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Calcium-binding phosphoprotein from pig brain: Identification as a calcium-dependent regulator of brain cyclic nucleotide phosphodiesterase
ARCHIVES
OF BIOCHEMISTRY
AND
Calcium-Binding
BIOPHYSICS
163, 349-358
(1974)
Phosphoprotein
as a Calcium-Dependent
from Pig Brain:
Regulator
Identification
of Brain Cyclic Nucleotide
Phosphodiesterase’ DONALD Department
of Pharmacology,
J. WOLFF
AND
CHARLES
0. BROSTROM
Rutgers Medical School, College of Medicine Piscataway, New Jersey 08854 Received January
and Dentistry
of New Jersey,
15, 1974
A homogeneous, acidic, Ca2+-binding phosphoprotein from pig brain has been identified as the mediator of a Cal+-dependent activation of a partially purified pig brain cyclic nucleotide phosphodiesterase. Crude extracts prepared from porcine brain have been resolved into two fractions by column chromatography with ECTEOLA-cellulose. One fraction possesses phosphodiesterase activity which is stimulated from 4. to 12-fold, depending upon the preparation and the assay conditions, by nanogram quantities of a purified Ca2+-binding phosphoprotein from pig brain at 5 x 10e5 M Ca*+. The stimulation is eliminated by ethylene glycol-bis( @-aminoethyl ether)-N, N’-tetraacetate. The second fraction contains an endogenous, Caz+-dependent activator of the phosphodiesterase activity. Further purification of the crude activator by hydroyxlapatite chromatography, Sephadex G-75 gel filtration, and acrylamide gel electrophoresis has established that the activator and the purified Ca 2+-binding phosphoprotein are identical with respect to chromatographic behavior, molecular weight, and electrophoretic migration. Both proteins bound Cal+ and were stable to boiling for 5 min. At saturating concentrations of the Ca2+ -dependent regulator, half-maximal stimulation of the phosphodiesterase was observed at a Ca2+ concentration of 4 x 10m6 M. Purified preparations of the acidic Ca*+-binding phosphoprotein from bovine adrenal medulla and testis were also active in stimulating the brain phosphodiesterase. Preparations of other known Ca*+-binding proteins, such as rabbit muscle troponin and pig brain S-100 protein, were nonstimulatory.
Cyclic nucleotide phosphodiesterase is a potential focal point through which CAMP levels may be modulated. Two forms of phosphodiesterase activity have been distinguished in brain on the basis of affinity for CAMP, one having a low Michaelis constant (10-6-10-5 M) and the other a higher value (lo-’ M) (1). The separation of multiple phosphodiesterases of varying molecular weight, Michaelis constants, and substrate specificities from brain extracts has been accomplished in several
laboratories (2-4). The relationships of these forms to each other, if any, are unclear. Kakiuchi et al. (5, 6) have reported that two-thirds of the soluble phosphodiesterase from rat brain is Ca2+ dependent. A heat-stable, nondialyzable factor from brain, similar to that reported by Cheung (7), increased the maximal velocity for CAMP hydrolysis (measured at l-2 mM CAMP) approximately 7-fold. A similar cyclic nucleotide phosphodiesterase has been reported from bovine heart by Teo et al. (8, 9) which is activated by a heat-staprotein purified to ap’ This work was supported by grants from the U. S. ble, Ca’+-binding Public Health Service: Rutgers Medical School Gen- parent homogeneity. Wolff and Siegel (10) have described the eral Research Support Grant No. RR 5576, and NS 10975. purification to homogeneity and the physi.^ 34Y Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
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AND BROSTROM
cal characterization of a Ca2+-binding phosphoprotein from pig brain. The phosphoprotein is highly acidic, exhibits a molecular weight of 11,500, and binds 1 mole of Ca2+ per mole of protein with an affinity of lo5 M-‘. A comparable Ca2+-binding protein has been purified from adrenal medulla (11) and testis (12). The specificity of the divalent metal cation binding site has been characterized by Wolff et al. (13). Subsequent investigations of the Ca2+binding phosphoprotein have been directed toward elucidating its function. The present report establishes that this protein mediates the Ca2+-dependent regulation of a brain cyclic nucleotide phosphodiesterase. Additional information is presented with respect to the specificity of the activation, the concentration of Ca2+ required, and the reversibility of the activation. MATERIALS
AND
METHODS
Materials. Calcium-45 (‘YZa) (14.9 Ci/g) and [SH]adenosine 3’, 5’cyclic phosphate ( [SH]cAMP) (22.1 Ci/mmole) were purchased from New England Nuclear Corp. Chelex-100, ECTEOLA-cellulose, hydroxylapatite (Bio-Gel HTP), and AG 1X8 resin (206400 mesh) were purchased from Bio-Rad. Calf intestinal mucosa alkaline phosphatase (350 units/mg protein) was purchased from Sigma Chemical Company. Spectrograde MgSO, ( <1 ppm CaZ+) was obtained from Jarrell Ash. Homogeneous Cal+-binding phosphoprotein from pig brain was prepared by the procedure of Wolff and Siegel (10). S-100 protein was prepared from porcine brain by a modification of the procedure of Moore (14). Rabbit skeletal muscle troponin was prepared by the procedure of Greaser and Gergely (15). Adrenal medullary and testicular Caz+-binding phosphoprotein were the kind gifts of Drs. Frank Siegel and Jack C. Brooks of the Joseph P. Kennedy, Jr., Laboratories, University of Wisconsin Center for Health Sciences. Assay of cyclic nucleotide phosphodiesterase. Phosphodiesterase measurements were performed by a modification of the procedure described by Brooker et al. (1). Standard assays were conducted at 30” C for 15 min. The reaction was initiated by the addition phosphodiesterase acof enzyme. Ca *+-independent tivity was assayed, unless otherwise indicated, in a total volume of 150 ~1 containing CAMP, 1 mM (1 Ci/mole); MgCl,, 5 mM; imidazole buffer, 20 mM, pH 7.5; and EGTA,2 0.05 msr. Cal+-dependent activity * Abbreviations: EGTA, ethylene glycol-bis(& aminoethyl ether)-N,N’-tetraacetic acid; CDR, calcium-dependent regulator.
was determined as the increment of phosphodiesterase activity produced by the addition of CaCl, (0.1 mM). Incubations were terminated by boiling and the 5’-AMP converted to adenosine by the addition of excess alkaline phosphatase (2 units for 15 min at 30” C). The samples were boiled, a slurry (1 ml) of AG 1X8 resin (1: 2 v/v in H,O) added to sequester unconverted CAMP, and the samples were centrifuged for 1OOOgmin. Aliquots (0.5 ml) of the supernatant liquid were removed for scintillation counting. The resultant values (cpm) were corrected for controls incubated without enzyme, and, when appropriate, expressed in terms of moles CAMP converted to Y-AMP. Boiled phosphodiesterase controls were identical to controls without enzyme. ECTEOLA-cellulose chromatography. ECTEOLA-cellulose (100 g) was suspended in 2 liters of 1 N NaOH and allowed to stand for 1 hr at room temperature. The resin was washed three times with 2 liters of double-distilled water and the fines decanted after each washing. The resin was then washed with 20 mM phosphate, pH 7.4, containing 20 mM NaCl and 2mMMg *+ , to a constant pH 7.4, and packed in a glass column (2.5 x 60 cm) under gravity. The column was equilibrated with this buffer and the protein samples applied and eluted as described in the legend of Fig. 1. Acrylamide gel electrophoresis. Analytical disc gel electrophoresis was conducted in Tris-glycine, pH 8.3, as described by Davis (16). Gels (6.0 x 0.6 cm) consisted of a &cm 15% acrylamide small-pore gel and a l-cm stacking gel. Protein samples were diluted with electrophoresis buffer containing 20% (w/v) sucrose and layered on the gels with a Hamilton syringe. Electrophoresis was conducted for 2i/4 hr at room temperature at 1.5 mA per gel employing bromophenol blue as a tracking dye. Gels were stained for 2 hr with a 1% solution of amido Schwartz in 7% acetic acid and destained in a Hoeffer gel-destaining apparatus against 7% acetic acid. Miscellaneous. Protein was determined by the method of Lowry (17). Nucleotide concentrations were standardized by extinction. Calcium concentrations were standardized by atomic absorption spectrophotometry.
RESULTS Resolution of phosphodiesterase.
the
Ca2+-dependent
Preliminary measurements of cyclic nucleotide phosphodiesterase activity in crude extracts of brain (prepared as described in the legend of Fig. 1) indicated that 40% of the enzyme was dependent on Ca2+. A sample of crude brain extract was applied to a column of ECTEOLA-cellulose, and eluted with a linear gradient of NaCl (Fig. 1). Cyclic
CALCIUM-DEPENDENT
"0
20
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30
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50
REGULATOR
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90
50 60 70 80 FRACTION NUMBER
90
60
70
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FIG. 1. ECTEOLA-cellulose chromatography of a crude extract of porcine brain. Adult porcine brains, obtained on ice from a local supplier, were homogenized in 2 vol of 50 mM TrisHCl, pH 7.4, in a Waring Blendor for 2 min at medium speed at 4’ C. The homogenate was clarified by centrifugation at 18,OOOg for 35 min and a portion (138 ml) applied to a (2.5 x 60.cm) column of ECTEOLA-cellulose previously equilibrated with 20 mM sodium phosphate, pH 7.4, containing 20 mM NaCl and 2 mM MgCl,. After the collection of 32 fractions of 9.2 ml, a 600.ml linear gradient to 0.4 M NaCl was applied. Plot A: Protein (04) was determined on the fractions by measuring light absorbance at 280 nm. Measurements of calcium-dependent cyclic nucleotide phosphodiesterase activity were conducted on 25.~1 aliquots of the fractions in the presence (04) and absence (W---m) of 1 pg of homogeneous Ca2+-binding phosphoprotein (see Materials and Methods). Values are expressed as moles CAMP hydrolyzed/ml of enzyme/min. Plot B: Aliquots of the ECTEOLA-cellulose fractions were boiled for 5 min and samples (1 ~1) tested for stimulation of the calcium-dependent cyclic nucleotide phosphodiesterase activity of fraction 54 (04). Values are expressed as the potential of 1 ml of test sample to stimulate CAMP hydrolysis by fraction 54. Aliquots of ECTEOLA-cellulose fractions were dialyzed against 2 liters of 50 mM Tris-HCl, pH 7.4, containing 25 PCi of %a. The system was allowed to come to equilibrium (24 hr), samples (0.2 ml) were withdrawn, and protein-bound %a determined as the difference in cpm inside and outside the dialysis bag as measured by liquid scintillation counting. Proteins were determined by the Lowry procedure (17). The specific activity, expressed as cpm of bound ‘%a per mg protein, is plotted in the form of a histogram
351
OF PHOSPHODIESTERASE
nucleotide phosphodiesterase activity was assayed in the presence of Ca2+ with and without the addition of the homogeneous Ca2+-binding phosphoprotein. A peak of cyclic nucleotide phosphodiesterase activity which was dependent on the presence of both Ca2+ and the Ca2+-binding phosphoprotein was observed (fraction 54). When the cyclic nucleotide phosphodiesterase activity was assayed in the presence of excess EGTA with and without the addition of the calcium-binding phosphoprotein, no such stimulation of activity was observed (data not shown). The cyclic nucleotide phosphodiesterase activity of fraction 54 was stimulated 6-fold by the concurrent addition of Ca2+ and homogeneous calcium-binding phosphoprotein. Samples of the Ca*+-binding phosphoprotein were boiled for 5 min under a variety of conditions as described in the legend of Table I. Boiling of the phosphoTABLE STABILIZATION OF Ca’+-BINDING AGAINST DENATURATION
Protective
agent
Control (unboiled) CDR Ca’+ EGTA Bovine serum albumin Human hemoglobin Rabbit serum proteins ECTEOLA fraction 27
I PHOSPHOPROTEIN BY BOILING’
% Control (unboiled) activity 100 63 38 100 109 94 99
“A lyophilized sample of calcium-binding phosphoprotein was diluted to 3.2 pg/ml in 10 mM imidazole buffer, pH 7.5. Aliquots (0.6 ml) were adjusted to contain (where indicated) the following additives: CaCl,, 0.2 mM; EGTA, 0.1 mM; bovine serum albumin, human hemoglobin, rabbit serum, or protein derived from ECTEOLA-cellulose chromatography of crude porcine brain extracts (fraction 27, Fig. lA), 400 fig/ml. The aliquots were boiled for 5 min and 50-bl portions of each (containing 160 ng of CDR) were assayed for activation of CDR-dependent phosphodiesterase activity as described under Materials and Methods. Phosphodiesterase controls incubated without added CDR hydrolyzed 1.1 x lo- I0 moles CAMP/ min/incubation and, when maximally stimulated with CDR, 3.8 x lo-‘” moles cAMP/min/incubation. One hundred sixty nanograms of CDR in this experiment activated the phosphodiesterase to 70% of the activity observed with the addition of 1 pg CDR.
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AND BROSTROM
protein with EGTA for 5 min destroyed more than half of the phosphodiesterase stimulatory activity; the addition of Ca2+ partially stabilized the protein to boiling. Crude protein mixtures, such as rabbit serum and porcine brain proteins which failed to bind to columns of ECTEOLA-cellulose, provided marked stabilization of the phosphodiesterase stimulatory activity of the phosphoprotein. Purifed proteins, in the form of human hemoglobin and bovine serum albumin, similarly protected the phosphodiesterase stimulatory activity from inactivation by boiling. Complete recovery of the stimulatory activity was observed in the presence of protein, indicating that the calcium-binding phosphoprotein did not adsorb strongly to the protein precipitates produced by boiling. Aliquots of the ECTEOLA-cellulose column fractions were boiled for 5 min to denature endogenous phosphodiesterase activity and l-cl1 samples were then assayed for their ability to stimulate the phosphodiesterase activity of fraction 54 in the presence of Ca2+. Such stimulation was noted for boiled fractions 75-84, with fraction 78 being the most effective (Fig. 1B). Additional aliquots were tested for their Ca2+-binding capacity by equilibrium dialysis. A strong correlation was observed between ‘%a binding and the fractions stimulating the phosphodiesterase. ECTEOLA-cellulose column chromatography thus resolved the Ca2+-dependent phosphodiesterase activity from crude extracts of brain into an endogenous Ca2+-dependent regulator (CDR) fraction and a fraction containing deactivated (CDR-dependent) phosphodiesterase. Occasional preparations of CDR-dependent phosphodiesterase from ECTEOLA-cellulose chromatography contained substantial amounts of CDR-independent phosphodiesterase activity. The independent activity could be selectively denatured by heating the preparation at 55” C for 5 min. Under these conditions, CDR-dependent activity was completely stable and a B-fold Ca*+dependent activity resulted. The ECTEOLA-cellulose fractions
which possessed the CDR activity were pooled and used to conduct further comparisons of the crude CDR with the homogeneous Ca2+-binding phosphoprotein. Hydroxylapatite chromatography of the crude CDR. The Ca2+-binding phosphoprotein had been noted in previous work to adsorb only weakly to columns of hydroxylapatite which had been equilibrated with 20 mM phosphate, pH 6.8, whereas almost all other proteins present in crude brain extracts adsorbed strongly under this condition (10). An aliquot of the pooled crude CDR (ECTEOLA-cellulose fractions 75-84) was chromatographed on a hydroxylapatite column (Fig. 2). The column fractions were assayed for protein and CDR activity. Only a very small amount of the 28. b
ZL24E k2 N 20. z
I;
OS-
a
04 -
IO
20
30
40
FRACTION NUMBER
FIG. 2. Hydroxylapatite chromatography of the crude calcium-dependent regulator. The fractions (75-84) containing the calcium-dependent phosphodiesterase regulator from ECTEOLA-cellulose column chromatography (Fig. 1) were dialyzed against 20 mM potassium phosphate, pH 6.8, and concentrated to 4 ml with a Diaflo apparatus fitted with a UM-10 membrane. The concentrate was applied to a column (1 x 60 cm) of hydroxylapatite and eluted stepwise with buffers of the following composition: 20 mM potassium phosphate, pH 6.8, 60 ml; and 500 mM potassium phosphate, pH 6.8, 60 ml. Fractions (3 ml) were collected and assayed for protein by measuring light absorbance at 230 nm (0-O). Aliquots (1 ~1) were assayed for calcium-dependent stimulation of the sensitive cyclic nucleotide phosphodiesterase of ECTELOA-cellulose column fraction 54 (O--O) as described in Materials and Methods. Values are expressed as the potential of 1 ml of activator to stimulate CAMP hydrolysis by the activator-deficient enzyme.
CALCIUM-DEPENDENT
REGULATOR
protein failed to adsorb to the column in 20 mM phosphate, pH 6.8, and this protein possessed very high CDR activity. Essentially, all of the protein applied to the column eluted when the column eluent was changed to 500 mM phosphate, pH 6.8; a small portion of CDR activity eluted at the leading edge of this fraction. The small amount of CDR activity at the leading edge of the 500 mM phosphate fraction represents trace amounts of CDR which adsorbed at 20 mM phosphate. Such behavior is customarily observed during purification of Ca2+-binding phosphoprotein on hydroxylapatite (10). The fractions comprising each of the protein peaks were pooled, concentrated, and subjected to electrophoresis on 15% acrylamide gels (Fig. 3). The electrophoretic patterns indicated that fractions 11-12, which contained most of the CDR activity, were apparently composed of two proteins, one
OF PHOSPHODIESTERASE
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of which was present only in trace amounts. The predominant protein was observed to co-migrate with a sample of the purified Ca2+-binding phosphoprotein. In addition, the trace impurities were apparently present in much higher concentration in hydroxylapatite column fractions 32-40 which possessed little CDR activity. Previous experience indicates that this trace contaminant is an S-100 protein fraction (18). Purified S-100 protein has been tested for CDR activity (Table II) and does not activate the phosphodiesterase. Sephadex G-75 gel filtration of the CDR. A second aliquot of the crude CDR activity (ECTEOLA-cellulose fractions 75-84) was dialyzed for 18 hr against two changes (4 liters total volume) of 50 mM Tris-HCl, pH 7.4, to remove NaCl, concentrated to 5 ml with a Diaflo apparatus (UM-10 membrane), incubated with 10 PCi of ‘%a and subjected to gel filtration on Sephadex
FIG. 3. Electrophoretic comparison of the crude Ca2+ -dependent regulator from hydroxylapatite chromatography and the homogeneous Ca*+-b’ m d’mg phosphoprotein. Electrophoresis was conducted on 15% acrylamide gels, pH 8.3, by the procedure of Davis (16). The samples were of the following compositions: Gel l-purified pig brain Caz+ -binding phosphoprotein; Gel 2-hydroxylapatite column fractions 11-12; Gel 3-samples 1 + sample 2; Gel 4-hydroxylapatite column fractions 32-40; Gel 5-sample 4 + sample 1; Gel 6-hydroxylapatite column fractions 32-40; Gel ‘i-sample 6 + sample 2.
354
WOLFF TABLE
AND BROSTROM
II
unresolvable. To further establish that the Ca’+-dependent activation of phosphodiesterase was specific to the Ca2+-binding phosphoprotein, other purified proteins Additives Specific activity possessing Ca 2+-binding properties were (% of control) examined for their ability to activate the CDR-dependent phosphodiesterase (Table Ca’+-binding phosphoprotein II). In addition, samples of the homogenePorcine brain 100 ous Ca2+-binding phosphoprotein from Bovine adrenal medulla 103 Bovine testis 133 species and tissue sources other than porS-100 protein 0.6 cine brain were tested. Rabbit muscle tropTroponin
OF CDR-DEPENDENT PHOSPHODIESTERASE BY Ca’+-BINDING PROTEINS~
CALCIUM-DEPENDENT
REGULATOR
OF PHOSPHODIESTERASE
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trations from 1 x 10m6M to 1 x lo-’ M. The concentration of Ca*+ providing half-maximal activation of CDR-dependent phosphodiesterase in the presence of saturating CDR was 4 x 10e6 M. The Ca2+ requirement of the phosphodiesterase was explored further to determine whether the enzyme was activated reversibly or irreversibly. A sample of phosphodiesterase was incubated with CAMP, Mg*+, 20 30 IO and saturating CDR for 45 min, with aliMINUTES quots being withdrawn each min for deterFIG. 4. Time dependence of the cyclic nucleotide mination of CAMP hydrolysis (Fig. 7). The phosphodiesterase activity. Brain phosphodiesterase initial incubation was 0.1 mM in EGTA. At purified through the ECTEOLA-cellulose chromatog6 min of incubation the mixture was diraphy stage was assayed as described under Materials and Methods. EGTA (0.05 mM) was present in all vided into two portions, one of which was a incubation tubes. The assays were initiated by the control to which no further additions were addition of CAMP. Values are expressed as moles of made and the other of which was adjusted CAMP converted/ml of phosphodiesterase (2.1 mg to 0.2 mM Ca*- with 20 mM CaCl,. An protein) in the presence of homogeneous CDR (1 pg) immediate 11-fold stimulation of phosphowith (04) and without (04) the addition of diesterase activity occurred. At 22 min the 0.1 mM CaCl,, and in the absence of CDR with addition of 1.3 mM EGTA resulted in an (W-H) and without (04) the addition of immediate cessation of CAMP conversion CaCI,. in the incubation. After 32 min of incubaHCI. Maximal CDR-dependent phospho- tion the Ca*’ concentration of the incubadiesterase activity was observed at pH T.5 tion was adjusted to 3 mM Ca2+; a marked in imidazole buffer (data not shown) and and immediate stimulation of the phosCDR dependence of the enzyme was main- phodiesterase activity was again observed. tained from pH 6-9. These data, combined with the observation Variation of phosphodiesterase activity of the linear response of the CDR-dependas a function of the CDR concentration was examined. Incubations of a fixed amount of CDR-dependent phosphodiesterase were conducted at rate-limiting concentrations of the CDR (standardized by extinction). The activity of the CDR-dependent phosphodiesterase increased linearly over the range from O-50 ng of homogeneous CDR (Fig. 5). In the presence of amounts of CDR greater t,han 50 ng the activity of the CDR-dependent phosphodiesterase asymptotically approached a maximum. FIG. 5. Variation of cyclic nucleotide phosphodiesThe quantitative sensitivity to Ca2+ of terase activity with concentration of homogeneous CDR. Phosphodiesterase was assayed as described in the CDR activation of the phosphodiesterMaterials and Methods. Phosphodiesterase controls ase was investigated with reagents purified incubated in the absence of CDR hydrolyzed 1.9 x of contaminating Ca2+ (Fig. 6). Controls and, when maxicontaining 0.05 mM EGTA exhibited phos- lo-” moles cAMP/min/incubation mally stimulated with CDR, 6.3 x lo- I” moles phodiesterase activity equivalent to that cAMP/min/incubation. The CDR was predialyzed observed for Ca2+-free reagents in the ab- against H,O and diluted for assay with H,O. Values sence of added EGTA. Increasing degrees are expressed in terms of the stimulation of phosphoof stimulation of the phosphodiesterase diesterase activity and are corrected for basal levels of were observed over a range of Ca*+ concen- CDR-independent activity.
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I 7
6
5
4
3
p CO2’
FIG. 6. Calcium dependence of the brain cyclic nucleotide phosphodiesterase. Phosphodiesterase assays were conducted as described in Materials and Methods with the exceptions that all components were first freed of trace contaminations of CaZ+ by passage through a column (1 x 30 cm) of Chelex-100 resin. Mg2+ was added as spectrograde MgSO, which contained < 1 ppm contaminating Ca*+. Homogeneous CDR (1 pg) and phosphodiesterase were stored over Chelex-100 resin at 2” C for 1 hr before use. Assays were conducted in new polypropylene tubes washed five times with double-distilled H,O. Ca*+ was added at the indicated concentrations as a solution of CaCl,. Values for the ordinate are expressed as percentages of the phosphodiesterase activity observed with the addition of 1 mM CaCl, and are corrected for CDR-independent activity. Controls incubated in the absence of added Ca2+ hydrolyzed 2.2 x 10-l” moles cAMP/min/incubation contents. Samples maximally stimulated with Ca2+ hydrolyzed 1.3 x 10mg moles cAMP/min/incubation contents. Controls to which 0.05 mM EGTA was added exhibited no further decreases in activity beyond that observed for Ca2+-free agents.
ent phosphodiesterase to CDR in the presence of Ca’+, provide compelling evidence that the CDR-dependent phosphodiesterase stimulation responds directly to changes in free Ca*+ in an immediate and reversible fashion. The Ca2+ sensitivity (K, = 4 x 10e6 M) of the effect is compatible with the notion that a substantial portion of the phosphodiesterase activity of brain may be regulated by intracellular Ca2+ fluxes. DISCUSSION
The results presented in this paper demonstrate that Ca2+-dependent activation is
conferred on a brain cyclic nucleotide phosphodiesterase by a previously purified and physically characterized Ca’+-binding phosphoprotein from brain. A Ca2+dependent activating factor endogenous to brain extracts was found to be identical to the characterized phosphoprotein. CDR-dependent phosphodiesterase exhibited a linear increase in activity in response to O-50 ng of the homogeneous phosphoprotein when assayed in the presence of excess Ca2+. Boiling dilute solutions of the phosphoprotein resulted in a loss of CDR activity; this loss of activity was decreased by the presence of Ca’+, and eliminated by the presence of nonspecific protein. Boiled tissue extracts would, therefore, appear suitable for surveying the phosphoprotein in tissue, cellular, and sub-
5
IO I5 20 25 30 35 40 45 MINUTES
FIG. ‘i. Regulation of brain cyclic nucleotide phosphodiesterase by Ca*+. The incubation mixture was constructed containing (in 5 ml) CAMP, 3 mM; MgCl,, 10 mM; imidazole, 50 mM, pH 7.5; EGTA, 0.1 mM; and homogeneous CDR, 10 fig/ml. After a 5-min preincubation of the mixture at 30” C, 200 ~1 of partially purified phosphodiesterase (0.3 mg protein from ECTEOLA-cellulose chromatography) was added to initiate the reaction. Aliquots of 100 11 were removed at specified time intervals and denatured immediately by boiling. At 6 min of incubation the reaction mixture was divided into two portions, one of which was adjusted to 0.2 mM Ca2+ with 20 mM CaCl,. Further additives to the Ca’+-treated tubes were 1.3 mM EGTA (final) at 20 min and 3 mM Ca2+ (final) at 32 min. Values are expressed as moles CAMP hydrolyzed/0.1 ml of enzyme. Open circles (O-O) represent aliquots from the sample containing the additives; closed circles (O--O) represent aliquots from the untreated samples.
CALCIUM-DEPENDENT
REGULATOR
cellular localization studies. Ca’+-binding phosphoproteins isolated from bovine adrenal medulla and testis possess comparable potency in conferring Ca*+-dependent activation on the porcine brain enzyme. CDR activity from different tissues and species can therefore be assayed with the CDRdependent enzyme from porcine brain. Calcium-dependent regulator-dependent phosphodiesterase activity was stimulated half-maximally at 4 x lOme M Ca*+ indicating that the affinity of the phosphoprotein for Ca *+ is increased 2- to S-fold when associated with the phosphodiesterase. Stimulation of phos.phodiesterase by Ca*+ was readily reversed by EGTA and reestablished by the subsequent addition of Ca*+ in excess EGTA. The immediate response of phosphodiesterase activity to the addition of Ca*+ or to the removal of Ca*+ by chelation provides strong evidence that this form of phosphodiesterase activity can be regulated by physiological Ca*+ flux. Hodgkin and Keynes (21) reported that the mobility and the diffusion constant of ‘%a introduced into squid giant axons by microinjection was consistent with the hypothesis that most intracellular Ca*+ is bound rather than free. Cytoplasmic concentrations of free Ca2+ in unstimulated axons were estimated to be considerably less than 10e5 M. Several groups (21,22) have reported that depolarization of the neurolemma in response to electrical stimulation and neurohumors result in a prompt increase in flux (20-fold) of Ca2+, with a resultant increase of free intracellular Ca’+. The concomitant increase of CAMP in response to neurohumoral stimulation is well documented (23-25). Kakiuchi et al. (5) have speculated that under these conditions the increased concentration of Ca*+ could stimulate the activity of Ca*+ plus Mg*+dependent phosphodiesterase, reducing intracellular CAMP to basal (unstimulated) levels. The investigations detailed in this report support these speculations. The Ca*+-dependent forms of cyclic nucleotide phosphodiesterase from bovine heart (8,9) and rat cerebral cortex (6) have been reported in which the effects of the
OF PHOSPHODIESTERASE
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activating factors have varied as a function of substrate concentrations. In these reports CAMP concentrations in the millimolar range were employed. Comparable concentrations were, therefore, utilized in the present investigations to establish that the homogeneous Ca*+-binding phosphoprotein from brain increases the activity of the Ca2+-dependent phosphodiesterase. Experiments are currently in progress to define the kinetics of the interaction of the homogeneous calcium-binding phosphoprotein with the Ca *+-dependent phosphodiesterase. The results of the present report strongly suggest that the sensitivity of the phosphodiesterase to Ca*+ is conferred by a Ca*+binding phosphoprotein (CDR). In unstimulated cells, CDR and CDR-deficient enzyme may not be associated at the concentrations of intracellular Ca2+. Upon stimulation, increased intracellular Ca*+ concentrations result in Ca2+ sequestration by CDR. This binding effects a change in the tertiary structure of the phosphoprotein, permitting interaction of the CDR with CDR-dependent enzyme. This interaction results in increased phosphodiesterase activity. The sensitivity of this interaction to Ca*+ may depend upon the molar ratio of CDR to CDR-dependent phosphodiesterase. A high molar ratio of CDR to phosphodiesterase would lead to an apparent increase in Ca*+ sensitivity of the phosphodiesterase activity, since only a small percentage of the CDR would be required t,o bind Ca 2+ in order to activate the phosphodiesterase maximally. Support which can be cited for this proposal is: (1) the observation of a marked transition in the far uv circular dichroic spectrum of the calcium-binding phosphoprotein (CDR) after removal of bound Ca2+ wit,h EGTA, reflecting a decrease in a-helical content with concomitant increase in random coil content (D. J. Wolff, unpublished work); (2) the ease with which the CDR is separated from the phosphodiesterase by ECTEOLA-cellulose chromatography; (3) the discrepancy between the affinity of CDR for Ca*+ and the Ca*+ concentration required for half-maximal phosphodiesterase
358
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activity; and (4) the presence in fresh brain extracts of an apparent excess of CDR. ACKNOWLEDGMENTS The authors express their gratitude for the excellent technical assistance provided throughout these investigations by Mrs. Maria Topal and Miss Sandra Wien. REFERENCES 1. BROOKER, G., THOMAS, L. J., AND APPLEMAN, M. M. (1968) Biochemistry 7, 4177-4181. 2. THOMPSON, W. J., AND APPLEMAN, M. M. (1971) Biochemistry 10, 311-316. 3. MOON, E., AND CHRISTIANSEN,R. 0. (1971) Science 173, 540-542. BiO4. UZUNOV, P., AND WEISS, B. (1972) BiOChim. phys. Acta 284, 220-226. 5. KAKIUCHI, S., YAMAZAKI, R., AND TESHIMA, Y. (1972) in Advances in Cyclic Nucleotide Research (Greengard, P., Robison, G. A., and Paoletti, P., eds.), Vol. 1, pp. 455-477, Raven Press, New York. 6. KAKIUCHI, S., YAMAZAKI, R., TESHIMA, Y., AND UENISHI, K. (1973) Proc. Nut. Acad. Sci. USA 70, 3526-3530. 7. CHEUNG, W. Y. (1971) J. Biol. Chem. 246, 2859-2869. 8. TEO, T. S., WANG, T. H., AND WANG, J. H. (1973) J. Biol. Chem. 248, 588-595. 9. TEO, T. S., AND WANG, J. H. (1973) J. Biol. Chem. 248, 5950-5955.
10. WOLFF, D. J., AND SIEGEL, F. (1972) J. Biol. Chem. 247, 4180-4185. 11. BROOKS, J. C., AND SIEGEL, F. L. (1973) J. Biol. Chem. 248, 4189-4193. 12. BROOKS, J. C., AND SIEGEL, F. L. (1973) Biochem. Biophys. Res. Commun. 55, 710-716. 13. WOLFF, D. J., HUEBNER, J. A., AND SIEGEL, F. L. (1972) J. Neurochem. 19, 2855-2862. 14. MOORE, B. W. (1965) Biochem. Biophys. Res. Commun. 19, 739-744. 15. GREASER, M. L., AND GERGELY, J. (1971) J. Biol. Chem. 246, 4226-4233. 16. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 17. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. UYEMUR~ K., VINCENDON, G., GOMBOS, G., AND MANDEL, P. (1971) J. Neurochem. l&429-438. 19. CICERO, T. J., COWAN, W. M., MOORE, B. W., AND SUNTZEFF, V. (1970) Brain Res. 18, 25-34. 20. CALISSANO, P., MOORE, B. W., AND FRIESEN, A. (1969) Biochemistry 8, 4318-4326. 21. HODGKIN, A. L., AND KEYNES, R. D. (1957) J. Physiol. 176, 261-283. 22. RAMSEY, R. L., AND MCILWAIN, H. (1970) J. Neurothem. 17, 781-787. 23. KAKIUCHI, S., RALL, T. H., AND MCILWAIN, H. (1969) J. Neurochem. 16, 485-491. 24. SHIMIZU, H., CREVELING, C. R., ANDDALY, J. (1970) Proc. Nat. Acad. Sci. USA 65, 1033-1040. 25. SHIMIZU, H., CREVELING, C. R., AND DALY, J. W. (1970) Mol. Pharmacol. 6, 184-188.
OF BIOCHEMISTRY
AND
Calcium-Binding
BIOPHYSICS
163, 349-358
(1974)
Phosphoprotein
as a Calcium-Dependent
from Pig Brain:
Regulator
Identification
of Brain Cyclic Nucleotide
Phosphodiesterase’ DONALD Department
of Pharmacology,
J. WOLFF
AND
CHARLES
0. BROSTROM
Rutgers Medical School, College of Medicine Piscataway, New Jersey 08854 Received January
and Dentistry
of New Jersey,
15, 1974
A homogeneous, acidic, Ca2+-binding phosphoprotein from pig brain has been identified as the mediator of a Cal+-dependent activation of a partially purified pig brain cyclic nucleotide phosphodiesterase. Crude extracts prepared from porcine brain have been resolved into two fractions by column chromatography with ECTEOLA-cellulose. One fraction possesses phosphodiesterase activity which is stimulated from 4. to 12-fold, depending upon the preparation and the assay conditions, by nanogram quantities of a purified Ca2+-binding phosphoprotein from pig brain at 5 x 10e5 M Ca*+. The stimulation is eliminated by ethylene glycol-bis( @-aminoethyl ether)-N, N’-tetraacetate. The second fraction contains an endogenous, Caz+-dependent activator of the phosphodiesterase activity. Further purification of the crude activator by hydroyxlapatite chromatography, Sephadex G-75 gel filtration, and acrylamide gel electrophoresis has established that the activator and the purified Ca 2+-binding phosphoprotein are identical with respect to chromatographic behavior, molecular weight, and electrophoretic migration. Both proteins bound Cal+ and were stable to boiling for 5 min. At saturating concentrations of the Ca2+ -dependent regulator, half-maximal stimulation of the phosphodiesterase was observed at a Ca2+ concentration of 4 x 10m6 M. Purified preparations of the acidic Ca*+-binding phosphoprotein from bovine adrenal medulla and testis were also active in stimulating the brain phosphodiesterase. Preparations of other known Ca*+-binding proteins, such as rabbit muscle troponin and pig brain S-100 protein, were nonstimulatory.
Cyclic nucleotide phosphodiesterase is a potential focal point through which CAMP levels may be modulated. Two forms of phosphodiesterase activity have been distinguished in brain on the basis of affinity for CAMP, one having a low Michaelis constant (10-6-10-5 M) and the other a higher value (lo-’ M) (1). The separation of multiple phosphodiesterases of varying molecular weight, Michaelis constants, and substrate specificities from brain extracts has been accomplished in several
laboratories (2-4). The relationships of these forms to each other, if any, are unclear. Kakiuchi et al. (5, 6) have reported that two-thirds of the soluble phosphodiesterase from rat brain is Ca2+ dependent. A heat-stable, nondialyzable factor from brain, similar to that reported by Cheung (7), increased the maximal velocity for CAMP hydrolysis (measured at l-2 mM CAMP) approximately 7-fold. A similar cyclic nucleotide phosphodiesterase has been reported from bovine heart by Teo et al. (8, 9) which is activated by a heat-staprotein purified to ap’ This work was supported by grants from the U. S. ble, Ca’+-binding Public Health Service: Rutgers Medical School Gen- parent homogeneity. Wolff and Siegel (10) have described the eral Research Support Grant No. RR 5576, and NS 10975. purification to homogeneity and the physi.^ 34Y Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
350
WOLFF
AND BROSTROM
cal characterization of a Ca2+-binding phosphoprotein from pig brain. The phosphoprotein is highly acidic, exhibits a molecular weight of 11,500, and binds 1 mole of Ca2+ per mole of protein with an affinity of lo5 M-‘. A comparable Ca2+-binding protein has been purified from adrenal medulla (11) and testis (12). The specificity of the divalent metal cation binding site has been characterized by Wolff et al. (13). Subsequent investigations of the Ca2+binding phosphoprotein have been directed toward elucidating its function. The present report establishes that this protein mediates the Ca2+-dependent regulation of a brain cyclic nucleotide phosphodiesterase. Additional information is presented with respect to the specificity of the activation, the concentration of Ca2+ required, and the reversibility of the activation. MATERIALS
AND
METHODS
Materials. Calcium-45 (‘YZa) (14.9 Ci/g) and [SH]adenosine 3’, 5’cyclic phosphate ( [SH]cAMP) (22.1 Ci/mmole) were purchased from New England Nuclear Corp. Chelex-100, ECTEOLA-cellulose, hydroxylapatite (Bio-Gel HTP), and AG 1X8 resin (206400 mesh) were purchased from Bio-Rad. Calf intestinal mucosa alkaline phosphatase (350 units/mg protein) was purchased from Sigma Chemical Company. Spectrograde MgSO, ( <1 ppm CaZ+) was obtained from Jarrell Ash. Homogeneous Cal+-binding phosphoprotein from pig brain was prepared by the procedure of Wolff and Siegel (10). S-100 protein was prepared from porcine brain by a modification of the procedure of Moore (14). Rabbit skeletal muscle troponin was prepared by the procedure of Greaser and Gergely (15). Adrenal medullary and testicular Caz+-binding phosphoprotein were the kind gifts of Drs. Frank Siegel and Jack C. Brooks of the Joseph P. Kennedy, Jr., Laboratories, University of Wisconsin Center for Health Sciences. Assay of cyclic nucleotide phosphodiesterase. Phosphodiesterase measurements were performed by a modification of the procedure described by Brooker et al. (1). Standard assays were conducted at 30” C for 15 min. The reaction was initiated by the addition phosphodiesterase acof enzyme. Ca *+-independent tivity was assayed, unless otherwise indicated, in a total volume of 150 ~1 containing CAMP, 1 mM (1 Ci/mole); MgCl,, 5 mM; imidazole buffer, 20 mM, pH 7.5; and EGTA,2 0.05 msr. Cal+-dependent activity * Abbreviations: EGTA, ethylene glycol-bis(& aminoethyl ether)-N,N’-tetraacetic acid; CDR, calcium-dependent regulator.
was determined as the increment of phosphodiesterase activity produced by the addition of CaCl, (0.1 mM). Incubations were terminated by boiling and the 5’-AMP converted to adenosine by the addition of excess alkaline phosphatase (2 units for 15 min at 30” C). The samples were boiled, a slurry (1 ml) of AG 1X8 resin (1: 2 v/v in H,O) added to sequester unconverted CAMP, and the samples were centrifuged for 1OOOgmin. Aliquots (0.5 ml) of the supernatant liquid were removed for scintillation counting. The resultant values (cpm) were corrected for controls incubated without enzyme, and, when appropriate, expressed in terms of moles CAMP converted to Y-AMP. Boiled phosphodiesterase controls were identical to controls without enzyme. ECTEOLA-cellulose chromatography. ECTEOLA-cellulose (100 g) was suspended in 2 liters of 1 N NaOH and allowed to stand for 1 hr at room temperature. The resin was washed three times with 2 liters of double-distilled water and the fines decanted after each washing. The resin was then washed with 20 mM phosphate, pH 7.4, containing 20 mM NaCl and 2mMMg *+ , to a constant pH 7.4, and packed in a glass column (2.5 x 60 cm) under gravity. The column was equilibrated with this buffer and the protein samples applied and eluted as described in the legend of Fig. 1. Acrylamide gel electrophoresis. Analytical disc gel electrophoresis was conducted in Tris-glycine, pH 8.3, as described by Davis (16). Gels (6.0 x 0.6 cm) consisted of a &cm 15% acrylamide small-pore gel and a l-cm stacking gel. Protein samples were diluted with electrophoresis buffer containing 20% (w/v) sucrose and layered on the gels with a Hamilton syringe. Electrophoresis was conducted for 2i/4 hr at room temperature at 1.5 mA per gel employing bromophenol blue as a tracking dye. Gels were stained for 2 hr with a 1% solution of amido Schwartz in 7% acetic acid and destained in a Hoeffer gel-destaining apparatus against 7% acetic acid. Miscellaneous. Protein was determined by the method of Lowry (17). Nucleotide concentrations were standardized by extinction. Calcium concentrations were standardized by atomic absorption spectrophotometry.
RESULTS Resolution of phosphodiesterase.
the
Ca2+-dependent
Preliminary measurements of cyclic nucleotide phosphodiesterase activity in crude extracts of brain (prepared as described in the legend of Fig. 1) indicated that 40% of the enzyme was dependent on Ca2+. A sample of crude brain extract was applied to a column of ECTEOLA-cellulose, and eluted with a linear gradient of NaCl (Fig. 1). Cyclic
CALCIUM-DEPENDENT
"0
20
30
40
20
30
40
50
REGULATOR
so
90
50 60 70 80 FRACTION NUMBER
90
60
70
2 $8
0 x
t7 ?6 g5 "4 23 :.2 $1 8
FIG. 1. ECTEOLA-cellulose chromatography of a crude extract of porcine brain. Adult porcine brains, obtained on ice from a local supplier, were homogenized in 2 vol of 50 mM TrisHCl, pH 7.4, in a Waring Blendor for 2 min at medium speed at 4’ C. The homogenate was clarified by centrifugation at 18,OOOg for 35 min and a portion (138 ml) applied to a (2.5 x 60.cm) column of ECTEOLA-cellulose previously equilibrated with 20 mM sodium phosphate, pH 7.4, containing 20 mM NaCl and 2 mM MgCl,. After the collection of 32 fractions of 9.2 ml, a 600.ml linear gradient to 0.4 M NaCl was applied. Plot A: Protein (04) was determined on the fractions by measuring light absorbance at 280 nm. Measurements of calcium-dependent cyclic nucleotide phosphodiesterase activity were conducted on 25.~1 aliquots of the fractions in the presence (04) and absence (W---m) of 1 pg of homogeneous Ca2+-binding phosphoprotein (see Materials and Methods). Values are expressed as moles CAMP hydrolyzed/ml of enzyme/min. Plot B: Aliquots of the ECTEOLA-cellulose fractions were boiled for 5 min and samples (1 ~1) tested for stimulation of the calcium-dependent cyclic nucleotide phosphodiesterase activity of fraction 54 (04). Values are expressed as the potential of 1 ml of test sample to stimulate CAMP hydrolysis by fraction 54. Aliquots of ECTEOLA-cellulose fractions were dialyzed against 2 liters of 50 mM Tris-HCl, pH 7.4, containing 25 PCi of %a. The system was allowed to come to equilibrium (24 hr), samples (0.2 ml) were withdrawn, and protein-bound %a determined as the difference in cpm inside and outside the dialysis bag as measured by liquid scintillation counting. Proteins were determined by the Lowry procedure (17). The specific activity, expressed as cpm of bound ‘%a per mg protein, is plotted in the form of a histogram
351
OF PHOSPHODIESTERASE
nucleotide phosphodiesterase activity was assayed in the presence of Ca2+ with and without the addition of the homogeneous Ca2+-binding phosphoprotein. A peak of cyclic nucleotide phosphodiesterase activity which was dependent on the presence of both Ca2+ and the Ca2+-binding phosphoprotein was observed (fraction 54). When the cyclic nucleotide phosphodiesterase activity was assayed in the presence of excess EGTA with and without the addition of the calcium-binding phosphoprotein, no such stimulation of activity was observed (data not shown). The cyclic nucleotide phosphodiesterase activity of fraction 54 was stimulated 6-fold by the concurrent addition of Ca2+ and homogeneous calcium-binding phosphoprotein. Samples of the Ca*+-binding phosphoprotein were boiled for 5 min under a variety of conditions as described in the legend of Table I. Boiling of the phosphoTABLE STABILIZATION OF Ca’+-BINDING AGAINST DENATURATION
Protective
agent
Control (unboiled) CDR Ca’+ EGTA Bovine serum albumin Human hemoglobin Rabbit serum proteins ECTEOLA fraction 27
I PHOSPHOPROTEIN BY BOILING’
% Control (unboiled) activity 100 63 38 100 109 94 99
“A lyophilized sample of calcium-binding phosphoprotein was diluted to 3.2 pg/ml in 10 mM imidazole buffer, pH 7.5. Aliquots (0.6 ml) were adjusted to contain (where indicated) the following additives: CaCl,, 0.2 mM; EGTA, 0.1 mM; bovine serum albumin, human hemoglobin, rabbit serum, or protein derived from ECTEOLA-cellulose chromatography of crude porcine brain extracts (fraction 27, Fig. lA), 400 fig/ml. The aliquots were boiled for 5 min and 50-bl portions of each (containing 160 ng of CDR) were assayed for activation of CDR-dependent phosphodiesterase activity as described under Materials and Methods. Phosphodiesterase controls incubated without added CDR hydrolyzed 1.1 x lo- I0 moles CAMP/ min/incubation and, when maximally stimulated with CDR, 3.8 x lo-‘” moles cAMP/min/incubation. One hundred sixty nanograms of CDR in this experiment activated the phosphodiesterase to 70% of the activity observed with the addition of 1 pg CDR.
352
WOLFF
AND BROSTROM
protein with EGTA for 5 min destroyed more than half of the phosphodiesterase stimulatory activity; the addition of Ca2+ partially stabilized the protein to boiling. Crude protein mixtures, such as rabbit serum and porcine brain proteins which failed to bind to columns of ECTEOLA-cellulose, provided marked stabilization of the phosphodiesterase stimulatory activity of the phosphoprotein. Purifed proteins, in the form of human hemoglobin and bovine serum albumin, similarly protected the phosphodiesterase stimulatory activity from inactivation by boiling. Complete recovery of the stimulatory activity was observed in the presence of protein, indicating that the calcium-binding phosphoprotein did not adsorb strongly to the protein precipitates produced by boiling. Aliquots of the ECTEOLA-cellulose column fractions were boiled for 5 min to denature endogenous phosphodiesterase activity and l-cl1 samples were then assayed for their ability to stimulate the phosphodiesterase activity of fraction 54 in the presence of Ca2+. Such stimulation was noted for boiled fractions 75-84, with fraction 78 being the most effective (Fig. 1B). Additional aliquots were tested for their Ca2+-binding capacity by equilibrium dialysis. A strong correlation was observed between ‘%a binding and the fractions stimulating the phosphodiesterase. ECTEOLA-cellulose column chromatography thus resolved the Ca2+-dependent phosphodiesterase activity from crude extracts of brain into an endogenous Ca2+-dependent regulator (CDR) fraction and a fraction containing deactivated (CDR-dependent) phosphodiesterase. Occasional preparations of CDR-dependent phosphodiesterase from ECTEOLA-cellulose chromatography contained substantial amounts of CDR-independent phosphodiesterase activity. The independent activity could be selectively denatured by heating the preparation at 55” C for 5 min. Under these conditions, CDR-dependent activity was completely stable and a B-fold Ca*+dependent activity resulted. The ECTEOLA-cellulose fractions
which possessed the CDR activity were pooled and used to conduct further comparisons of the crude CDR with the homogeneous Ca2+-binding phosphoprotein. Hydroxylapatite chromatography of the crude CDR. The Ca2+-binding phosphoprotein had been noted in previous work to adsorb only weakly to columns of hydroxylapatite which had been equilibrated with 20 mM phosphate, pH 6.8, whereas almost all other proteins present in crude brain extracts adsorbed strongly under this condition (10). An aliquot of the pooled crude CDR (ECTEOLA-cellulose fractions 75-84) was chromatographed on a hydroxylapatite column (Fig. 2). The column fractions were assayed for protein and CDR activity. Only a very small amount of the 28. b
ZL24E k2 N 20. z
I;
OS-
a
04 -
IO
20
30
40
FRACTION NUMBER
FIG. 2. Hydroxylapatite chromatography of the crude calcium-dependent regulator. The fractions (75-84) containing the calcium-dependent phosphodiesterase regulator from ECTEOLA-cellulose column chromatography (Fig. 1) were dialyzed against 20 mM potassium phosphate, pH 6.8, and concentrated to 4 ml with a Diaflo apparatus fitted with a UM-10 membrane. The concentrate was applied to a column (1 x 60 cm) of hydroxylapatite and eluted stepwise with buffers of the following composition: 20 mM potassium phosphate, pH 6.8, 60 ml; and 500 mM potassium phosphate, pH 6.8, 60 ml. Fractions (3 ml) were collected and assayed for protein by measuring light absorbance at 230 nm (0-O). Aliquots (1 ~1) were assayed for calcium-dependent stimulation of the sensitive cyclic nucleotide phosphodiesterase of ECTELOA-cellulose column fraction 54 (O--O) as described in Materials and Methods. Values are expressed as the potential of 1 ml of activator to stimulate CAMP hydrolysis by the activator-deficient enzyme.
CALCIUM-DEPENDENT
REGULATOR
protein failed to adsorb to the column in 20 mM phosphate, pH 6.8, and this protein possessed very high CDR activity. Essentially, all of the protein applied to the column eluted when the column eluent was changed to 500 mM phosphate, pH 6.8; a small portion of CDR activity eluted at the leading edge of this fraction. The small amount of CDR activity at the leading edge of the 500 mM phosphate fraction represents trace amounts of CDR which adsorbed at 20 mM phosphate. Such behavior is customarily observed during purification of Ca2+-binding phosphoprotein on hydroxylapatite (10). The fractions comprising each of the protein peaks were pooled, concentrated, and subjected to electrophoresis on 15% acrylamide gels (Fig. 3). The electrophoretic patterns indicated that fractions 11-12, which contained most of the CDR activity, were apparently composed of two proteins, one
OF PHOSPHODIESTERASE
353
of which was present only in trace amounts. The predominant protein was observed to co-migrate with a sample of the purified Ca2+-binding phosphoprotein. In addition, the trace impurities were apparently present in much higher concentration in hydroxylapatite column fractions 32-40 which possessed little CDR activity. Previous experience indicates that this trace contaminant is an S-100 protein fraction (18). Purified S-100 protein has been tested for CDR activity (Table II) and does not activate the phosphodiesterase. Sephadex G-75 gel filtration of the CDR. A second aliquot of the crude CDR activity (ECTEOLA-cellulose fractions 75-84) was dialyzed for 18 hr against two changes (4 liters total volume) of 50 mM Tris-HCl, pH 7.4, to remove NaCl, concentrated to 5 ml with a Diaflo apparatus (UM-10 membrane), incubated with 10 PCi of ‘%a and subjected to gel filtration on Sephadex
FIG. 3. Electrophoretic comparison of the crude Ca2+ -dependent regulator from hydroxylapatite chromatography and the homogeneous Ca*+-b’ m d’mg phosphoprotein. Electrophoresis was conducted on 15% acrylamide gels, pH 8.3, by the procedure of Davis (16). The samples were of the following compositions: Gel l-purified pig brain Caz+ -binding phosphoprotein; Gel 2-hydroxylapatite column fractions 11-12; Gel 3-samples 1 + sample 2; Gel 4-hydroxylapatite column fractions 32-40; Gel 5-sample 4 + sample 1; Gel 6-hydroxylapatite column fractions 32-40; Gel ‘i-sample 6 + sample 2.
354
WOLFF TABLE
AND BROSTROM
II
unresolvable. To further establish that the Ca’+-dependent activation of phosphodiesterase was specific to the Ca2+-binding phosphoprotein, other purified proteins Additives Specific activity possessing Ca 2+-binding properties were (% of control) examined for their ability to activate the CDR-dependent phosphodiesterase (Table Ca’+-binding phosphoprotein II). In addition, samples of the homogenePorcine brain 100 ous Ca2+-binding phosphoprotein from Bovine adrenal medulla 103 Bovine testis 133 species and tissue sources other than porS-100 protein 0.6 cine brain were tested. Rabbit muscle tropTroponin
OF CDR-DEPENDENT PHOSPHODIESTERASE BY Ca’+-BINDING PROTEINS~
CALCIUM-DEPENDENT
REGULATOR
OF PHOSPHODIESTERASE
355
trations from 1 x 10m6M to 1 x lo-’ M. The concentration of Ca*+ providing half-maximal activation of CDR-dependent phosphodiesterase in the presence of saturating CDR was 4 x 10e6 M. The Ca2+ requirement of the phosphodiesterase was explored further to determine whether the enzyme was activated reversibly or irreversibly. A sample of phosphodiesterase was incubated with CAMP, Mg*+, 20 30 IO and saturating CDR for 45 min, with aliMINUTES quots being withdrawn each min for deterFIG. 4. Time dependence of the cyclic nucleotide mination of CAMP hydrolysis (Fig. 7). The phosphodiesterase activity. Brain phosphodiesterase initial incubation was 0.1 mM in EGTA. At purified through the ECTEOLA-cellulose chromatog6 min of incubation the mixture was diraphy stage was assayed as described under Materials and Methods. EGTA (0.05 mM) was present in all vided into two portions, one of which was a incubation tubes. The assays were initiated by the control to which no further additions were addition of CAMP. Values are expressed as moles of made and the other of which was adjusted CAMP converted/ml of phosphodiesterase (2.1 mg to 0.2 mM Ca*- with 20 mM CaCl,. An protein) in the presence of homogeneous CDR (1 pg) immediate 11-fold stimulation of phosphowith (04) and without (04) the addition of diesterase activity occurred. At 22 min the 0.1 mM CaCl,, and in the absence of CDR with addition of 1.3 mM EGTA resulted in an (W-H) and without (04) the addition of immediate cessation of CAMP conversion CaCI,. in the incubation. After 32 min of incubaHCI. Maximal CDR-dependent phospho- tion the Ca*’ concentration of the incubadiesterase activity was observed at pH T.5 tion was adjusted to 3 mM Ca2+; a marked in imidazole buffer (data not shown) and and immediate stimulation of the phosCDR dependence of the enzyme was main- phodiesterase activity was again observed. tained from pH 6-9. These data, combined with the observation Variation of phosphodiesterase activity of the linear response of the CDR-dependas a function of the CDR concentration was examined. Incubations of a fixed amount of CDR-dependent phosphodiesterase were conducted at rate-limiting concentrations of the CDR (standardized by extinction). The activity of the CDR-dependent phosphodiesterase increased linearly over the range from O-50 ng of homogeneous CDR (Fig. 5). In the presence of amounts of CDR greater t,han 50 ng the activity of the CDR-dependent phosphodiesterase asymptotically approached a maximum. FIG. 5. Variation of cyclic nucleotide phosphodiesThe quantitative sensitivity to Ca2+ of terase activity with concentration of homogeneous CDR. Phosphodiesterase was assayed as described in the CDR activation of the phosphodiesterMaterials and Methods. Phosphodiesterase controls ase was investigated with reagents purified incubated in the absence of CDR hydrolyzed 1.9 x of contaminating Ca2+ (Fig. 6). Controls and, when maxicontaining 0.05 mM EGTA exhibited phos- lo-” moles cAMP/min/incubation mally stimulated with CDR, 6.3 x lo- I” moles phodiesterase activity equivalent to that cAMP/min/incubation. The CDR was predialyzed observed for Ca2+-free reagents in the ab- against H,O and diluted for assay with H,O. Values sence of added EGTA. Increasing degrees are expressed in terms of the stimulation of phosphoof stimulation of the phosphodiesterase diesterase activity and are corrected for basal levels of were observed over a range of Ca*+ concen- CDR-independent activity.
356
WOLFF
AND BROSTROM
I 7
6
5
4
3
p CO2’
FIG. 6. Calcium dependence of the brain cyclic nucleotide phosphodiesterase. Phosphodiesterase assays were conducted as described in Materials and Methods with the exceptions that all components were first freed of trace contaminations of CaZ+ by passage through a column (1 x 30 cm) of Chelex-100 resin. Mg2+ was added as spectrograde MgSO, which contained < 1 ppm contaminating Ca*+. Homogeneous CDR (1 pg) and phosphodiesterase were stored over Chelex-100 resin at 2” C for 1 hr before use. Assays were conducted in new polypropylene tubes washed five times with double-distilled H,O. Ca*+ was added at the indicated concentrations as a solution of CaCl,. Values for the ordinate are expressed as percentages of the phosphodiesterase activity observed with the addition of 1 mM CaCl, and are corrected for CDR-independent activity. Controls incubated in the absence of added Ca2+ hydrolyzed 2.2 x 10-l” moles cAMP/min/incubation contents. Samples maximally stimulated with Ca2+ hydrolyzed 1.3 x 10mg moles cAMP/min/incubation contents. Controls to which 0.05 mM EGTA was added exhibited no further decreases in activity beyond that observed for Ca2+-free agents.
ent phosphodiesterase to CDR in the presence of Ca’+, provide compelling evidence that the CDR-dependent phosphodiesterase stimulation responds directly to changes in free Ca*+ in an immediate and reversible fashion. The Ca2+ sensitivity (K, = 4 x 10e6 M) of the effect is compatible with the notion that a substantial portion of the phosphodiesterase activity of brain may be regulated by intracellular Ca2+ fluxes. DISCUSSION
The results presented in this paper demonstrate that Ca2+-dependent activation is
conferred on a brain cyclic nucleotide phosphodiesterase by a previously purified and physically characterized Ca’+-binding phosphoprotein from brain. A Ca2+dependent activating factor endogenous to brain extracts was found to be identical to the characterized phosphoprotein. CDR-dependent phosphodiesterase exhibited a linear increase in activity in response to O-50 ng of the homogeneous phosphoprotein when assayed in the presence of excess Ca2+. Boiling dilute solutions of the phosphoprotein resulted in a loss of CDR activity; this loss of activity was decreased by the presence of Ca’+, and eliminated by the presence of nonspecific protein. Boiled tissue extracts would, therefore, appear suitable for surveying the phosphoprotein in tissue, cellular, and sub-
5
IO I5 20 25 30 35 40 45 MINUTES
FIG. ‘i. Regulation of brain cyclic nucleotide phosphodiesterase by Ca*+. The incubation mixture was constructed containing (in 5 ml) CAMP, 3 mM; MgCl,, 10 mM; imidazole, 50 mM, pH 7.5; EGTA, 0.1 mM; and homogeneous CDR, 10 fig/ml. After a 5-min preincubation of the mixture at 30” C, 200 ~1 of partially purified phosphodiesterase (0.3 mg protein from ECTEOLA-cellulose chromatography) was added to initiate the reaction. Aliquots of 100 11 were removed at specified time intervals and denatured immediately by boiling. At 6 min of incubation the reaction mixture was divided into two portions, one of which was adjusted to 0.2 mM Ca2+ with 20 mM CaCl,. Further additives to the Ca’+-treated tubes were 1.3 mM EGTA (final) at 20 min and 3 mM Ca2+ (final) at 32 min. Values are expressed as moles CAMP hydrolyzed/0.1 ml of enzyme. Open circles (O-O) represent aliquots from the sample containing the additives; closed circles (O--O) represent aliquots from the untreated samples.
CALCIUM-DEPENDENT
REGULATOR
cellular localization studies. Ca’+-binding phosphoproteins isolated from bovine adrenal medulla and testis possess comparable potency in conferring Ca*+-dependent activation on the porcine brain enzyme. CDR activity from different tissues and species can therefore be assayed with the CDRdependent enzyme from porcine brain. Calcium-dependent regulator-dependent phosphodiesterase activity was stimulated half-maximally at 4 x lOme M Ca*+ indicating that the affinity of the phosphoprotein for Ca *+ is increased 2- to S-fold when associated with the phosphodiesterase. Stimulation of phos.phodiesterase by Ca*+ was readily reversed by EGTA and reestablished by the subsequent addition of Ca*+ in excess EGTA. The immediate response of phosphodiesterase activity to the addition of Ca*+ or to the removal of Ca*+ by chelation provides strong evidence that this form of phosphodiesterase activity can be regulated by physiological Ca*+ flux. Hodgkin and Keynes (21) reported that the mobility and the diffusion constant of ‘%a introduced into squid giant axons by microinjection was consistent with the hypothesis that most intracellular Ca*+ is bound rather than free. Cytoplasmic concentrations of free Ca2+ in unstimulated axons were estimated to be considerably less than 10e5 M. Several groups (21,22) have reported that depolarization of the neurolemma in response to electrical stimulation and neurohumors result in a prompt increase in flux (20-fold) of Ca2+, with a resultant increase of free intracellular Ca’+. The concomitant increase of CAMP in response to neurohumoral stimulation is well documented (23-25). Kakiuchi et al. (5) have speculated that under these conditions the increased concentration of Ca*+ could stimulate the activity of Ca*+ plus Mg*+dependent phosphodiesterase, reducing intracellular CAMP to basal (unstimulated) levels. The investigations detailed in this report support these speculations. The Ca*+-dependent forms of cyclic nucleotide phosphodiesterase from bovine heart (8,9) and rat cerebral cortex (6) have been reported in which the effects of the
OF PHOSPHODIESTERASE
357
activating factors have varied as a function of substrate concentrations. In these reports CAMP concentrations in the millimolar range were employed. Comparable concentrations were, therefore, utilized in the present investigations to establish that the homogeneous Ca*+-binding phosphoprotein from brain increases the activity of the Ca2+-dependent phosphodiesterase. Experiments are currently in progress to define the kinetics of the interaction of the homogeneous calcium-binding phosphoprotein with the Ca *+-dependent phosphodiesterase. The results of the present report strongly suggest that the sensitivity of the phosphodiesterase to Ca*+ is conferred by a Ca*+binding phosphoprotein (CDR). In unstimulated cells, CDR and CDR-deficient enzyme may not be associated at the concentrations of intracellular Ca2+. Upon stimulation, increased intracellular Ca*+ concentrations result in Ca2+ sequestration by CDR. This binding effects a change in the tertiary structure of the phosphoprotein, permitting interaction of the CDR with CDR-dependent enzyme. This interaction results in increased phosphodiesterase activity. The sensitivity of this interaction to Ca*+ may depend upon the molar ratio of CDR to CDR-dependent phosphodiesterase. A high molar ratio of CDR to phosphodiesterase would lead to an apparent increase in Ca*+ sensitivity of the phosphodiesterase activity, since only a small percentage of the CDR would be required t,o bind Ca 2+ in order to activate the phosphodiesterase maximally. Support which can be cited for this proposal is: (1) the observation of a marked transition in the far uv circular dichroic spectrum of the calcium-binding phosphoprotein (CDR) after removal of bound Ca2+ wit,h EGTA, reflecting a decrease in a-helical content with concomitant increase in random coil content (D. J. Wolff, unpublished work); (2) the ease with which the CDR is separated from the phosphodiesterase by ECTEOLA-cellulose chromatography; (3) the discrepancy between the affinity of CDR for Ca*+ and the Ca*+ concentration required for half-maximal phosphodiesterase
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activity; and (4) the presence in fresh brain extracts of an apparent excess of CDR. ACKNOWLEDGMENTS The authors express their gratitude for the excellent technical assistance provided throughout these investigations by Mrs. Maria Topal and Miss Sandra Wien. REFERENCES 1. BROOKER, G., THOMAS, L. J., AND APPLEMAN, M. M. (1968) Biochemistry 7, 4177-4181. 2. THOMPSON, W. J., AND APPLEMAN, M. M. (1971) Biochemistry 10, 311-316. 3. MOON, E., AND CHRISTIANSEN,R. 0. (1971) Science 173, 540-542. BiO4. UZUNOV, P., AND WEISS, B. (1972) BiOChim. phys. Acta 284, 220-226. 5. KAKIUCHI, S., YAMAZAKI, R., AND TESHIMA, Y. (1972) in Advances in Cyclic Nucleotide Research (Greengard, P., Robison, G. A., and Paoletti, P., eds.), Vol. 1, pp. 455-477, Raven Press, New York. 6. KAKIUCHI, S., YAMAZAKI, R., TESHIMA, Y., AND UENISHI, K. (1973) Proc. Nut. Acad. Sci. USA 70, 3526-3530. 7. CHEUNG, W. Y. (1971) J. Biol. Chem. 246, 2859-2869. 8. TEO, T. S., WANG, T. H., AND WANG, J. H. (1973) J. Biol. Chem. 248, 588-595. 9. TEO, T. S., AND WANG, J. H. (1973) J. Biol. Chem. 248, 5950-5955.
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