- Email: [email protected]
[72] Purification of cAMP phosphodiesterase from platelets
772
PHOSPHODIESTERASE ISOZYME METHODS
[72]
soluble low-Km cAMP activity is unaltered by GTP and N6-PIA 19have led to the suggestion that membrane-bound guanine nucleotide regulatory proteins analogous to those controlling adenylate cyclase activity are involved in the mechanisms by which GTP and N6-PIA modulate the activity of the particulate enzyme in brain. As cAMP is the second messenger of various neurotransmitters, it is almost certain that a better knowledge of the regulation of this enzyme will not only contribute to improve our understanding of how external stimuli modulate neuronal function but also provide new therapeutic issues. Acknowledgments Part of this work was supported by Grant 824005from the Institut National de la Sant6 et de la Recherche M6dicale.
[72] P u r i f i c a t i o n o f c A M P P h o s p h o d i e s t e r a s e f r o m P l a t e l e t s
By PAUL G. GRANT and ROBERT W. COLMAN The cyclic nucleotide phosphodiesterases (EC 3.1.4.17) form the only known enzymatic pathway for the hydrolysis of the important regulatory nucleotides cAMP and cGMP. The enzyme activity has been reported to exist in multiple forms in a wide variety of tissues and cell types. 1,2 These forms differ in their substrate specificities, in their kinetic and physical properties, and in their responses to natural and pharmacologic effectors. The physiological roles of these multiple forms within cells and their relationships to each other are not well understood. Purification and physical and immunological characterization of these enzymes should advance our understanding of these enzymes and their roles in cells. In human platelets, cAMP plays an important role in the regulation of platelet activation and thus platelet participation in hemostasis and thrombosis. An increase in the intracellular concentration of cAMP is closely associated with inhibition of platelet shape change, aggregation, adhesion, and secretion of granule contents. 3-5 Three forms of cyclic nucleotide phosphodiesterase activity have been separated by DEAE-Cellulose chromatography from the cytosol of huJ. A. Beavo, R. S. Hansen, S. A. Harrison, R. L. Hurwitz, T. J. Martins, and M. C. Mumby, Mol. Cell. Endocrinol. 28, 387 (1982). 2 M. M. Appleman, M. Ariano, D. J. Takemoto, and R. H. Whitson, Handb. Exp. Pharmacol. 58, 261 (1982). 3 E. W. Salzman and H. Weisinberger, Adv. Cyclic Nucleotide Res. 1, 231 (1972). 4 R. J. Haslam, Ser. Haematol. 6, 333 (1973). 5 D. C. B. Mills, Handb. Exp. Pharmacol. 58/II, 725 (1982).
METHODS IN ENZYMOLOGY,VOL. 159
Copyright © 1988by Academic Press. Inc. All rights of reproduction in any form reserved.
[72]
PLATELETcAMP PHOSPHODIESTERASE
773
man platelets. 6 One form was reported to be specific for cGMP, another relatively nonspecific, and the third form relatively specific for cAMP. The cytosolic fraction contains at least 75 to 80% of the cAMP hydrolytic activity in human platelets. 7,s This chapter describes the purification of the cAMP-specific form, or low-Kin cAMP form, of cyclic nucleotide phosphodiesterase from the cytosolic fraction of human platelets. The purification of this phosphodiesterase consists basically of three steps: preparation of a 150,000 g supernatant fraction from disrupted platelets, DEAE-Cellulose chromatography, and chromatography on blue dextran-Sepharose. Preparation of Blue Dextran-Sepharose Blue dextran-Sepharose was prepared using a modification of a procedure described by Ryan and Vestling. 9 Five grams of cyanogen bromideactivated Sepharose 4B (Pharmacia) was rehydrated in 25 to 50 ml of ! mM HC1 for 30 rain at room temperature (20 to 25°). The rehydrated gel was filtered and washed repeatedly with aliquots of 1 mM HCI (a total of 1 liter). The gel was then washed repeatedly with aliquots of 0.1 M NaHCO3, pH 8.5 (250 ml total). The washed gel was then resuspended in 37.5 ml of 0.1 M NaHCO3, pH 8.5 containing 0.35 g of blue dextran 2000 (Pharmacia), and mixed on a rocking table for 5 hr at room temperature. The blue dextran-containing buffer was removed by filtration. The gel was then resuspended in 50 ml of 0.1 M NaHCO3, pH 8.5, containing 1 M ethanolamine and mixed for 1.5 hr at room temperature to derivatize any remaining active groups on the gel. The gel was then filtered, washed repeatedly with water, and stored at 4° in 50 mM Tris-HCl, pH 7.5, containing 20 m M MgC12 and 0.02% NAN3.
Regeneration of Blue Dextran-Sepharose After use the blue dextran-Sepharose was regenerated by washing the column with 6 M urea. The column was washed with two to three column volumes of 6 M urea and then allowed to sit overnight in 6 M urea. The next day the column was washed with an additional 2 vol of 6 M urea and then reequilibrated by washing with several column volumes of 50 mM Tris-HC1, pH 7.5, containing 20 mM MgCI2. When the gel was to be stored at 4 °, 0.02% NaN3 was added to the buffer. 6 H. Hidaka and T. Asano, Biochim. Biophys. Acta 429, 485 (1976). 7 p. G. Grant and R. W. Colman, Biochemist~ 23, 1801 (1984). H. Hidaka and T. Endo, Adv. Cyclic Nucleotide Protein Phospho©'lation Res. 16, 245 (1984). L. D. Ryan and C. S. Vestling, Arch. Biochem. Biophys. 160, 279 (1974).
774
P H O S P H O D I E S T E R A S E ISOZYME M E T H O D S
[72]
Purification of Low-KIn cAMP Phosphodiesterase from Human Platelets
Preparation of Platelets Outdated platelet concentrates were obtained from the local American Red Cross blood bank. Each unit of platelet concentrate was derived from one unit (450 ml) of blood and contained about 10N platelets. The sealed bags were opened and the contents drained into polycarbonate centrifuge bottles. The suspensions were made 1 mM in EDTA by the addition of neutralized 0.5 M EDTA solution. The platelet suspensions were then centrifuged at 5 to 10° for 2.5 rain at 1500 g (3000 rpm) in a Sorvall GS-3 rotor. This centrifugation pelleted most of the contaminating erythrocytes with minimal loss of platelets. The supernatant platelet suspension was carefully decanted and centrifuged at 10,000 g (8000 rpm) for 20 min in a GS-3 rotor to pellet the platelets. The plasma was discarded and the platelet pellet resuspended in 50 mM Tris-acetate, pH 6.0, containing 1 m M EDTA and 0.15 M NaC1. Platelets were resuspended by carefully removing the upper part of the pellet with a rubber policeman while leaving the lower portion of the pellet, which contained some erythrocyte contamination, undisturbed. The wash was repeated a second time and the platelet pellets were then frozen in a dry-ice bath and stored at - 7 0 °.
Disruption of Platelets The washed, frozen platelet pellets from 150 to 250 units of blood were resuspended in 200 ml of 100 m M Tris-acetate, pH 6.0, containing 20 m M MgCI2, 20 m M benzamidine hydrochloride, 10 m M e-amino-n-caproic acid (EACA), 4 m M EGTA, 0.4 mg/ml soybean trypsin inhibitor, 0.4 U/ ml hirudin, 50 kallikrein inhibitory U/ml aprotinin, 10 mM diisopropyl fluorophosphate (DFP), 4 m M phenylmethylsulfonyl fluoride, 40/zM Np-tosyl-L-lysine chlorometyl ketone (TLCK), 20 /zM leupeptin, and 10 ~ M pepstatin A. All succeeding steps were done at 0 to 4 ° unless indicated. The resuspended platelets (about 350 ml total volume) were placed in a plastic beaker inside a nitrogen bomb (Paar Instrument Company, Moline, IL) and pressurized to 1200 to 1300 psi. The sample was stirred for 45 min while pressure was maintained to allow the nitrogen to dissolve in the sample. The sample was then released from the bomb and centrifuged at 27,000 g (15,000 rpm) for I0 min in a Sorvall SS-34 rotor. The supernatant was decanted and retained. The pellets were resuspended in 50 to 100 ml of the resuspension buffer and recentrifuged. The supernatants from the initial centrifugation and the wash were combined and centrifuged for 1 hr at 50,000 rpm (150,000 g) in a Beckman Ti-60 rotor. The supernatant from
[72]
PLATELETcAMP PHOSPHODIESTERASE
775
this centrifugation was carefully removed, avoiding the cloudy region that was just above the pellet.
DEAE-Cellulose Chromatography The 150,000 g supernatant was applied to a 200- to 250-ml DEAECellulose column (Whatman DE-52) preequilibrated in 50 mM Tris-acetate, pH 6.0, containing 20 m M MgCI2, 10 mM benzamidine-HC1, 5 mM EACA, 2 m M EGTA, 0.02% soybean trypsin inhibitor, 20/xM TLCK, 10 /zM leupeptin, and 5 /zM pepstatin A. After the 150,000 g supernatant sample had been applied, the column was washed with two to four bed volumes of buffer at a flow rate of 1 ml/min. The column was eluted with a linear gradient of sodium acetate (0 to 0.6 M) in a total volume of 1 liter. Fractions (4 ml) were collected at a flow rate of 1 ml/min. The column fractions were assayed for cAMP phosphodiesterase activity at both l and 100/zM cAMP using a barium sulfate precipitation assay. J0Two peaks of cAMP phosphodiesterase activity were eluted from the DEAE-Cellulose column. The first peak eluted at about 0.25 to 0.3 M sodium acetate and the second peak at about 0.4 to 0.45 M sodium acetate. Peak II had a lower Km for cAMP hydrolysis than peak I and hydrolyzed cAMP more rapidly than cGMP. The first peak had higher Km values for both cAMP and cGMP and appeared to hydrolyze both cAMP and cGMP equally. 6,11The second, low-Kin cAMP phosphodiesterase was chosen for further purification.
Blue Dextran-Sepharose Chromatography The fractions from the DEAE-Cellulose column containing the second peak of cAMP phosphodiesterase activity were pooled and applied directly to a I0- to 15-ml column of blue dextran-Sepharose equilibrated in 50 m M Tris-HC1, pH 7.5, containing 20 m M MgCI2, 10 m M benzamidineHCI, 5 m M EACA, 2 m M EGTA, 20 p,M TLCK, l0/zM leupeptin, and 5 /z M pepstatin A at a flow rate of 0.5 ml/min. The column was then washed with 3 to 5 vol of the equilibration buffer. Under these conditions the cAMP phosphodiesterase activity found in the first peak from the DEAE column did not bind to the blue dextran-Sepharose and any contamination by this enzyme was removed during the wash of the blue dextranSepharose column. After washing the column was eluted with a 100 ml linear gradient of cAMP (0 to 1 mM). When the fractions (2 ml) were assayed, one peak of cAMP phosphodiesterase activity was observed. to A. K. Sinha, S. J. Shattil, and R. W. Colman, J. Biol. Chem. 252, 3360 (1977). u p. G. Grant, A. F. Mannarino, and R. W. Colman, submitted for publication (1988).
776
[72]
P H O S P H O D I E S T E R AISOZYME SE METHODS TABLE l PURIFICATION OF cAMP PHOSPHODIESTERASE FROM HUMAN PLATELETS
Total activity
Recovery
Specific activity
Purification
Step
(nmol/min)
(%)
(nmol/min/mg)"
(-fold)
150,000 g supernatant DEAE-cellulose peak Blue dextran-Sepharose peak
2780 1560 873
100 56 31
0.98 5.8 2530
1 5.9 2580
Specific activity was determined at 1 /xM cAMP.
TABLE I1 PROPERTIES OF cAMP PHOSPHODIESTERASE FROM HUMAN PLATELETS
Property
Value
Molecular weight Monomer (SDS)
61,000 140,000
Gel filtration
Kinetics of cyclic nucleotide hydrolysis (Michaelis-Menten) Km for cAMP Vmax for cAMP Km for cGMP Vmaxfor cGMP
Response to Ca2+-calmodulin
0.18 ± 0.06/xM 3000 pmol/min//zg 0.020 + 0.007/zM 310 pmol/min//zg
Insensitive
The peak fractions were pooled, placed in dialysis tubing, and concentrated against dry sucrose. The concentrated enzyme was stable on storage at 0 to 4 ° with little lose of activity for periods of up to 6 months. Table I shows the results of a typical purification. The overall purification was about 2500-fold, with most of the purification occurring in the blue dextran-Sepharose step. The purification is rapid with good recovery of activity. Table II is a summary of some of the properties of the purified cAMP phosphodiesterase. A more detailed description of the characteristics of this enzyme have been published elsewhere. 7 Some preparations of the cAMP phosphodiesterase have been contaminated with actin. We have found that rechromatography of the enzyme on blue dextran-Sepharose with reelution with cAMP has been quite useful in removing this contamination. The actin contamination can also be removed by DNase I affinity chromatography as described by Lazarides and Lindberg.12 I2 E. Lazarides and U. Lindberg, Proc. Natl. Acad. Sci. U.S.A. 71, 4742 (1974).
[73]
YEAST ZINC-CONTAINING PHOSPHODIESTERASES
777
Recent immunological evidence indicates that the native form of this phosphodiesterase has a monomer molecular weight of 110 kDa. j3 Proteolysis of the enzyme occurs during the purification procedure. We have recently found that passage of the 150,000 g supernatant fraction directly through a blue dextran-Sepharose column resulted in the isolation of a 110 kDa form of the phosphodiesterase as determined by immunoblot analysis.ll Additional protease inhibitors or a change in the order of the purification procedure may be necessary to avoid this proteolysis. The physiological roles of the various forms of cyclic nucleotide phosphodiesterase activity found in cells are not well understood. Purification and characterization of these multiple forms should aid in our understanding of the roles of these various forms in the regulation of cyclic nucleotide metabolism in cells and in our understanding of the relationships of the various phosphodiesterases to each other. Acknowledgment Supported by a ProgramProject Grant from NIH HL36579and by a Special Investigator Award (To P.G.G.) and a Grant-in-Aidfrom the Southeastern Pennsylvaniachapter of the American Heart Association. ~ C. H. Macphee, S. A. Harrison,and J. A. Beavo.Proc.
Natl. Acad. Sci. U.S.A.
83, 6660
(1986).
[73] Z i n c - C o n t a i n i n g C y c l i c N u c l e o t i d e P h o s p h o d i e s t e r a s e s from Bakers' Yeast By JOHN LONDESBOROUGH and KARl SUORANTA
Bakers' yeast contains at least two cyclic nucleotide phosphodiesterases, A high-Kin, free bivalent metal ion-independent cytosolic enzyme and a low-Kin, free bivalent metal ion-dependent cAMP-specific enzyme that is loosely bound to microsomal particles have been purified to apparent homogeneity. 1,2 Both contain tightly bound zinc: two atoms/subunit (43 kDa) of high-Kin enzyme and probably one atom/molecule (61 kDa) of low-Kin enzyme. Removal of the zinc causes reversible losses of catalytic activity. The activity of the high-Km phosphodiesterase 3 was highest in stationary phase cells, such as commercial bakers' yeast. Highest activities of J. Londesborough and K. Suoranta, J. Biol. Chem. 258, 2966 (1983). z K. Suoranta and J. Londesborough, J. Biol. Chem. 259, 6964 (1984). 3K. Suoranta, J. Cyclic Nucleotide Protein Phospho~lation Res. 10, 121 (1985).
METHODS IN ENZYMOLOGY,VOL. 159
Copyright ¢3 1988by Academic Press, Inc. All rights of reproductionin any form reserved.
PHOSPHODIESTERASE ISOZYME METHODS
[72]
soluble low-Km cAMP activity is unaltered by GTP and N6-PIA 19have led to the suggestion that membrane-bound guanine nucleotide regulatory proteins analogous to those controlling adenylate cyclase activity are involved in the mechanisms by which GTP and N6-PIA modulate the activity of the particulate enzyme in brain. As cAMP is the second messenger of various neurotransmitters, it is almost certain that a better knowledge of the regulation of this enzyme will not only contribute to improve our understanding of how external stimuli modulate neuronal function but also provide new therapeutic issues. Acknowledgments Part of this work was supported by Grant 824005from the Institut National de la Sant6 et de la Recherche M6dicale.
[72] P u r i f i c a t i o n o f c A M P P h o s p h o d i e s t e r a s e f r o m P l a t e l e t s
By PAUL G. GRANT and ROBERT W. COLMAN The cyclic nucleotide phosphodiesterases (EC 3.1.4.17) form the only known enzymatic pathway for the hydrolysis of the important regulatory nucleotides cAMP and cGMP. The enzyme activity has been reported to exist in multiple forms in a wide variety of tissues and cell types. 1,2 These forms differ in their substrate specificities, in their kinetic and physical properties, and in their responses to natural and pharmacologic effectors. The physiological roles of these multiple forms within cells and their relationships to each other are not well understood. Purification and physical and immunological characterization of these enzymes should advance our understanding of these enzymes and their roles in cells. In human platelets, cAMP plays an important role in the regulation of platelet activation and thus platelet participation in hemostasis and thrombosis. An increase in the intracellular concentration of cAMP is closely associated with inhibition of platelet shape change, aggregation, adhesion, and secretion of granule contents. 3-5 Three forms of cyclic nucleotide phosphodiesterase activity have been separated by DEAE-Cellulose chromatography from the cytosol of huJ. A. Beavo, R. S. Hansen, S. A. Harrison, R. L. Hurwitz, T. J. Martins, and M. C. Mumby, Mol. Cell. Endocrinol. 28, 387 (1982). 2 M. M. Appleman, M. Ariano, D. J. Takemoto, and R. H. Whitson, Handb. Exp. Pharmacol. 58, 261 (1982). 3 E. W. Salzman and H. Weisinberger, Adv. Cyclic Nucleotide Res. 1, 231 (1972). 4 R. J. Haslam, Ser. Haematol. 6, 333 (1973). 5 D. C. B. Mills, Handb. Exp. Pharmacol. 58/II, 725 (1982).
METHODS IN ENZYMOLOGY,VOL. 159
Copyright © 1988by Academic Press. Inc. All rights of reproduction in any form reserved.
[72]
PLATELETcAMP PHOSPHODIESTERASE
773
man platelets. 6 One form was reported to be specific for cGMP, another relatively nonspecific, and the third form relatively specific for cAMP. The cytosolic fraction contains at least 75 to 80% of the cAMP hydrolytic activity in human platelets. 7,s This chapter describes the purification of the cAMP-specific form, or low-Kin cAMP form, of cyclic nucleotide phosphodiesterase from the cytosolic fraction of human platelets. The purification of this phosphodiesterase consists basically of three steps: preparation of a 150,000 g supernatant fraction from disrupted platelets, DEAE-Cellulose chromatography, and chromatography on blue dextran-Sepharose. Preparation of Blue Dextran-Sepharose Blue dextran-Sepharose was prepared using a modification of a procedure described by Ryan and Vestling. 9 Five grams of cyanogen bromideactivated Sepharose 4B (Pharmacia) was rehydrated in 25 to 50 ml of ! mM HC1 for 30 rain at room temperature (20 to 25°). The rehydrated gel was filtered and washed repeatedly with aliquots of 1 mM HCI (a total of 1 liter). The gel was then washed repeatedly with aliquots of 0.1 M NaHCO3, pH 8.5 (250 ml total). The washed gel was then resuspended in 37.5 ml of 0.1 M NaHCO3, pH 8.5 containing 0.35 g of blue dextran 2000 (Pharmacia), and mixed on a rocking table for 5 hr at room temperature. The blue dextran-containing buffer was removed by filtration. The gel was then resuspended in 50 ml of 0.1 M NaHCO3, pH 8.5, containing 1 M ethanolamine and mixed for 1.5 hr at room temperature to derivatize any remaining active groups on the gel. The gel was then filtered, washed repeatedly with water, and stored at 4° in 50 mM Tris-HCl, pH 7.5, containing 20 m M MgC12 and 0.02% NAN3.
Regeneration of Blue Dextran-Sepharose After use the blue dextran-Sepharose was regenerated by washing the column with 6 M urea. The column was washed with two to three column volumes of 6 M urea and then allowed to sit overnight in 6 M urea. The next day the column was washed with an additional 2 vol of 6 M urea and then reequilibrated by washing with several column volumes of 50 mM Tris-HC1, pH 7.5, containing 20 mM MgCI2. When the gel was to be stored at 4 °, 0.02% NaN3 was added to the buffer. 6 H. Hidaka and T. Asano, Biochim. Biophys. Acta 429, 485 (1976). 7 p. G. Grant and R. W. Colman, Biochemist~ 23, 1801 (1984). H. Hidaka and T. Endo, Adv. Cyclic Nucleotide Protein Phospho©'lation Res. 16, 245 (1984). L. D. Ryan and C. S. Vestling, Arch. Biochem. Biophys. 160, 279 (1974).
774
P H O S P H O D I E S T E R A S E ISOZYME M E T H O D S
[72]
Purification of Low-KIn cAMP Phosphodiesterase from Human Platelets
Preparation of Platelets Outdated platelet concentrates were obtained from the local American Red Cross blood bank. Each unit of platelet concentrate was derived from one unit (450 ml) of blood and contained about 10N platelets. The sealed bags were opened and the contents drained into polycarbonate centrifuge bottles. The suspensions were made 1 mM in EDTA by the addition of neutralized 0.5 M EDTA solution. The platelet suspensions were then centrifuged at 5 to 10° for 2.5 rain at 1500 g (3000 rpm) in a Sorvall GS-3 rotor. This centrifugation pelleted most of the contaminating erythrocytes with minimal loss of platelets. The supernatant platelet suspension was carefully decanted and centrifuged at 10,000 g (8000 rpm) for 20 min in a GS-3 rotor to pellet the platelets. The plasma was discarded and the platelet pellet resuspended in 50 mM Tris-acetate, pH 6.0, containing 1 m M EDTA and 0.15 M NaC1. Platelets were resuspended by carefully removing the upper part of the pellet with a rubber policeman while leaving the lower portion of the pellet, which contained some erythrocyte contamination, undisturbed. The wash was repeated a second time and the platelet pellets were then frozen in a dry-ice bath and stored at - 7 0 °.
Disruption of Platelets The washed, frozen platelet pellets from 150 to 250 units of blood were resuspended in 200 ml of 100 m M Tris-acetate, pH 6.0, containing 20 m M MgCI2, 20 m M benzamidine hydrochloride, 10 m M e-amino-n-caproic acid (EACA), 4 m M EGTA, 0.4 mg/ml soybean trypsin inhibitor, 0.4 U/ ml hirudin, 50 kallikrein inhibitory U/ml aprotinin, 10 mM diisopropyl fluorophosphate (DFP), 4 m M phenylmethylsulfonyl fluoride, 40/zM Np-tosyl-L-lysine chlorometyl ketone (TLCK), 20 /zM leupeptin, and 10 ~ M pepstatin A. All succeeding steps were done at 0 to 4 ° unless indicated. The resuspended platelets (about 350 ml total volume) were placed in a plastic beaker inside a nitrogen bomb (Paar Instrument Company, Moline, IL) and pressurized to 1200 to 1300 psi. The sample was stirred for 45 min while pressure was maintained to allow the nitrogen to dissolve in the sample. The sample was then released from the bomb and centrifuged at 27,000 g (15,000 rpm) for I0 min in a Sorvall SS-34 rotor. The supernatant was decanted and retained. The pellets were resuspended in 50 to 100 ml of the resuspension buffer and recentrifuged. The supernatants from the initial centrifugation and the wash were combined and centrifuged for 1 hr at 50,000 rpm (150,000 g) in a Beckman Ti-60 rotor. The supernatant from
[72]
PLATELETcAMP PHOSPHODIESTERASE
775
this centrifugation was carefully removed, avoiding the cloudy region that was just above the pellet.
DEAE-Cellulose Chromatography The 150,000 g supernatant was applied to a 200- to 250-ml DEAECellulose column (Whatman DE-52) preequilibrated in 50 mM Tris-acetate, pH 6.0, containing 20 m M MgCI2, 10 mM benzamidine-HC1, 5 mM EACA, 2 m M EGTA, 0.02% soybean trypsin inhibitor, 20/xM TLCK, 10 /zM leupeptin, and 5 /zM pepstatin A. After the 150,000 g supernatant sample had been applied, the column was washed with two to four bed volumes of buffer at a flow rate of 1 ml/min. The column was eluted with a linear gradient of sodium acetate (0 to 0.6 M) in a total volume of 1 liter. Fractions (4 ml) were collected at a flow rate of 1 ml/min. The column fractions were assayed for cAMP phosphodiesterase activity at both l and 100/zM cAMP using a barium sulfate precipitation assay. J0Two peaks of cAMP phosphodiesterase activity were eluted from the DEAE-Cellulose column. The first peak eluted at about 0.25 to 0.3 M sodium acetate and the second peak at about 0.4 to 0.45 M sodium acetate. Peak II had a lower Km for cAMP hydrolysis than peak I and hydrolyzed cAMP more rapidly than cGMP. The first peak had higher Km values for both cAMP and cGMP and appeared to hydrolyze both cAMP and cGMP equally. 6,11The second, low-Kin cAMP phosphodiesterase was chosen for further purification.
Blue Dextran-Sepharose Chromatography The fractions from the DEAE-Cellulose column containing the second peak of cAMP phosphodiesterase activity were pooled and applied directly to a I0- to 15-ml column of blue dextran-Sepharose equilibrated in 50 m M Tris-HC1, pH 7.5, containing 20 m M MgCI2, 10 m M benzamidineHCI, 5 m M EACA, 2 m M EGTA, 20 p,M TLCK, l0/zM leupeptin, and 5 /z M pepstatin A at a flow rate of 0.5 ml/min. The column was then washed with 3 to 5 vol of the equilibration buffer. Under these conditions the cAMP phosphodiesterase activity found in the first peak from the DEAE column did not bind to the blue dextran-Sepharose and any contamination by this enzyme was removed during the wash of the blue dextranSepharose column. After washing the column was eluted with a 100 ml linear gradient of cAMP (0 to 1 mM). When the fractions (2 ml) were assayed, one peak of cAMP phosphodiesterase activity was observed. to A. K. Sinha, S. J. Shattil, and R. W. Colman, J. Biol. Chem. 252, 3360 (1977). u p. G. Grant, A. F. Mannarino, and R. W. Colman, submitted for publication (1988).
776
[72]
P H O S P H O D I E S T E R AISOZYME SE METHODS TABLE l PURIFICATION OF cAMP PHOSPHODIESTERASE FROM HUMAN PLATELETS
Total activity
Recovery
Specific activity
Purification
Step
(nmol/min)
(%)
(nmol/min/mg)"
(-fold)
150,000 g supernatant DEAE-cellulose peak Blue dextran-Sepharose peak
2780 1560 873
100 56 31
0.98 5.8 2530
1 5.9 2580
Specific activity was determined at 1 /xM cAMP.
TABLE I1 PROPERTIES OF cAMP PHOSPHODIESTERASE FROM HUMAN PLATELETS
Property
Value
Molecular weight Monomer (SDS)
61,000 140,000
Gel filtration
Kinetics of cyclic nucleotide hydrolysis (Michaelis-Menten) Km for cAMP Vmax for cAMP Km for cGMP Vmaxfor cGMP
Response to Ca2+-calmodulin
0.18 ± 0.06/xM 3000 pmol/min//zg 0.020 + 0.007/zM 310 pmol/min//zg
Insensitive
The peak fractions were pooled, placed in dialysis tubing, and concentrated against dry sucrose. The concentrated enzyme was stable on storage at 0 to 4 ° with little lose of activity for periods of up to 6 months. Table I shows the results of a typical purification. The overall purification was about 2500-fold, with most of the purification occurring in the blue dextran-Sepharose step. The purification is rapid with good recovery of activity. Table II is a summary of some of the properties of the purified cAMP phosphodiesterase. A more detailed description of the characteristics of this enzyme have been published elsewhere. 7 Some preparations of the cAMP phosphodiesterase have been contaminated with actin. We have found that rechromatography of the enzyme on blue dextran-Sepharose with reelution with cAMP has been quite useful in removing this contamination. The actin contamination can also be removed by DNase I affinity chromatography as described by Lazarides and Lindberg.12 I2 E. Lazarides and U. Lindberg, Proc. Natl. Acad. Sci. U.S.A. 71, 4742 (1974).
[73]
YEAST ZINC-CONTAINING PHOSPHODIESTERASES
777
Recent immunological evidence indicates that the native form of this phosphodiesterase has a monomer molecular weight of 110 kDa. j3 Proteolysis of the enzyme occurs during the purification procedure. We have recently found that passage of the 150,000 g supernatant fraction directly through a blue dextran-Sepharose column resulted in the isolation of a 110 kDa form of the phosphodiesterase as determined by immunoblot analysis.ll Additional protease inhibitors or a change in the order of the purification procedure may be necessary to avoid this proteolysis. The physiological roles of the various forms of cyclic nucleotide phosphodiesterase activity found in cells are not well understood. Purification and characterization of these multiple forms should aid in our understanding of the roles of these various forms in the regulation of cyclic nucleotide metabolism in cells and in our understanding of the relationships of the various phosphodiesterases to each other. Acknowledgment Supported by a ProgramProject Grant from NIH HL36579and by a Special Investigator Award (To P.G.G.) and a Grant-in-Aidfrom the Southeastern Pennsylvaniachapter of the American Heart Association. ~ C. H. Macphee, S. A. Harrison,and J. A. Beavo.Proc.
Natl. Acad. Sci. U.S.A.
83, 6660
(1986).
[73] Z i n c - C o n t a i n i n g C y c l i c N u c l e o t i d e P h o s p h o d i e s t e r a s e s from Bakers' Yeast By JOHN LONDESBOROUGH and KARl SUORANTA
Bakers' yeast contains at least two cyclic nucleotide phosphodiesterases, A high-Kin, free bivalent metal ion-independent cytosolic enzyme and a low-Kin, free bivalent metal ion-dependent cAMP-specific enzyme that is loosely bound to microsomal particles have been purified to apparent homogeneity. 1,2 Both contain tightly bound zinc: two atoms/subunit (43 kDa) of high-Kin enzyme and probably one atom/molecule (61 kDa) of low-Kin enzyme. Removal of the zinc causes reversible losses of catalytic activity. The activity of the high-Km phosphodiesterase 3 was highest in stationary phase cells, such as commercial bakers' yeast. Highest activities of J. Londesborough and K. Suoranta, J. Biol. Chem. 258, 2966 (1983). z K. Suoranta and J. Londesborough, J. Biol. Chem. 259, 6964 (1984). 3K. Suoranta, J. Cyclic Nucleotide Protein Phospho~lation Res. 10, 121 (1985).
METHODS IN ENZYMOLOGY,VOL. 159
Copyright ¢3 1988by Academic Press, Inc. All rights of reproductionin any form reserved.