- Email: [email protected]
[70] Preparation of dog kidney high-affinity cAMP phosphodiesterase
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.
[70]
DOG KIDNEYcAMP PHOSPHODIESTERASE
761
regulatory properties. Thus, high-affinity cyclic nucleotide phosphodiesterases, which characteristically show a lower apparent Km for cAMP than do the other forms of the enzyme, and may be regulated by a variety of hormones, including insulin, were termed type IV enzymes. Their biochemical properties and ability to be influenced by several hormones and pharmacological agents, 2 together with a ubiquitous distribution in tissues, suggest that type IV PDE isozymes play a pivotal role in the regulation of cAMP turnover in cells. Since then, recent data from several laboratories using a combination of biochemical, immunological, and pharmacological approaches have indicated the presence of at least two types of type IV enzyme of high affinity. These two types both show high affinity for cAMP as substrate but differ in their ability to demonstrate allosteric regulation via cGMP inhibition, sensitivity to certain pharmacologic agents, and the utilization of cGMP as an alternate substrate in the hydrolytic reaction. This chapter deals with the purification procedures and properties for dog kidney cAMP phosphodiesterase. This enzyme shows an apparent Km for cAMP as substrate in the micromolar range, whereas cGMP is a poor substrate and will inhibit cAMP hydrolysis only at very high concentration (K~ = 312/zM). Dog, pig, and rat kidneys contain a similar enzyme and the properties of the purified canine kidney enzyme (Epstein et al. 3) are similar to those reported for type IV PDE for lung and skeletal muscle. 2 In contrast to these tissues, liver, heart, and platelet enzymes demonstrate cGMP inhibition of cAMP hydrolysis at low substrate or inhibitor concentrations (Ki -~ 0.5 /zM). Moreover, we have been able to show marked differences in the sensitivity of the kidney type IV enzyme to pharmacological inhibitors as compared to effects on the liver type IV activity. For example, the kidney type IV activity is 500 times more sensitive to the etazolate compound, SQ 65442, with submicromolar cAMP as substrate, than is the liver membrane type IV activity (unpublished observations). Conversely, the kidney enzyme is 15,000 times less sensitive to inhibition by cilostamide (OPC 3689; Hidaka et al. 4) than is the liver enzyme. Therefore, the comparative effects of drugs, the relative effects of cGMP to inhibit cAMP hydrolysis, or the ratio of cGMP (1 /zM substrate) to cAMP hydrolysis (0.25/zM) can be used to identify, characterize, and monitor type IV activity during purification.
2 W. J. Thompson, M. L. Pratt, and S. J. Strada, Adv. Cyclic Nucleotide Protein Phosphorylation Res. 16, 137 (1984). 3 p. M. Epstein, S. J. Strada, K. Sarada, and W. J. Thompson, Arch. Biochem. Biophys. 218, 119 (1982). 4 H. Hidaka, T. Tanaka, and H. Itoh, Trends Biochem. Sci. 5, 237 (1984).
762
P H O S P H O D I E S T E R AISOZYME SE METHODS
[70]
Purification Information from Previous Studies Earlier attempts to purify the dog kidney high-affinity enzyme (Thompson et al. 5) by conventional biochemical procedures yielded an activity associated with a 60,000-Da protein, which appeared homogeneous by classical criteria. Canine or porcine kidney provide a rich source of the type IV enzyme that can be recovered easily in high- or low-speed supernatant fractions after centrifugation. A buffer system was developed to help stabilize the enzyme, which facilitated use of purification strategies designed to separate types I and II activities from the type IV enzyme during the initial stages. DEAE-ce|lulose, granulated hydroxyapatite (GHA), anion-exchange rechromatography, and gel filtration were employed subsequently in this buffer and resulted in a relatively "specific" cAMP phosphodiesterase. The specific activity of the enzyme preparation obtained with these procedures was near 30 nmol/min/mg with 0.25/xM cAMP as substrate (Vmax = 95 nmol/min/mg). These rates were much less than those observed for types ! and II PDE. 1 Similar or lower values were also observed by others for lung and skeletal muscle using modified procedures to purify the enzymes. 1 As pointed out ear|ier, 2 several factors could account for the relatively low specific activity values of these preparations, including enzyme modification during bulk processing. Earlier procedures were of necessity slow and the potential for enzyme modification great. Unfortunately, until recently, no separation of cAMP hydrolysis from the 60-kDa protein could be obtained by a variety of concomitant chromatographic/electrophoretic procedures. Our development of ovine polyclonal antisera (Sarada et al. 6) has greatly aided the preparation of dog kidney high-affinity activity with some of our preparations now showing up to 200-fold higher maximum velocities. By monitoring both immunoreactivity and catalytic activity, major modifications of published procedures were made possible. Major procedural changes included more rapid bulk processing and the use of Affi-Gel Blue in the purification scheme. The latter enabled separation of high-affinity activity from the 60,000-Da protein, the major antigen to which our antiserum was directed and affinity purified. The 60-kDa protein binds the enzyme avidly. This is probably due to both hydrophobic and ionic interactions, which seem to be potentiated with the activity stabilizing buffer. While all of the kinetic and pharmacologic data are identical to that 5 W. J. Thompson, P. M. Epstein, and S. J. Strada, Biochemistry 18, 5228 (1979). 6 K. Sarada, P. M. Epstein, S. J. Strada, and W. J. Thompson, Arch. Biochem. Biophys. 215, 183 (1982).
[70]
DOG KIDNEY c A M P PHOSPHODIESTERASE
763
published except for the maximum velocity, the physical properties of the enzyme do appear different. The high-affinity activity obtained by the procedures detailed below shows a specific activity near 1800 nmol/min/ mg with 0.25 /zM cAMP substrate and Michaelis-Menten type kinetic behavior. Maximum velocities for cAMP range from 10 to 20/zmol/min/ mg with Km values from 1 to 2/zM. Sephacryl S-200 gel filtration yields an apparent native size of 71-74 kDa; I0% SDS-acrylamide gel electrophoresis indicates an 82-kDa protein with greater than 90% purity. Protein yields still remain a problem with this type of purification. Maximum activity stabilization of the final preparation requires storage in 10 mM K2PO4, 30% ethylene glycol, 0.1 mM DTT, and 10/zM TLCK. While the simplest method of purification might be to prepare milligram quantities of the 60,000-Da protein with enzymatic activity bound to it, followed by elution of activity by the procedures shown below, estimates are that approximately 20/zg of enzyme are bound per milligram of 60kDa protein. The procedure reported here is reliable, fast, and relatively easy, thus minimizing the potential for covalent modification, including proteolysis, of the enzyme during purification or storage. Materials/Buffers 1. Frozen dog kidneys are purchased conveniently from Pelfreeze, Inc., Rogers, Arkansas. Alternatively, the most reliable tissue source is kidneys obtained locally after surgical removal and perfusion with cold saline. The outer thin capsuled lining is removed along with adherent fat. The tissue is quick frozen in liquid nitrogen and stored at -70 ° until adequate quantities are accumulated. We have had less successful experiences with bovine and porcine kidneys, though useful for preparative level quantity, than our success with canine kidneys. 2. DE-52 (300 ml packed volume) and DEAE-Sephacel (35 ml packed volume) are purchased from Whatman and Pharmacia, respectively. Each is prepared according to manufacturer's specifications and the pH of the slurry adjusted with 2 M MES to pH 6.5. Affi-Gel Blue (350 ml packed volume) is purchased from Bio-Rad Laboratories. The gel is prepared in a coarse glass funnel by extensive washing with 6 M urea (BRL, ultrapure), 2 N NaCI, and equilibrating TMB buffer (see below). Hydroxylapatite (Calbiochem, fast flow dry powder) is charged according to manufacturer's procedures (20 g). Sephacryl SF S-200 (Pharmacia) is equilibrated in S-200 buffer which is 500 mM MEDGANT (see below) with the ethylene glycol reduced to 5% and glucose absent. The column is packed with high pressure according to manufacturer's recommendations in a 2.6 x 100 cm column (Pharmacia) to a bed volume of 390 ml.
764
P H O S P H O D I E S T E R AISOZYME SE METHODS
[70]
3. The following buffers are prepared and filtered using 0.45-tzm m e m branes. D T T (dithiothreitol) and T L C K ( N - a - p - t o s y l - l - l y s i n e chloromethyl ketone) are weighed separately and added just before use. We r e c o m m e n d that the initial homogenization buffer, the Affi-Gel Blue enz y m e elution buffer, and the final storage buffer be supplemented with a general protease inhibitor cocktail consisting of 2 m M benzamidine, I000 K I U aprotinin/liter, 2 / x M leupeptin, and 2 / z M pepstatin. Concentration terms used with the buffer e p o n y m indicate the concentration of the anion. 5 mM MES-30% ethylene glycol-0.1 mM DTT-5% glucose (w/v)-15 mM sodium acetate-50 mM NaF-20/zM TLCK (ph 6.5); also prepared are MEDGANT with 100, 250, and 1200 mM sodium acetate S-200 buffer 500 mM MEDGANT containing 5% ethylene glycol instead of 30% ethylene glycol and deletion of the glucose 10 mM KHPO4/KH:PO4-1 mM DTT-30% ethylene glycol-20/zM TLCK HA buffer (pH 7.0); also prepared is 350 mM HA buffer 20 mM Tris-5 mM MgCI2-2 mM 2-mercaptoethanol (pH 7.4) TMB 20 mM Tris-5 mM EDTA-0.1 M NaC1 (pH 7.4); also prepared is 2 M TENB TENB (dissolve the EDTA before adding NaCI) Triton/TENB TENB above plus 0.2% Triton X-100 TENB above plus 30% ethylene glycol EG/TENB MEDGANT
Experimental Procedure All steps are at 4 ° except where indicated. Step 1. T w e n t y kidneys ( - 5 0 0 g frozen weight) are thawed briefly in cold tap water and ground in a commercial meat chopper (Chop-Rite Mfg. Co.). The mince is homogenized in a commercial Waring blender (CB-6) at the low-speed setting for 20 sec. This is repeated three times with 2-rain intervals to minimize heating. The homogenate is brought to 6 liters with M E D G A N T and poured through two layers of cheesecloth fixed with a h e a v y rubber band o v e r 4-liter beakers. Step 2. The h o m o g e n a t e is centrifuged at 4500 g in six l-liter bottles (R = 26 cm, max) for 30 rain with a B e c k m a n J-6B centrifuge. Step 3. After fat cake removal the supernatant ( - 5 4 0 0 ml) is mixed with 250 ml (settled volume) of DE-52 equilibrated in M E D G A N T . The mixture is stirred for 30 min and centrifuged as in step 2. The supernatant is decanted and the DE-52 resuspended in 1200 ml of M E D G A N T and centrifuged as in step 2. This DE-52 washing procedure is repeated with 1200 ml of I00 m M M E D G A N T , twice. The e n z y m e is eluted by mixing with 600 ml of 1200 m M M E D G A N T , collecting the DE-52 mixture into one rotor bottle, and centrifuging as in step 2.
[70]
DOG KIDNEYcAMP PHOSPHODIESTERASE
765
Step 4. The supernatant from step 3 ( - 6 5 0 ml) is diluted to 6 liters with TMB and 300 ml of Affi-Gel Blue added. The gel is collected in a coarse glass funnel. High-affinity cAMP phosphodiesterase binds avidly to this gel and is not released at 4 ° . Therefore, extensive washing of the gel before enzyme elution is possible. Low-affinity phosphodiesterase activity and the 60-kDa binding protein are released by washing consecutively with five bed volumes of TMB, TENB, 2 M TENB, EG/TENB, Triton/ TENB, TMB, and EG/2 M TENB with the gel in the funnel. The gel is suspended in an equal volume of TMB and transferred to a Pharmacia column or its equivalent, water jacketed to 30 °. Approximately 20% of the applied activity measured with 0.25/~M cAMP as substrate (about 5-10% of the homogenate activity) is eluted with 1 liter of EG/2 M TENB at 30 °. The eluted enzyme activity is collected in a flask cooled to 4 °. Steps 1-4 can be performed in approximately 6 hr and should yield near 1 ~mol/min activity with 0.25 p~M cAMP as substrate. Step 5. The eluted enzyme fraction from step 4 is dialyzed against 12 liters of TMB overnight and pumped onto DEAE-Sephacel (5 ml). The column is washed with 50 ml of MEDGANT and high-affinity activity eluted isocratically with 40 ml of 1200 mM MEDGANT. Two-milliliter fractions are collected and the most active pooled ( - 1 0 ml) for gel filtration. TABLE I PURIFICATION PROCEDURES FOR HIGH-AFFINITY, cAMP-SPECIFIC, TYPE IV DOG KIDNEY c A M P PHOSPHODIESTERASEa
Step Homogenate 4500 g s u p e r n a t a n t 4500 g pellet DE-52 "batch" chromatography
(1200 mM MEDGANT eluate) Affi-Gel Blue "batch" absorb (EG/2M T E N B , 30 ° eluate) DEAE-Sephacel Sephacryl S-200 gel filtration Analytical HA chromatography
Protein (mg)
Activity (nmol/min)
Specific activity (mU/mg)
Purification (-fold)
27,000 16,900 9,000 1,600
9,100 6,700 3,000 3,710
44
910
21
69
406 180
185 360
615 1,200
110
1,800
6,100
2.2 0.5 0.06
0.3 0.4 0.3 2.3
I 1.3 -7.7
" Data are compiled from several different preparations. Activities were determined using 0.25 ~ M c A M P as substrate, 5 mM MgCl2, 3.75 mM 2-mercaptoethanol, 30 # g BSA, and 40 mM Tris-HCl (pH 8.0) in a total volume of 400/~1. One milliunit equals 1
nmol cAMP hydrolyzed/min.
766
PHOSPHODIESTERASE
ISOZYME METHODS
[71]
Step 6. The activity is fractionated by gel filtration with Sephacryl S-200 (bed volume = 390 ml) using the S-200 buffer. The flow rate for the column is 1.0 ml/min and 2.5-ml fractions are collected. The majority of the enzyme fractionates just ahead of bovine serum albumin. Step 7. An analytical hydroxylapatite column (1 ml) is charged with 350 mM HA buffer and equilibrated in 10 mM HA buffer. The peak activity fractions from the S-200 gel filtration column are pooled and applied directly ( - 4 0 ml). The column is washed with 20 ml of HA buffer and the nearly homogeneous enzyme eluted with a linear gradient (20 ml total volume) of 10 to 350 mM HA buffer. The enzyme should be dialyzed against 10 mM HA buffer for stable storage at 4° for several weeks. Purification of the enzyme is summarized in Table I. Acknowledgment Research supported by USPHS Grant GM 33538.
[71] B r a i n L o w - K i n c A M P P h o s p h o d i e s t e r a s e By PHILIPPE DE MAZANCOURT a n d YVES GIUDICELLI Multiple forms of cyclic nucleotide phosphodiesterase activities can be isolated from brain cortex extracts. Among these, three separable and kinetically distinct fractions are predominant: the extensively studied calmodulin-sensitive form, which has low affinity for cAMP, the Ca 2+dependent, calmodulin-insensitive enzyme, which hydrolyzes both cAMP and cGMP with low affinity, and a third form referred as the low-Km cAMP phosphodiesterase, which displays high affinity for cAMP and marginal responsiveness to calmodulin and Ca 2+. The first evidence for the existence of low-Kin cAMP phosphodiesterase activity in the brain was provided by Brooker et al.,~ who showed that the cAMP phosphodiesterase activity of rat brain could be kinetically resolved into a high(Km = 1/xM) and a low-affinity (Km = 100/xM) component. Three years later, Thompson and Appleman 2 applied the agarose gel filtration technique to rat brain extracts and found two cAMP phosphodiesterase fractions, one with high molecular weight and high Km (100/xM) and the other G. Brooker, L. Thomas, Jr., and M. Appleman, Biochemistry 7, 4177 (1968). 2 W. J. Thompson and M. M. Appleman, Biochemistry 10, 311 (1971).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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.
[70]
DOG KIDNEYcAMP PHOSPHODIESTERASE
761
regulatory properties. Thus, high-affinity cyclic nucleotide phosphodiesterases, which characteristically show a lower apparent Km for cAMP than do the other forms of the enzyme, and may be regulated by a variety of hormones, including insulin, were termed type IV enzymes. Their biochemical properties and ability to be influenced by several hormones and pharmacological agents, 2 together with a ubiquitous distribution in tissues, suggest that type IV PDE isozymes play a pivotal role in the regulation of cAMP turnover in cells. Since then, recent data from several laboratories using a combination of biochemical, immunological, and pharmacological approaches have indicated the presence of at least two types of type IV enzyme of high affinity. These two types both show high affinity for cAMP as substrate but differ in their ability to demonstrate allosteric regulation via cGMP inhibition, sensitivity to certain pharmacologic agents, and the utilization of cGMP as an alternate substrate in the hydrolytic reaction. This chapter deals with the purification procedures and properties for dog kidney cAMP phosphodiesterase. This enzyme shows an apparent Km for cAMP as substrate in the micromolar range, whereas cGMP is a poor substrate and will inhibit cAMP hydrolysis only at very high concentration (K~ = 312/zM). Dog, pig, and rat kidneys contain a similar enzyme and the properties of the purified canine kidney enzyme (Epstein et al. 3) are similar to those reported for type IV PDE for lung and skeletal muscle. 2 In contrast to these tissues, liver, heart, and platelet enzymes demonstrate cGMP inhibition of cAMP hydrolysis at low substrate or inhibitor concentrations (Ki -~ 0.5 /zM). Moreover, we have been able to show marked differences in the sensitivity of the kidney type IV enzyme to pharmacological inhibitors as compared to effects on the liver type IV activity. For example, the kidney type IV activity is 500 times more sensitive to the etazolate compound, SQ 65442, with submicromolar cAMP as substrate, than is the liver membrane type IV activity (unpublished observations). Conversely, the kidney enzyme is 15,000 times less sensitive to inhibition by cilostamide (OPC 3689; Hidaka et al. 4) than is the liver enzyme. Therefore, the comparative effects of drugs, the relative effects of cGMP to inhibit cAMP hydrolysis, or the ratio of cGMP (1 /zM substrate) to cAMP hydrolysis (0.25/zM) can be used to identify, characterize, and monitor type IV activity during purification.
2 W. J. Thompson, M. L. Pratt, and S. J. Strada, Adv. Cyclic Nucleotide Protein Phosphorylation Res. 16, 137 (1984). 3 p. M. Epstein, S. J. Strada, K. Sarada, and W. J. Thompson, Arch. Biochem. Biophys. 218, 119 (1982). 4 H. Hidaka, T. Tanaka, and H. Itoh, Trends Biochem. Sci. 5, 237 (1984).
762
P H O S P H O D I E S T E R AISOZYME SE METHODS
[70]
Purification Information from Previous Studies Earlier attempts to purify the dog kidney high-affinity enzyme (Thompson et al. 5) by conventional biochemical procedures yielded an activity associated with a 60,000-Da protein, which appeared homogeneous by classical criteria. Canine or porcine kidney provide a rich source of the type IV enzyme that can be recovered easily in high- or low-speed supernatant fractions after centrifugation. A buffer system was developed to help stabilize the enzyme, which facilitated use of purification strategies designed to separate types I and II activities from the type IV enzyme during the initial stages. DEAE-ce|lulose, granulated hydroxyapatite (GHA), anion-exchange rechromatography, and gel filtration were employed subsequently in this buffer and resulted in a relatively "specific" cAMP phosphodiesterase. The specific activity of the enzyme preparation obtained with these procedures was near 30 nmol/min/mg with 0.25/xM cAMP as substrate (Vmax = 95 nmol/min/mg). These rates were much less than those observed for types ! and II PDE. 1 Similar or lower values were also observed by others for lung and skeletal muscle using modified procedures to purify the enzymes. 1 As pointed out ear|ier, 2 several factors could account for the relatively low specific activity values of these preparations, including enzyme modification during bulk processing. Earlier procedures were of necessity slow and the potential for enzyme modification great. Unfortunately, until recently, no separation of cAMP hydrolysis from the 60-kDa protein could be obtained by a variety of concomitant chromatographic/electrophoretic procedures. Our development of ovine polyclonal antisera (Sarada et al. 6) has greatly aided the preparation of dog kidney high-affinity activity with some of our preparations now showing up to 200-fold higher maximum velocities. By monitoring both immunoreactivity and catalytic activity, major modifications of published procedures were made possible. Major procedural changes included more rapid bulk processing and the use of Affi-Gel Blue in the purification scheme. The latter enabled separation of high-affinity activity from the 60,000-Da protein, the major antigen to which our antiserum was directed and affinity purified. The 60-kDa protein binds the enzyme avidly. This is probably due to both hydrophobic and ionic interactions, which seem to be potentiated with the activity stabilizing buffer. While all of the kinetic and pharmacologic data are identical to that 5 W. J. Thompson, P. M. Epstein, and S. J. Strada, Biochemistry 18, 5228 (1979). 6 K. Sarada, P. M. Epstein, S. J. Strada, and W. J. Thompson, Arch. Biochem. Biophys. 215, 183 (1982).
[70]
DOG KIDNEY c A M P PHOSPHODIESTERASE
763
published except for the maximum velocity, the physical properties of the enzyme do appear different. The high-affinity activity obtained by the procedures detailed below shows a specific activity near 1800 nmol/min/ mg with 0.25 /zM cAMP substrate and Michaelis-Menten type kinetic behavior. Maximum velocities for cAMP range from 10 to 20/zmol/min/ mg with Km values from 1 to 2/zM. Sephacryl S-200 gel filtration yields an apparent native size of 71-74 kDa; I0% SDS-acrylamide gel electrophoresis indicates an 82-kDa protein with greater than 90% purity. Protein yields still remain a problem with this type of purification. Maximum activity stabilization of the final preparation requires storage in 10 mM K2PO4, 30% ethylene glycol, 0.1 mM DTT, and 10/zM TLCK. While the simplest method of purification might be to prepare milligram quantities of the 60,000-Da protein with enzymatic activity bound to it, followed by elution of activity by the procedures shown below, estimates are that approximately 20/zg of enzyme are bound per milligram of 60kDa protein. The procedure reported here is reliable, fast, and relatively easy, thus minimizing the potential for covalent modification, including proteolysis, of the enzyme during purification or storage. Materials/Buffers 1. Frozen dog kidneys are purchased conveniently from Pelfreeze, Inc., Rogers, Arkansas. Alternatively, the most reliable tissue source is kidneys obtained locally after surgical removal and perfusion with cold saline. The outer thin capsuled lining is removed along with adherent fat. The tissue is quick frozen in liquid nitrogen and stored at -70 ° until adequate quantities are accumulated. We have had less successful experiences with bovine and porcine kidneys, though useful for preparative level quantity, than our success with canine kidneys. 2. DE-52 (300 ml packed volume) and DEAE-Sephacel (35 ml packed volume) are purchased from Whatman and Pharmacia, respectively. Each is prepared according to manufacturer's specifications and the pH of the slurry adjusted with 2 M MES to pH 6.5. Affi-Gel Blue (350 ml packed volume) is purchased from Bio-Rad Laboratories. The gel is prepared in a coarse glass funnel by extensive washing with 6 M urea (BRL, ultrapure), 2 N NaCI, and equilibrating TMB buffer (see below). Hydroxylapatite (Calbiochem, fast flow dry powder) is charged according to manufacturer's procedures (20 g). Sephacryl SF S-200 (Pharmacia) is equilibrated in S-200 buffer which is 500 mM MEDGANT (see below) with the ethylene glycol reduced to 5% and glucose absent. The column is packed with high pressure according to manufacturer's recommendations in a 2.6 x 100 cm column (Pharmacia) to a bed volume of 390 ml.
764
P H O S P H O D I E S T E R AISOZYME SE METHODS
[70]
3. The following buffers are prepared and filtered using 0.45-tzm m e m branes. D T T (dithiothreitol) and T L C K ( N - a - p - t o s y l - l - l y s i n e chloromethyl ketone) are weighed separately and added just before use. We r e c o m m e n d that the initial homogenization buffer, the Affi-Gel Blue enz y m e elution buffer, and the final storage buffer be supplemented with a general protease inhibitor cocktail consisting of 2 m M benzamidine, I000 K I U aprotinin/liter, 2 / x M leupeptin, and 2 / z M pepstatin. Concentration terms used with the buffer e p o n y m indicate the concentration of the anion. 5 mM MES-30% ethylene glycol-0.1 mM DTT-5% glucose (w/v)-15 mM sodium acetate-50 mM NaF-20/zM TLCK (ph 6.5); also prepared are MEDGANT with 100, 250, and 1200 mM sodium acetate S-200 buffer 500 mM MEDGANT containing 5% ethylene glycol instead of 30% ethylene glycol and deletion of the glucose 10 mM KHPO4/KH:PO4-1 mM DTT-30% ethylene glycol-20/zM TLCK HA buffer (pH 7.0); also prepared is 350 mM HA buffer 20 mM Tris-5 mM MgCI2-2 mM 2-mercaptoethanol (pH 7.4) TMB 20 mM Tris-5 mM EDTA-0.1 M NaC1 (pH 7.4); also prepared is 2 M TENB TENB (dissolve the EDTA before adding NaCI) Triton/TENB TENB above plus 0.2% Triton X-100 TENB above plus 30% ethylene glycol EG/TENB MEDGANT
Experimental Procedure All steps are at 4 ° except where indicated. Step 1. T w e n t y kidneys ( - 5 0 0 g frozen weight) are thawed briefly in cold tap water and ground in a commercial meat chopper (Chop-Rite Mfg. Co.). The mince is homogenized in a commercial Waring blender (CB-6) at the low-speed setting for 20 sec. This is repeated three times with 2-rain intervals to minimize heating. The homogenate is brought to 6 liters with M E D G A N T and poured through two layers of cheesecloth fixed with a h e a v y rubber band o v e r 4-liter beakers. Step 2. The h o m o g e n a t e is centrifuged at 4500 g in six l-liter bottles (R = 26 cm, max) for 30 rain with a B e c k m a n J-6B centrifuge. Step 3. After fat cake removal the supernatant ( - 5 4 0 0 ml) is mixed with 250 ml (settled volume) of DE-52 equilibrated in M E D G A N T . The mixture is stirred for 30 min and centrifuged as in step 2. The supernatant is decanted and the DE-52 resuspended in 1200 ml of M E D G A N T and centrifuged as in step 2. This DE-52 washing procedure is repeated with 1200 ml of I00 m M M E D G A N T , twice. The e n z y m e is eluted by mixing with 600 ml of 1200 m M M E D G A N T , collecting the DE-52 mixture into one rotor bottle, and centrifuging as in step 2.
[70]
DOG KIDNEYcAMP PHOSPHODIESTERASE
765
Step 4. The supernatant from step 3 ( - 6 5 0 ml) is diluted to 6 liters with TMB and 300 ml of Affi-Gel Blue added. The gel is collected in a coarse glass funnel. High-affinity cAMP phosphodiesterase binds avidly to this gel and is not released at 4 ° . Therefore, extensive washing of the gel before enzyme elution is possible. Low-affinity phosphodiesterase activity and the 60-kDa binding protein are released by washing consecutively with five bed volumes of TMB, TENB, 2 M TENB, EG/TENB, Triton/ TENB, TMB, and EG/2 M TENB with the gel in the funnel. The gel is suspended in an equal volume of TMB and transferred to a Pharmacia column or its equivalent, water jacketed to 30 °. Approximately 20% of the applied activity measured with 0.25/~M cAMP as substrate (about 5-10% of the homogenate activity) is eluted with 1 liter of EG/2 M TENB at 30 °. The eluted enzyme activity is collected in a flask cooled to 4 °. Steps 1-4 can be performed in approximately 6 hr and should yield near 1 ~mol/min activity with 0.25 p~M cAMP as substrate. Step 5. The eluted enzyme fraction from step 4 is dialyzed against 12 liters of TMB overnight and pumped onto DEAE-Sephacel (5 ml). The column is washed with 50 ml of MEDGANT and high-affinity activity eluted isocratically with 40 ml of 1200 mM MEDGANT. Two-milliliter fractions are collected and the most active pooled ( - 1 0 ml) for gel filtration. TABLE I PURIFICATION PROCEDURES FOR HIGH-AFFINITY, cAMP-SPECIFIC, TYPE IV DOG KIDNEY c A M P PHOSPHODIESTERASEa
Step Homogenate 4500 g s u p e r n a t a n t 4500 g pellet DE-52 "batch" chromatography
(1200 mM MEDGANT eluate) Affi-Gel Blue "batch" absorb (EG/2M T E N B , 30 ° eluate) DEAE-Sephacel Sephacryl S-200 gel filtration Analytical HA chromatography
Protein (mg)
Activity (nmol/min)
Specific activity (mU/mg)
Purification (-fold)
27,000 16,900 9,000 1,600
9,100 6,700 3,000 3,710
44
910
21
69
406 180
185 360
615 1,200
110
1,800
6,100
2.2 0.5 0.06
0.3 0.4 0.3 2.3
I 1.3 -7.7
" Data are compiled from several different preparations. Activities were determined using 0.25 ~ M c A M P as substrate, 5 mM MgCl2, 3.75 mM 2-mercaptoethanol, 30 # g BSA, and 40 mM Tris-HCl (pH 8.0) in a total volume of 400/~1. One milliunit equals 1
nmol cAMP hydrolyzed/min.
766
PHOSPHODIESTERASE
ISOZYME METHODS
[71]
Step 6. The activity is fractionated by gel filtration with Sephacryl S-200 (bed volume = 390 ml) using the S-200 buffer. The flow rate for the column is 1.0 ml/min and 2.5-ml fractions are collected. The majority of the enzyme fractionates just ahead of bovine serum albumin. Step 7. An analytical hydroxylapatite column (1 ml) is charged with 350 mM HA buffer and equilibrated in 10 mM HA buffer. The peak activity fractions from the S-200 gel filtration column are pooled and applied directly ( - 4 0 ml). The column is washed with 20 ml of HA buffer and the nearly homogeneous enzyme eluted with a linear gradient (20 ml total volume) of 10 to 350 mM HA buffer. The enzyme should be dialyzed against 10 mM HA buffer for stable storage at 4° for several weeks. Purification of the enzyme is summarized in Table I. Acknowledgment Research supported by USPHS Grant GM 33538.
[71] B r a i n L o w - K i n c A M P P h o s p h o d i e s t e r a s e By PHILIPPE DE MAZANCOURT a n d YVES GIUDICELLI Multiple forms of cyclic nucleotide phosphodiesterase activities can be isolated from brain cortex extracts. Among these, three separable and kinetically distinct fractions are predominant: the extensively studied calmodulin-sensitive form, which has low affinity for cAMP, the Ca 2+dependent, calmodulin-insensitive enzyme, which hydrolyzes both cAMP and cGMP with low affinity, and a third form referred as the low-Km cAMP phosphodiesterase, which displays high affinity for cAMP and marginal responsiveness to calmodulin and Ca 2+. The first evidence for the existence of low-Kin cAMP phosphodiesterase activity in the brain was provided by Brooker et al.,~ who showed that the cAMP phosphodiesterase activity of rat brain could be kinetically resolved into a high(Km = 1/xM) and a low-affinity (Km = 100/xM) component. Three years later, Thompson and Appleman 2 applied the agarose gel filtration technique to rat brain extracts and found two cAMP phosphodiesterase fractions, one with high molecular weight and high Km (100/xM) and the other G. Brooker, L. Thomas, Jr., and M. Appleman, Biochemistry 7, 4177 (1968). 2 W. J. Thompson and M. M. Appleman, Biochemistry 10, 311 (1971).
METHODS IN ENZYMOLOGY, VOL. 159
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