer B (2 x, 1 liter) (yield 72%, purification x 3). Step 3. The dialysate was recentrifuged as per step 2 prior to the supernatant being applied, at a flow rate of 0.8 ml/min, to a column (3.5 x 40 cm) of DEAE-cellulose previously equilibrated in buffer B. The column was then washed with 200 ml of buffer B prior to eluting PDE activity (4-ml fractions) with a 300-ml linear NaCl gradient (0.075-0.4 M) in buffer B, final pH 7.4. Two peaks of activity were eluted between 0.2 and 0.275 M NaCl [peaks A and B, respectively (Fig. la)]. The fractions of peak A which exhibited the highest specific activity for PDE were taken for the next step (yield 45%, purification x27). Step 4. Peak A fractions were applied (0.75 ml/min) to a butyl-agarose column (2 x 5 cm) equilibrated in buffer C (Fig. 1b). Over 90% of the PDE activity failed to bind to this column, and this eluted activity was collected and taken for step 5 (yield 40%, purification x 30). Step 5. The pooled eluate was applied (0.5 ml/min) to a column (0.5 x 3 cm) of Affi-Gel blue previously equilibrated with buffer C (Fig. lc). After loading, this column was washed with 100 ml of buffer C, followed by 100 ml of buffer D, followed by 50 ml of buffer D containing 1 mM ATP, 1 m M NAD +, with l m M EDTA in place of the MgC12. A final wash of 200 ml of buffer D was made prior to enzyme elution with 15 ml of buffer D containing 1 m M cAMP (elution buffer). This was done by running 5 ml of this elution buffer into the column, stopping the flow, leaving the column for 1 hr at 4° and then restarting the flow (0.2 ml/min) while collecting fractions (0.2 ml). Active fractions (normally 5 ml), from step 5, were applied to a column (2.5 x 50 cm) of Sephadex G-25 equilibrated lZ R. J. M a r c h m o n t , S. Ayad, and M. D. Houslay, Biochem. J. 195, 645 (1981).
[69]
HIGH-AFFINITY LIVER cAMP PHOSPHODIESTERASE B
755
04
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FIG. 1. Purification of the peripheral plasma membrane enzyme. (a) DEAE-cellulose column with peak A being pooled for further study and arrow (i) being the start of the NaCl gradient, (b) butyryl-agarose chromatography of peak A, (c) Affi-Gel blue chromatography step with arrows (ii), (iii), and (iv) indicating buffer changes. PDE activity was assessed at 1 ~M [cAMP] at 30°. Activity is expressed as nmol/min/ml fraction. Protein is given as OD280. with buffer B (15 ml/hr) and fractions o f highest specific activity collected (yield 18%, purification ×3246). In s o m e instances this final stage had small a m o u n t s ( < 10%) o f l o w - m o l e c u l a r - w e i g h t impurities which could be r e m o v e d b y gel filtration on S e p h a c r y l 200 or a similar type o f c o l u m n equilibrated in buffer B. Purification of the "Dense-Vesicle" c A M P Phosphodiesterase W e h a v e used t w o distinct m e t h o d s , with similar success, to purify this e n z y m e to a p p a r e n t h o m o g e n e i t y . One o f these is described below.
756
PHOSPHODIESTERASE ISOZYME METHODS
[69]
The specific solubilization of this enzyme takes advantage of the hypotonic shock procedure devised by Loten et al.~3 involving the proteolytic release of this membrane-bound enzyme 14from an as yet undefined intracellular vesicle fraction. 2 Step 1. Three rat livers (1 vol) are rapidly excised, rinsed in ice-cold buffer E, and blotted dry. They are then diced with scissors prior to homogenization in 8 vol of buffer E using a glass homogenizing vessel with a Teflon pestle rotating at 250 rpm for eight up and down strokes. The homogenate is filtered through cheesecloth and centrifuged at 1500 gay for 10 min at 4°. Step 2. The supernatant is carefully removed and recentrifuged at 25,000 gav for 20 min at 4°. The resulting pellet is resuspended in 2-3 vol of ice-cold buffer F and left on ice for 40 min. This allows for the solubilization of the dense-vesicle cAMP phosphodiesterase. After this time the solution is centrifuged at 48,000 gay for 30 min and the supernatant taken for the next step. In some instances, from this point onward, and not before, solutions are made 0.1 m M with PMSF and 2 mM benzamidine (yield 100%, purification x 3.8). Step 3. A column of ECTEOLA-cellulose is equilibrated with buffer G prior to application (0.5 ml/min) of the supernatant extract (Fig. 2, top). Buffer G is then passed through the column until no further protein (constant OD280) is eluted. The column is then washed with buffer H until no further protein comes off prior to the elution of the dense vesicle enzyme using buffer H containing 0.4 M NaCl (final pH 7.4) (yield 71%, purification x51). Step 4. Fractions of highest specific activity are diluted 1 : 1 (v/v) with buffer G and applied (0.4 ml/min) to a column (1 x 5 cm) of aminopentylagarose that has been preequilibrated with buffer G (Fig. 2, middle). Buffer G is used to wash the column until no further protein elutes and then with buffer H containing 0.275 M NaCI until no further protein is eluted. The dense-vesicle enzyme is then eluted using buffer H containing 0.5 M NaCI and fractions of highest specific activity are collected (yield 48%, purification x 1068). Step 5. The enzyme sample is desalted on a Sephadex G-25 column which has been previously equilibrated with buffer G prior to application (0.1 ml/min) of the sample to a guanine-Sepharose column. This guanine° Sepharose column is equilibrated with buffer G 15 prior to the loading of the sample. ~3 E. G. Loten, F. D. A s s i m a c o p o u l o s - J e a n n e t , J. H. Exton, and C. R. Park, J. Biol. Chem. 253, 746 (1978). ~4 E. G. Loten, S. H. Francis, and J. D. Corbin, J. Biol. Chem. 255, 7838 (1980). ~5 R. H. W h i t s o n and M. M. Appleman, Biochim. Biophys. Acta 714, 279 (1982).
[69]
HIGH-AFFINITY LIVERcAMP PHOSPHODIESTERASE
757
The preparation of this guanine-Sepharose column involves, first, the synthesis of ethylamine guanine. For this 2 mmol of chloroguanine plus 2 mmol of ethylenediamine are added to 5 ml HzO. Then I0 M NaOH is added dropwise, with stirring, until the ethylenediamine goes into solution. This mixture is refluxed with extreme care in a protected area for 12 hr until a yellow paste forms. This product is then coupled to CNBractivated Sepharose 4B. To achieve this, 6 g of lyophilized CNBrSepharose is taken up in water to give a 20-ml gel slurry. To 20 ml of gel is added 5 ml of the refluxed product and 35 ml of coupling buffer. These components are mixed and then left at room temperature (18-21 °) for 20 h or at 4° for 3 days. The resultant gel mixture is washed with 20 vol of coupling buffer before adding 2 vol of coupling buffer containing 1 M ethanolamine per volume of gel. This is left for 20 hr at 23 ° before washing sequentially with (1) 10 vol coupling buffer, (2) 10 vol of 0.5 M NaC1, 0. I M sodium acetate, final pH 4.0, (3) 4 vol of coupling buffer. The gel can now be stored at 4 °. Just before use it is washed (4 vol) with buffer G and then thoroughly equilibrated with buffer G prior to its use. This column is washed with buffer G until no further protein elutes. The dense-vesicle enzyme is then eluted with buffer H containing 0.5 M NaC1 (Fig. 2, bottom). Fractions of highest specific activity are collected (yield 18%, purification × 2773).
Properties of the Two cAMP Phosphodiesterases (PDE) Both procedures yield preparations exhibiting a single protein band which comigrated with PDE activity on nondenaturing gels. The plasma membrane enzyme was purified some 3246-fold, over the specific activity of the membrane fraction from which it was isolated, with an 18% yield. This enzyme, assayed at 30°, exhibited a Vma×of 3.6 U/rag protein and showed kinetics indicative of apparent negative cooperativity with an apparent Km of 0.9/zM and Hill coefficient (h) of 0.54. As only a single PDE species was present then the kinetic mechanism for cAMP hydrolysis is evidently unusual. One possibility is that it obeys a mnemonical type of kinetic mechanism. J2 This PDE showed a high specificity for cAMP, as cGMP was hydrolyzed only slowly ( Vma×0.2 U/rag protein, Km 105/zM) obeying, apparently, Michaelis kinetics. In accord with this, the hydrolysis of cAMP was relatively insensitive to inhibition by cGMP. This PDE had a subunit molecular mass of 52.4 kDa and appeared as a monomer (51.2 kDa) on sucrose density gradient centrifugation although a dimeric species (104 kDa) could be found on molecular sieving using either Sephadex G-100 or Sepharose S-200. Tryptic iodinated peptide
758
PHOSPHODIESTERASE ISOZYME METHODS 0"3
[69]
8
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fraction N~ FIG. 2. Purification of the dense-vesicle enzyme. (a) ECTEOLA-cellulose step with the PDE activity being eluted in the third and final step, after the buffer change [arrow (i)], being taken for further study, (b) aminopentyl-agarose chromatography with arrows signifying buffer changes, and (c) guanine-Sepharose chromatography with arrows signifying buffer changes. Assays were performed at 0.1 ttM [cAMP] at 30°. Activity is expressed as nmol cAMP hydrolyzed/min/mg protein. Protein is given at Azs0(arbitrary units).
m a p p i n g s h o w e d it to b e d i s t i n c t f r o m o t h e r P D E s 16 as well as t h e d e n s e v e s i c l e e n z y m e ( u n p u b l i s h e d ) . P o l y c l o n a l a n t i b o d i e s p r e p a r e d in r a b b i t a g a i n s t this p u r i f i e d e n z y m e d i d n o t c r o s s - r e a c t w i t h e i t h e r t h e d e n s e v e s i c l e o r c G M P - s t i m u l a t e d P D E s p r e p a r e d f r o m rat liver. 17 I n s u l i n elicits t h e p h o s p h o r y l a t i o n o f this e n z y m e w h i c h l e a d s to a c t i v a t i o n r e f l e c t e d in a d e c r e a s e in t h e Hill c o e f f i c i e n t 6 f o r c A M P h y d r o l y s i s • T h e r e a s o n t h a t this P D E is f o u n d a s s o c i a t e d e x c l u s i v e l y w i t h t h e p l a s m a m e m b r a n e is t h a t it b i n d s to a specific i n t e g r a l p r o t e i n in this m e m b r a n e . 5 16D. J. Takemoto, J. Hansen, L. J. Takemoto, and M. D. Houslay, J. Biol. Chem. 257, 14597 (1982). ~7N. J. Pyne, M. E. Cooper, and M. D. Houslay, Biochem. J. 242, 33 (1984).
[69]
HIGH-AFFINITY LIVERcAMP PHOSPHODIESTERASE
759
The dense-vesicle enzyme was purified some 2773-fold, over the specific activity of the homogenate from which it was isolated, with an 18% yield. The enzyme, as purified, is a proteolytically clipped version of the native enzyme, as endogenous lysosomal proteases, which are released by the hypotonic shock procedure, affect its solubilization.13,14 The solubilized enzyme can now be purified by standard techniques. The clipped enzyme behaves like a normal soluble enzyme and still exhibits enhanced activity after activation by insulin and glucagon. ~.~3Thus, it is highly likely that the functional globular mass of this PDE is not normally embedded in the bilayer but is found without the bilayer anchored by a small hydrophobic pedicle which spans the bilayer. The dense-vesicle PDE, assayed at 30°, had a Vmaxof 0.7 U/mg protein and exhibited kinetics indicative of apparent negative cooperativity with an apparent Km of 0.3 /xM and a Hill coefficient (h) of 0.43. The enzyme preparation contained only one species of PDE and thus it remains to be seen whether either the negative cooperative kinetics or some other type of mechanism yielding apparent negative cooperativity is present. The enzyme hydrolyzed cGMP slowly with a Vmaxof only 4 milliU/mg protein. However, it still succeeded in binding cGMP with high affinity as the Km for cGMP was 10/xM. Indeed the hydrolysis of cAMP by this enzyme is very sensitive to inhibition by cGMP, which is in contrast to the plasma membrane enzyme. This feature can be used diagnostically to distinguish these two enzymes. The dense-vesicle PDE, as isolated, has a subunit mass of 57 kDa, although a species of 51 kDa may also be found upon further proteolysis during purification. The enzyme appears as a dimer of 114 kDa upon either sucrose density gradient analysis or gel filtration. Tryptic-iodinated peptide mapping shows this enzyme to be distinct from the plasma membrane species and, furthermore, a polyclonal antibody raised against this enzyme did not cross-react with the plasma membrane or any other rat liver PDE. However, such an antibody can be used to identify the native, membrane-bound dense-vesicle PDE as an integral membrane protein of subunit molecular mass 63 kDa. Both of these two PDEs exhibit similar kinetics as regards the hydrolysis of cAMP, but not cGMP. However, various PDE inhibitors can be seen to exhibit different (selective) potencies as inhibitors of cAMP hydrolysis by these two enzymes (Table I). These two enzymes are also expressed differentially in various rat tissues. 18 In summary, two distinct high-affinity, membrane-bound cAMP phosphodiesterases can be isolated from rat liver. These are a peripheral plasma membrane enzyme which is activated by insulin alone I and the t8 N. J. Pyne, N. Anderson, B. E. Lavan, G. Milligan, H. G. Nimmo, and M. D. Houslay, Biochem. J. 248, 897 (1987).
760
PHOSPHODIESTERASE ISOZYME METHODS
[70]
TABLE I SELECTIVE INHIBITION OF THE TWO HORMONE-STIMULATED MEMBRANE-BOUND c A M P PHOSPHODIESTERASES FROM RAT L1VERa Inhibition (%)
Drug
Peripheral PDE
Dense-vesicle PDE
Milrinone Zaprinast Dipyridamole RO-1724 IC1 63,197
61 19 35 58 56
0 31 9 38 0
" A s s a y s were performed using the purified enz y m e s with 1 ~ M c A M P substrate concentration and 1 0 / z M drug.
dense-vesicle enzyme which is activated by both insulin and glucagon through apparently distinct mechanisms. 1,7,~3,14 Acknowledgments This work was supported by grants from the Medical Research Council (UK), California Metabolic Research Foundation, British Diabetic Association, British Heart Foundation, Agriculture & Food Research Council, and the Scottish H o m e and Health Department.
[70] Preparation of Dog Kidney High-Affinity c A M P Phosphodiesterase
By W. J. THOMPSON, C.-C. SHEN, and S. J. STRADA Introduction
A nomenclature developed at a recent symposium on cyclic nucleotide phosphohydrolases (EC 3.1.4.17, see Strada and Thompson 1) attempted to categorize the enzyme system on the basis of substrate preference and S. J. Strada and W. J. T h o m p s o n (eds.), Adv. Cyclic" Nucleotide Protein Phosphorylation Res. 16 (1984).
METHODS IN ENZYMOLOGY,VOL. 159
Copyright © 1988by AcademicPress, Inc. All rights of reproduction in any form reserved.
HIGH-AFFINITY LIVER cAMP PHOSPHODIESTERASE
751
presence of 1 m M EDTA. 7 The Stokes radius of the solubilized basal e n z y m e is 8.7 nm, 7 that of the solubilized plus-insulin enzyme is 9.4 nm, 7 and that of the catalytic domain is 4.6-5.4 nm. 9 Acknowledgments The methods presented above have been tested and improved over a period of several years in collaboration with F. W. Robinson, J. A. Sarver, Dr. R. H. Pointer, Dr. P. M. de Buschiazzo, Dr, H. Makino, J. E. Jordan, and Dr. M. Ueda. Our original studies on this subject were supported by NIH Grant 5R01 AM 06725.
[69] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f I n s u l i n - S t i m u l a t e d , High-Affinity cAMP
Phosphodiesterases
from Rat Liver
By MILES D. HOUSLAY, NIGEL J. PYNE, and MICHAEL E. COOPER Introduction The ability of glucagon to elevate intracellular cAMP concentrations in hepatocytes is antagonized by insulin.l This effect is achieved through inhibition of adenylate cyclasC and activation of specific cAMP phosphodiesterases.l,3,4 The activation of two specific cAMP phosphodiesterases can readily be assessed using a rapid Percoll gradient fractionation procedure to resolve the various membrane fractions. 1 Both of these enzymes express a high affinity and specificity for cAMP. One of them is a peripheral protein found associated exclusively with the plasma membrane 5 and is activated by its phosphorylation, which is triggered by insulin. 6 The other is associated with an as yet undefined intracellular dense-vesicle fraction.~ The mechanism of its activation has yet to be defined but may involve m e m b r a n e processing/translocation reactions. 7 The dense-vesicle e n z y m e can also be activated by glucagon through a process which is distinct from that whereby insulin elicits its activation. 1,7 i C. M. Heyworth, A. V. Wallace, and M. D. Houslay, Biochem. J. 214, 99 (1983). 2 C. M. Heyworth and M. D. Houslay, Biochem. J. 214, 547 (1983). 3 M. D. Houslay, A. V. Wallace, S. R. Wilson, R. J. Marchmont, and C. M. Heyworth, Horm. Cell Regul. 7, 105 (1983). 4 M. D. Houslay, Mol. Aspects Cell. Regul. 4, 279 (1985). 5 M. D. Houslay and R. J. Marchmont, Biochem. J. 198, 703 (1981). 6 R. J. Marchmont and M. D. Houslay, Biochem. J. 195, 653 (1981). 7 S. R. Wilson, A. V. Wallace, and M. D. Houslay, Biochem. J. 216, 245 (1983).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
752
PHOSPHODIESTERASE ISOZYME METHODS
[69]
Reagents All buffers were prepared in glass-distilled water which had been deionized and were then kept at 0-4 °. Adult Sprague-Dawley rats, 200-300 g weight Bicarbonate buffer: 1 mM potassium bicarbonate, pH 7.4 Buffer A: 0.5 M NaCI, 20 mM Tris-HC1 buffer, final pH 7.4, at 4° Buffer B: 0.075 M NaC1, 5 m M MgCl2, 2 mM 2-mercaptoethanol, 20 m M Tris-HC1, final pH 7.4. Also a solution of this buffer but with 0.4 M NaC1, final pH 7.4 Buffer C: As buffer B but with 0.2 M NaCI, final pH 7.4 Buffer D: As buffer B but with 1.0 M NaCI, final pH 7.4 Buffer E: 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris-HC1 buffer, final pH 7.4 at 4° Buffer F: 1 m M EDTA, 10 mM Tris-HCl buffer, final pH 7.4 Buffer G: 1 m M MgCI2, 10 mM Tris-HCl buffer, final pH 7.4 Buffer H: 1 m M MgC12, 0.15 M NaCl, 20% ethylene glycol, 10 mM Tris-HC1 buffer, final pH 7.4 Coupling buffer: 0.1 M NaHCO3 + 0.5 M NaCl, final pH 9.8 0.5 M NaC1, 0.! M sodium acetate, final pH 4.0 Whatman DEAE-cellulose Butyl-agarose Aminopentyl-agarose Affi-Gel blue Sephadex G-25 ECTEOLA-cellulose 6-Chloroguanine Ethylenediamine Ethanolamine CNBr-activated Sepharose 4B PMSF Dowex 1 (200-400 mesh) Ophiophagus hannah venom [3H]cAMP [3H]Adenosine A s s a y of c A M P P h o s p h o d i e s t e r a s e c A M P p h o s p h o d i e s t e r a s e ( P D E ) a c t i v i t y in p u r i f i e d / p a r t i a l l y purified p r e p a r a t i o n s w a s m e a s u r e d b y a m o d i f i c a t i o n 8 o f the t w o - s t e p p r o c e d u r e o f T h o m p s o n a n d A p p l e m a n . 9 W h e n P D E a c t i v i t y w a s m e a s u r e d in ho8 R. J. Marchmont and M. D. Houslay, Biochem. J. 187, 381 (1980). 9 W. J. Thompson and M. M. Appleman, Biochemistry 10, 311 (1981).
[69]
HIGH-AFFINITY LIVERcAMP PHOSPHODIESTERASE
753
mogenates, in order to compensate for any underestimation of activity owing to adenosine deaminase ~° activity being present, the Dowex resin was prepared fresh as a l : 1 : 1 (v/v/v) slurry of resin : H20 : ethanol and used thus. ~1Routinely a [cAMP] of 1.0/zM was employed for the peripheral PDE and 0.1/zM for the dense-vesicIe PDE, both with an [Mg2~] of 5 mM. It was checked that cAMP degradation was linear with respect to time and initial rates of degradation were assessed. Activity is expressed in international units (i.u.) when 1 U is the production of 1/zmol product/ min/mg protein. A 100-/zl reaction mixture, in Eppendorf (I .8-ml) tubes, contained final concentrations of 5 m M MgCI2, [3H]cAMP ( - 100,000 cpm), 20 mM TrisHCI, final pH 7.4, and an appropriate amount of enzyme. During kinetic analysis [cAMP] range from 4 x 10 -7 to 1! X 10-3 M. Reaction tubes were vortexed and incubated at 30° for an appropriate time interval, usually 15 min (linearity should be ascertained under all conditions for initial rate analysis). Termination of assays was achieved by removal of tubes to a boiling H20 bath for 2 rain. The tubes were then cooled to 4° before the addition of 25 p~g of Ophiophagus hannah venom (l mg/ml). This was then incubated for 10 min at 30° in order to allow for the conversion of 5'AMP to adenosine; 0.4 ml of resin slurry (Dowex 1; 200-400 mesh; C1form as resin : H20, ! : 2 (v/v) mixture) was then added to the tubes. They were then vortexed several times over a 15-rain period before being centrifuged at 14,000 gay for 4 rain in order to sediment the resin thoroughly. A 150-/.d aliquot of the supernatant was taken for liquid scintillation counting of [3H]adenosine. The resin will bind some adenosine, often as much as 50%, and this can be evaluated by control experiments by assessing the binding of [3H]adenosine over a range of concentrations. In all cases blanks were performed with no enzyme added and these counts subtracted from test experiments. The value of the blank can be affected by changes in buffer constituents (e.g., NaC[). This needs careful attention, especially during purification. Also high substrate [cAMP] may affect [3H]adenosine binding, which again can be tested for. Product inhibition studies, using 5'-AMP, require careful evaluation of the completion of conversion of 5'-AMP to adenosine when treating with the snake venom. Purification of the Peripheral cAMP Phosphodiesterase from Rat Liver Plasma Membranes The method described below uses a crude membrane fraction for the extraction of this enzyme. This obviates the need to purify liver plasma ~0 W. J. Rutten, B. M. Schoot, and J. S. H. Dupont, Biochim. Biophys. Acta 315, 378 (1983). H j. L o n d e s b o r o u g h , Anal. Biochem. 71, 623 (1976).
754
PHOSPHODIESTERASE ISOZYME METHODS
[69]
membranes for the starting material, as has been used for an alternative purification strategy described elsewhere by us.12 In that procedure the plasma membrane PDE was eluted from Affi-Gel blue by high salt only. However, batches used over the last 2 years demand the addition of cAMP + MgCI2 to elicit its elution. Step 1. Using a glass homogenizer and rotating Teflon pestle, six freshly isolated rat livers were homogenized in ice-cold bicarbonate buffer [3 : 1 (v/v), liver : buffer) using six to eight additional strokes at 400 rpm after dispersion. This was centrifuged for 10 min at 1500 gay, the resultant supernatant discarded, and the pellet resuspended in ice-cold bicarbonate buffer [1 : 3 (v/v), pellet : buffer]. This was recentrifuged at 10,000 gay for I0 min and the resuspension and centrifugation process repeated once more to yield a washed pellet fraction. Step 2. The washed pellet fraction was resuspended in 120-150 ml of ice-cold buffer A and left at 4° for 1 hr before being centrifuged at 48,000 gay for 30 min at 4°. The supernatant was then dialyzed overnight at 4 ° against two changes of buffer B (2 x, 1 liter) (yield 72%, purification x 3). Step 3. The dialysate was recentrifuged as per step 2 prior to the supernatant being applied, at a flow rate of 0.8 ml/min, to a column (3.5 x 40 cm) of DEAE-cellulose previously equilibrated in buffer B. The column was then washed with 200 ml of buffer B prior to eluting PDE activity (4-ml fractions) with a 300-ml linear NaCl gradient (0.075-0.4 M) in buffer B, final pH 7.4. Two peaks of activity were eluted between 0.2 and 0.275 M NaCl [peaks A and B, respectively (Fig. la)]. The fractions of peak A which exhibited the highest specific activity for PDE were taken for the next step (yield 45%, purification x27). Step 4. Peak A fractions were applied (0.75 ml/min) to a butyl-agarose column (2 x 5 cm) equilibrated in buffer C (Fig. 1b). Over 90% of the PDE activity failed to bind to this column, and this eluted activity was collected and taken for step 5 (yield 40%, purification x 30). Step 5. The pooled eluate was applied (0.5 ml/min) to a column (0.5 x 3 cm) of Affi-Gel blue previously equilibrated with buffer C (Fig. lc). After loading, this column was washed with 100 ml of buffer C, followed by 100 ml of buffer D, followed by 50 ml of buffer D containing 1 mM ATP, 1 m M NAD +, with l m M EDTA in place of the MgC12. A final wash of 200 ml of buffer D was made prior to enzyme elution with 15 ml of buffer D containing 1 m M cAMP (elution buffer). This was done by running 5 ml of this elution buffer into the column, stopping the flow, leaving the column for 1 hr at 4° and then restarting the flow (0.2 ml/min) while collecting fractions (0.2 ml). Active fractions (normally 5 ml), from step 5, were applied to a column (2.5 x 50 cm) of Sephadex G-25 equilibrated lZ R. J. M a r c h m o n t , S. Ayad, and M. D. Houslay, Biochem. J. 195, 645 (1981).
[69]
HIGH-AFFINITY LIVER cAMP PHOSPHODIESTERASE B
755
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FIG. 1. Purification of the peripheral plasma membrane enzyme. (a) DEAE-cellulose column with peak A being pooled for further study and arrow (i) being the start of the NaCl gradient, (b) butyryl-agarose chromatography of peak A, (c) Affi-Gel blue chromatography step with arrows (ii), (iii), and (iv) indicating buffer changes. PDE activity was assessed at 1 ~M [cAMP] at 30°. Activity is expressed as nmol/min/ml fraction. Protein is given as OD280. with buffer B (15 ml/hr) and fractions o f highest specific activity collected (yield 18%, purification ×3246). In s o m e instances this final stage had small a m o u n t s ( < 10%) o f l o w - m o l e c u l a r - w e i g h t impurities which could be r e m o v e d b y gel filtration on S e p h a c r y l 200 or a similar type o f c o l u m n equilibrated in buffer B. Purification of the "Dense-Vesicle" c A M P Phosphodiesterase W e h a v e used t w o distinct m e t h o d s , with similar success, to purify this e n z y m e to a p p a r e n t h o m o g e n e i t y . One o f these is described below.
756
PHOSPHODIESTERASE ISOZYME METHODS
[69]
The specific solubilization of this enzyme takes advantage of the hypotonic shock procedure devised by Loten et al.~3 involving the proteolytic release of this membrane-bound enzyme 14from an as yet undefined intracellular vesicle fraction. 2 Step 1. Three rat livers (1 vol) are rapidly excised, rinsed in ice-cold buffer E, and blotted dry. They are then diced with scissors prior to homogenization in 8 vol of buffer E using a glass homogenizing vessel with a Teflon pestle rotating at 250 rpm for eight up and down strokes. The homogenate is filtered through cheesecloth and centrifuged at 1500 gay for 10 min at 4°. Step 2. The supernatant is carefully removed and recentrifuged at 25,000 gav for 20 min at 4°. The resulting pellet is resuspended in 2-3 vol of ice-cold buffer F and left on ice for 40 min. This allows for the solubilization of the dense-vesicle cAMP phosphodiesterase. After this time the solution is centrifuged at 48,000 gay for 30 min and the supernatant taken for the next step. In some instances, from this point onward, and not before, solutions are made 0.1 m M with PMSF and 2 mM benzamidine (yield 100%, purification x 3.8). Step 3. A column of ECTEOLA-cellulose is equilibrated with buffer G prior to application (0.5 ml/min) of the supernatant extract (Fig. 2, top). Buffer G is then passed through the column until no further protein (constant OD280) is eluted. The column is then washed with buffer H until no further protein comes off prior to the elution of the dense vesicle enzyme using buffer H containing 0.4 M NaCl (final pH 7.4) (yield 71%, purification x51). Step 4. Fractions of highest specific activity are diluted 1 : 1 (v/v) with buffer G and applied (0.4 ml/min) to a column (1 x 5 cm) of aminopentylagarose that has been preequilibrated with buffer G (Fig. 2, middle). Buffer G is used to wash the column until no further protein elutes and then with buffer H containing 0.275 M NaCI until no further protein is eluted. The dense-vesicle enzyme is then eluted using buffer H containing 0.5 M NaCI and fractions of highest specific activity are collected (yield 48%, purification x 1068). Step 5. The enzyme sample is desalted on a Sephadex G-25 column which has been previously equilibrated with buffer G prior to application (0.1 ml/min) of the sample to a guanine-Sepharose column. This guanine° Sepharose column is equilibrated with buffer G 15 prior to the loading of the sample. ~3 E. G. Loten, F. D. A s s i m a c o p o u l o s - J e a n n e t , J. H. Exton, and C. R. Park, J. Biol. Chem. 253, 746 (1978). ~4 E. G. Loten, S. H. Francis, and J. D. Corbin, J. Biol. Chem. 255, 7838 (1980). ~5 R. H. W h i t s o n and M. M. Appleman, Biochim. Biophys. Acta 714, 279 (1982).
[69]
HIGH-AFFINITY LIVERcAMP PHOSPHODIESTERASE
757
The preparation of this guanine-Sepharose column involves, first, the synthesis of ethylamine guanine. For this 2 mmol of chloroguanine plus 2 mmol of ethylenediamine are added to 5 ml HzO. Then I0 M NaOH is added dropwise, with stirring, until the ethylenediamine goes into solution. This mixture is refluxed with extreme care in a protected area for 12 hr until a yellow paste forms. This product is then coupled to CNBractivated Sepharose 4B. To achieve this, 6 g of lyophilized CNBrSepharose is taken up in water to give a 20-ml gel slurry. To 20 ml of gel is added 5 ml of the refluxed product and 35 ml of coupling buffer. These components are mixed and then left at room temperature (18-21 °) for 20 h or at 4° for 3 days. The resultant gel mixture is washed with 20 vol of coupling buffer before adding 2 vol of coupling buffer containing 1 M ethanolamine per volume of gel. This is left for 20 hr at 23 ° before washing sequentially with (1) 10 vol coupling buffer, (2) 10 vol of 0.5 M NaC1, 0. I M sodium acetate, final pH 4.0, (3) 4 vol of coupling buffer. The gel can now be stored at 4 °. Just before use it is washed (4 vol) with buffer G and then thoroughly equilibrated with buffer G prior to its use. This column is washed with buffer G until no further protein elutes. The dense-vesicle enzyme is then eluted with buffer H containing 0.5 M NaC1 (Fig. 2, bottom). Fractions of highest specific activity are collected (yield 18%, purification × 2773).
Properties of the Two cAMP Phosphodiesterases (PDE) Both procedures yield preparations exhibiting a single protein band which comigrated with PDE activity on nondenaturing gels. The plasma membrane enzyme was purified some 3246-fold, over the specific activity of the membrane fraction from which it was isolated, with an 18% yield. This enzyme, assayed at 30°, exhibited a Vma×of 3.6 U/rag protein and showed kinetics indicative of apparent negative cooperativity with an apparent Km of 0.9/zM and Hill coefficient (h) of 0.54. As only a single PDE species was present then the kinetic mechanism for cAMP hydrolysis is evidently unusual. One possibility is that it obeys a mnemonical type of kinetic mechanism. J2 This PDE showed a high specificity for cAMP, as cGMP was hydrolyzed only slowly ( Vma×0.2 U/rag protein, Km 105/zM) obeying, apparently, Michaelis kinetics. In accord with this, the hydrolysis of cAMP was relatively insensitive to inhibition by cGMP. This PDE had a subunit molecular mass of 52.4 kDa and appeared as a monomer (51.2 kDa) on sucrose density gradient centrifugation although a dimeric species (104 kDa) could be found on molecular sieving using either Sephadex G-100 or Sepharose S-200. Tryptic iodinated peptide
758
PHOSPHODIESTERASE ISOZYME METHODS 0"3
[69]
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fraction N~ FIG. 2. Purification of the dense-vesicle enzyme. (a) ECTEOLA-cellulose step with the PDE activity being eluted in the third and final step, after the buffer change [arrow (i)], being taken for further study, (b) aminopentyl-agarose chromatography with arrows signifying buffer changes, and (c) guanine-Sepharose chromatography with arrows signifying buffer changes. Assays were performed at 0.1 ttM [cAMP] at 30°. Activity is expressed as nmol cAMP hydrolyzed/min/mg protein. Protein is given at Azs0(arbitrary units).
m a p p i n g s h o w e d it to b e d i s t i n c t f r o m o t h e r P D E s 16 as well as t h e d e n s e v e s i c l e e n z y m e ( u n p u b l i s h e d ) . P o l y c l o n a l a n t i b o d i e s p r e p a r e d in r a b b i t a g a i n s t this p u r i f i e d e n z y m e d i d n o t c r o s s - r e a c t w i t h e i t h e r t h e d e n s e v e s i c l e o r c G M P - s t i m u l a t e d P D E s p r e p a r e d f r o m rat liver. 17 I n s u l i n elicits t h e p h o s p h o r y l a t i o n o f this e n z y m e w h i c h l e a d s to a c t i v a t i o n r e f l e c t e d in a d e c r e a s e in t h e Hill c o e f f i c i e n t 6 f o r c A M P h y d r o l y s i s • T h e r e a s o n t h a t this P D E is f o u n d a s s o c i a t e d e x c l u s i v e l y w i t h t h e p l a s m a m e m b r a n e is t h a t it b i n d s to a specific i n t e g r a l p r o t e i n in this m e m b r a n e . 5 16D. J. Takemoto, J. Hansen, L. J. Takemoto, and M. D. Houslay, J. Biol. Chem. 257, 14597 (1982). ~7N. J. Pyne, M. E. Cooper, and M. D. Houslay, Biochem. J. 242, 33 (1984).
[69]
HIGH-AFFINITY LIVERcAMP PHOSPHODIESTERASE
759
The dense-vesicle enzyme was purified some 2773-fold, over the specific activity of the homogenate from which it was isolated, with an 18% yield. The enzyme, as purified, is a proteolytically clipped version of the native enzyme, as endogenous lysosomal proteases, which are released by the hypotonic shock procedure, affect its solubilization.13,14 The solubilized enzyme can now be purified by standard techniques. The clipped enzyme behaves like a normal soluble enzyme and still exhibits enhanced activity after activation by insulin and glucagon. ~.~3Thus, it is highly likely that the functional globular mass of this PDE is not normally embedded in the bilayer but is found without the bilayer anchored by a small hydrophobic pedicle which spans the bilayer. The dense-vesicle PDE, assayed at 30°, had a Vmaxof 0.7 U/mg protein and exhibited kinetics indicative of apparent negative cooperativity with an apparent Km of 0.3 /xM and a Hill coefficient (h) of 0.43. The enzyme preparation contained only one species of PDE and thus it remains to be seen whether either the negative cooperative kinetics or some other type of mechanism yielding apparent negative cooperativity is present. The enzyme hydrolyzed cGMP slowly with a Vmaxof only 4 milliU/mg protein. However, it still succeeded in binding cGMP with high affinity as the Km for cGMP was 10/xM. Indeed the hydrolysis of cAMP by this enzyme is very sensitive to inhibition by cGMP, which is in contrast to the plasma membrane enzyme. This feature can be used diagnostically to distinguish these two enzymes. The dense-vesicle PDE, as isolated, has a subunit mass of 57 kDa, although a species of 51 kDa may also be found upon further proteolysis during purification. The enzyme appears as a dimer of 114 kDa upon either sucrose density gradient analysis or gel filtration. Tryptic-iodinated peptide mapping shows this enzyme to be distinct from the plasma membrane species and, furthermore, a polyclonal antibody raised against this enzyme did not cross-react with the plasma membrane or any other rat liver PDE. However, such an antibody can be used to identify the native, membrane-bound dense-vesicle PDE as an integral membrane protein of subunit molecular mass 63 kDa. Both of these two PDEs exhibit similar kinetics as regards the hydrolysis of cAMP, but not cGMP. However, various PDE inhibitors can be seen to exhibit different (selective) potencies as inhibitors of cAMP hydrolysis by these two enzymes (Table I). These two enzymes are also expressed differentially in various rat tissues. 18 In summary, two distinct high-affinity, membrane-bound cAMP phosphodiesterases can be isolated from rat liver. These are a peripheral plasma membrane enzyme which is activated by insulin alone I and the t8 N. J. Pyne, N. Anderson, B. E. Lavan, G. Milligan, H. G. Nimmo, and M. D. Houslay, Biochem. J. 248, 897 (1987).
760
PHOSPHODIESTERASE ISOZYME METHODS
[70]
TABLE I SELECTIVE INHIBITION OF THE TWO HORMONE-STIMULATED MEMBRANE-BOUND c A M P PHOSPHODIESTERASES FROM RAT L1VERa Inhibition (%)
Drug
Peripheral PDE
Dense-vesicle PDE
Milrinone Zaprinast Dipyridamole RO-1724 IC1 63,197
61 19 35 58 56
0 31 9 38 0
" A s s a y s were performed using the purified enz y m e s with 1 ~ M c A M P substrate concentration and 1 0 / z M drug.
dense-vesicle enzyme which is activated by both insulin and glucagon through apparently distinct mechanisms. 1,7,~3,14 Acknowledgments This work was supported by grants from the Medical Research Council (UK), California Metabolic Research Foundation, British Diabetic Association, British Heart Foundation, Agriculture & Food Research Council, and the Scottish H o m e and Health Department.
[70] Preparation of Dog Kidney High-Affinity c A M P Phosphodiesterase
By W. J. THOMPSON, C.-C. SHEN, and S. J. STRADA Introduction
A nomenclature developed at a recent symposium on cyclic nucleotide phosphohydrolases (EC 3.1.4.17, see Strada and Thompson 1) attempted to categorize the enzyme system on the basis of substrate preference and S. J. Strada and W. J. T h o m p s o n (eds.), Adv. Cyclic" Nucleotide Protein Phosphorylation Res. 16 (1984).
METHODS IN ENZYMOLOGY,VOL. 159
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