ARCHIVES

OF BIOCHEYI8TRY

AND

BIOPHYSICS

172, 301-311 (1976)

Calcium-Dependent Cyclic Nucleotide Phosphodiesterase from Brain: Comparison of Adenosine 3’,5’-Monophosphate and Guanosine 3’,5’Monophosphate as Substrates1 CHARLES 0. BROSTROM AND DONALD J. WOLFF Department

of Pharmacology,

Rutgers

Medical School, College of Medicine Piscataway, New Jersey 08854

and Dentistry

of New Jersey,

Received June 30, 1975 A Ca2+-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca*+ and microgram quantities of a purified Ca2+-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3’,5’-monophosphate (cyclic AMP) and guanosine 3’,5’-monophosphate (cyclic GMP), with apparent K, values of 180 and 8 PM, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca*+. This activity exhibited apparent K, values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca’+-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior. Apparent K, values of the Ca2+-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K, for Mg2+ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of MgZ+. At low cyclic nucleotide substrate concentrations, i.e., 2.5 FM, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.

Transient fluctuation of free intracellular Caz+concentration has been implicated as a potential determinant of various enzymatic activities involved in the metabolism of adenosine 3’,5’-monophosphate (cyclic AMP) and guanosine 3’,5’-monophosphate (cyclic GMP) in neural tissue. Ca*+-dependent activation of brain adenylate cyclase (l-3) and cyclic nucleotide phos-

phodiesterase (4-7) activities have been reported from a number of laboratories. Recently, a Ca*+-binding protein purified from brain has been demonstrated by Brostrom et al. (8) to act in vitro as a Ca*+dependent regulator (CDRJ2 of both enzymes. Although the adenylate cyclase and the phosphodiesterase each consti-

2 Abbreviations used: CDR, Ca*+-dependent regu’ This work was supported by grants from the U. lator of cyclic nucleotide phosphodiesterase activiS. Public Health Service, No. NS 11340, NS 11252, ties; EGTA, ethylene glycol-bis@aminoethyl and NS 10975. etherW,N’-tetraacetate. 301 Copyright 0 1976by Academic Press, Inc. All rights of reproduction in any form reserved.

302

BROSTROM

AND

tutes a potential focal point for the regulation of intracellular cyclic nucleotide concentrations by Ca2+, the two enzymes may not necessarily represent a system of concerted control. Kakiuchi et al. (9) have proposed on the basis of kinetic evidence that the Ca2+dependent cyclic nucleotide phosphodiesterase in crude extracts prepared from rat brain hydrolyzes cyclic GMP in preference to cyclic AMP at substrate concentrations in the micromolar range. At higher substrate concentrations, however, cyclic AMP was hydrolyzed at more rapid rates than cyclic GMP. Since cyclic AMP tends to occur at greater intracellular concentrations than cyclic GMP (10, 111, it is difficult to ascertain whether the Ca’+-dependent phosphodiesterase functions predominantly in.uico to hydrolyze cyclic GMP or cyclic AMP. A previous report from our laboratory (12) established that Ca’+ and CDR form a complex that subsequently activates the phosphodiesterase of C-6 glioma cells. Variations of Ca2+concentration did not affect substrate specificity. In the present communication the effects of CDR, Mg2+, and cyclic nucleotide substrate concentrations on the relative rates of cyclic AMP and cyclic GMP hydrolysis have been examined utilizing a partially purified Ca2+-dependent phosphodiesterase preparation from porcine brain. MATERIALS

AND

METHODS

Materials. [G-3H]adenosine 3’:5’-cyclic phosphate (39 Ci/mmol) and [G-3Hlguanosine 3’:5’-cyclic phosphate (11 Ci/mmol) were purchased from New England Nuclear Corp., Boston, Mass. ECTEOLA-cellulose and AG 1-X8 resin (200-400 mesh, chloride form) were obtained from Bio-Rad Laboratories, Richmond, Calif. The AG 1-X8 resin was washed thoroughly with Hz0 and used in the chloride form. Sepharose 6 B and Sephadex G-200 were obtained from the Pharmacia Company, Piscataway, N.J. Molecular weight calibration sets for gel filtration chromatography were purchased from SchwarziMann, Orangeburg, N.Y. Salmon protamine (Grade I), human hemoglobin (2x crystallized), and calf intestinal mucosa alkaline phosphatase were obtained from the Sigma Chemical Co., St. Louis, MO. Cal+-dependent regulator. Homogeneous CDR was prepared from porcine brain by the procedure of Wolff and Siegel (131 and standardized by light ab-

WOLFF

sorbance at 280 nm. CDR in tissue extracts was assayed as described previously (12). Cyclic nucleotide phosphodiesterase assay. Cyclic nucleotide phosphodiesterase measurements were conducted at 37°C as described previously (12). The reaction was initiated by the addition of enzyme diluted for assay in 10 mM imidazole, pH 7.5, and 100 @g/ml of hemoglobin. Reaction mixtures were adjusted to contain 2 x lo” cpm of tritiated cyclic nucleotide and varying concentrations ofnonradioactive cyclic nucleotide in a total volume of 150 ~1. Ca2+-independent phosphodiesterase activity was assayed, unless otherwise specified, with MgCl,, 5 mM; imidazole buffer, 20 mM, pH 7.5; and EGTA, 0.1 mM. Ca2+-dependent activity was determined as the increment of phosphodiesterase activity produced by the addition of CaCl, (0.3 mM) in excess of the EGTA. Conversion of cyclic nucleotide to product was linear with time and was not allowed to exceed 20% over the incubation period. Values in terms of counts per minute were converted to picomoles per milligram of protein per minute and corrected for the absorption of nucleoside to the AG l-X8 resin. Adsorption of 13H]nucleoside, either adenosine or guanosine, in control experiments was constant at 40 _C 2% of the total nucleoside in the system at concentrations from lo-fold above to lo-fold below the concentration of product formed in the system. In later experiments. AG l-X8 resin suspended in 1:l isopropanol:HpO (v/v) was substituted for AG lX8 resin suspended in HzO. Under this condition the binding of nucleoside to the resin was almost completely eliminated. Miscellaneous procedures. Tritiated cyclic nucleotides were purified by thin-layer chromatography with the system of Huang and Kemp (14). Analytical acrylamide-gel electrophoresis was conducted in Tris/glycine, pH 8.3, by the procedure of Davis (151, as described previously (13). Protein was determined by the Method of Lowry et al. (161. ProtamineSepharose 6 B was prepared by reacting 1 g of protamine dissolved in 0.1 M NaHC03 with 100 ml of settled Sepharose 6 B which had been activated with cyanogen bromide, as described by Cuatrecasas (17). Isoelectric focusing was conducted with an LKB Model 1801 isoelectric focusing column employing LKB ampholytes, pH range 3-10 for 48 h at 10°C and 400-500 V. The column was operated in accordance with the manufacturer’s instructions with a 5-20% linear sucrose gradient as a stabilizer. Gel filtration chromatography was conducted on a 1.5 x loo-cm column of Sephadex G-200. The elution volumes of blue dextran, human y-globulin, bovine serum albumin, bovine chymotrypsinogen, and equine cytochrome c were determined on the column. The ratio of the elution volumes of the individual proteins to the exclusion volume of the gel bed (V,No) versus the respective logarithms of the molecular weights

ml) was dialyzed for 16 h against two 4liter changes of 2.5 mM dithiothreitol/lO mM Tris buffer, pH 7.5, divided into aliquots, frozen, and lyophilized. Losses of phosphodiesterase activity were not experienced during lyophilization. Following lyRESULTS ophilization the activity was stable for pePurification of the Cyclic Nucleotide Phos- riods up to 12 months when stored at phodiesterase -80°C under vacuum with CaSO, as a Porcine brains obtained on ice from a drying agent. The Ca2+-dependent phosphodiesterase local supplier were homogenized in two volumes of 50 mM Tris-HCl, pH 7.4, in a was purified eightfold from the starting Waring Blendor at high speed for 1 min at extract with an overall recovery of 30% as with either cyclic AMP or 4°C. All subsequent procedures were per- determined formed at 2-4°C. The homogenate was clarcyclic GMP as substrate (Table I). CDR ified by centrifugation at 18,OOOg for 35 was totally removed from the preparation, min. Protamine-Sepharose 6 B (1 liter of and the phosphodiesterase no longer remoist gel) was suspended in the resulting sponded with detectable activation to the extract (6 liters) and the mixture agitated addition of Ca2+. Since this phosphodiesterwith an overhead stirring device for 30 ase preparation was quite impure, no atmin. Under these conditions, approxitempt was made to establish the number mately 70% of the Ca2+-dependent phosphoor amounts of contaminating proteins diesterase in the extract adhered to the present. gel. The gel was subsequently separated Characterization of the Phosphodiesterase from the extract by filtration on a Buchner Preparation funnel, washed with 2 liters of 0.25 M NaCl Aliquots of the enzyme from ECTEOLAin 10 mM Tris buffer, pH 7.4, and the enzyme eluted with 1.5 liters of 1 M NaCl in cellulose chromatography was applied to a 10 mM Tris buffer. The gel was washed Sephadex G-200 column (1.5 x 100 cm) with 10 rnM Tris buffer, added to the ex- equilibrated with 0.5 M NaC1/2 mM dithiomM Tris buffer, pH 7.5. One tract, and the washing procedure re- threitol/lO major peak of phosphodiesterase activity, peated. Th.e pooled 1 M salt washes were adjusted to 30% saturation with solid which hydrolyzed both cyclic AMP and ammonium sulfate. After 30 min the solu- cyclic GMP, was eluted (not illustrated). tion was clarified by centrifugation at lO,- The enzyme was activated sixfold by the addition of Ca2+ and CDR. An estimated OOOgfor 30 min. The pellet was discarded and the supernatant fraction adjusted to molecular weight of 140,000 was deter50% saturation with additional ammo- mined for the phosphodiesterase when the nium sulfate. After 30 min the precipitate ratio of the elution volume of the enzyme was collected by centrifugation at 10,OOOg to the exclusion volume of the column W,/V,J was calculated (see Materials and for 30 min, dissolved in a minimal volume of 100 mM NaC1/25 mM Tris buffer, 2.5 mM Methods). Recoveries of 80-100% were obdithiothreitol, 3 mM MgC12, and 0.1 mM tained from this procedure, provided that folEGTA, pH 7.5, and dialyzed against two 4- analyses were conducted immediately liter changes of the same buffer during a lowing completion of chromatography. 16-h period. The dialysate was applied to One form of phosphodiesterase was oban ECTEOLA-cellulose column (2.5 x 60 served in isoelectric focusing experiments cm), equilibrated with the same buffer and (not illustrated). This form exhibited an eluted with more of the buffer at a flow isoelectric point at pH 5.0, was activated rate of 60 ml/h. Under these conditions the by CDR, and hydrolyzed both cyclic AMP 30% of the phosphodiesterase was collected in the pro- and cyclic GMP. Approximately tein fraction that did not adhere to the total activity applied to the isoelectric focolumn. The fraction (approximately 300 cusing device was recovered in the experiof the proteins were graphed in order to calibrate the column for estimation of the molecular weight of the phosphodiesterase. ECTEOLA-cellulose was prepared for chromatography as described previously (7).

304

BROSTROM

AND

WOLFF

TABLE I PURIFICATION OF CDR-DEPENDENT CYCLIC NUCLEOTIDE PHOSPHODIESTERASE” Fraction

Total volume (ml)

Total protei n (d

Protein

Cyclic AMP

Cyclic GMP

COWZIP

tration bag/ml)

Totalactivity (pm01 hydrolyzed/min)

fg?Ei bmol

mg-’ min-‘)

e”,“;- z;“(“g;tii-Y

(96)

drdlyzedlmin)

6340 2500

114 30

ReyiT

-I__

___Crude extract Protamine-Sepharose 30-50% (NH&SO, precipitate ECTEOLA-cellulose

“Fz&ac(nmol mg-’ min-‘1 Ind. Dep.

Dep.

Ind. Dep.

Ind. Dep.

Dep.

Ind. Dep.

18 12

800 1250 210 570

7 11 7 19

100 45

3770 4560 630 1980

33 21

40 66

100 44

242

13.3

55

190

480

14 36

38

630 2060

47

155

44

260

4.4

17

75

380

17 85

34

290 1410

65

318

30

a Phosphodiesterase activity was assayed with (total activity) and without (independent (Ind.) activity) the addition of 0.2 mM free Ca*+ and 1 pg of CDR (Ca*+-dependent regulator). Substrate concentrations were 1 mM cyclic AMP or 26 PM cyclic GMP. Ca2+-dependent (Dep.) activity represents the difference between total activity and independent activity. Recoveries of dependent activity from the original homogenate into the 18,OOOg supernatant fraction (crude extract) were 55 and 56% with cyclic GMP and cyclic AMP, respectively, as substrates. The extract contained 17% of the protein from the homogenate.

ment. A heavy precipitate was observed in the fractions with enzyme. The preparation contained a single form of phosphodiesterase by the criterion of electrophoretic migration on 5% acrylamide gels in 2 mM dithiothreitoV50 mM Tris/O.27 mM glycine buffer, pH 8.3 (not illustrated). Following electrophoresis at 1.5 mA/gel for 2 h, the gels were removed, frozen at -8o”C, and sliced into l-mm sections and the slices incubated overnight at 4°C in 3 mM MgClJlO rnM imidazole buffer, pH 7.5. The electrophoretic migration of the enzyme was then established in a subsequent phosphodiesterase assay of the slice extracts. The single enzymic form which was observed hydrolyzed both cyclic nucleotides and was activated sixfold by CDR. Approximately 60% of the activity applied was recovered. Heat-stability studies were conducted with the phosphodiesterase preparation (Fig. 1). Aliquots of the enzyme were diluted to a protein concentration of 1 mg/ml in 3 mM MgClJO.1 mM EGTA/lO mM imidazole, pH 7.5, and heated for 5 min at various temperatures ranging from 40 to 80°C. The enzyme was later diluted further for assay of phosphodiesterase activity, as de-

scribed in Materials and Methods. With added CDR and either cyclic AMP or cyclic GMP as substrate, identical degrees of thermal denaturation were observed with increasing temperatures during the preincubation. Approximately 90% of the phosphodiesterase was denatured between 60 and 8o”C!, with half of the activity denatured at 70°C. Similar thermal denaturation curves were found for the basal (CDRindependent) activity associated with the preparation. CDR, in the presence of excess Ca2+ when added to the preincubation, was found to lower the thermal stability of the enzyme some 8-10°C as assayed with either substrate. Competitive substrate kinetics demonstrated that increasing concentrations of cyclic AMP inhibited the hydrolysis of cyclic GMP by the CDR-dependent phosphodiesterase. These data are illustrated in the Dixon plot of Fig. 2. An apparent inhibition constant of 160 ,UM was determined for cyclic AMP. Similarly, cyclic GMP was observed to inhibit competitively the hydrolysis of cyclic AMP by the enzyme, with an apparent inhibition constant of 8 PM (Fig. 3). These values correspond closely to the apparent K, values of

the enzyme for cyclic AMP (180 PM) and cyclic GMP (8 PM) determined at nonlimiting concentrations of CDR and Mg2+. The K, data are described below (Figs. 4 and 5).

The single form of phosphodiesterase activity in the purified preparation was compared to the Ca2’-dependent phosphodiesterase activity in crude extracts of porcine brain. The enzymes were identical by the criteria of gel filtration chromatography on Sephadex G-200 and by electrophoretic mobility on 5% acrylamide gels. Apparent K, values for cyclic AMP and cyclic GMP, 185 and 8 PM, respectively, determined for the crude Ca2+-dependent phosphodiesterI I I ase when supplemented with CDR, were 50 60 40 70 80 similar .to those found for the purified enTEMFERATUREW zyme. The velocity of cyclic GMP hydrolyE‘IG. 1. Temperature stability of the Cae+-dependsis relative to cyclic AMP hydrolysis of the ent phosphodiesterase. Aliquots of the CDR-dependent phosphodiesterase purified through the EC- Ca’+-dependent phosphodiesterase was exTEOLA-cellulose stage were diluted to 1 mg of pro- amined for a brain homogenate, a crude tein/ml with 10 mM imidazole/O.l rnM EGTA/l mM extract, and the purified enzyme preparaMgCIZ, pH 7.5. The aliquots were preincubated for 5 tion (Table II). At concentrations of 2.5 min at various temperatures. No CDR was present PM, cyclic GMP was hydrolyzed fourfold in the preincubation. The heated enzyme was then more rapidly by all preparations. At 25 PM assayed for Ca2+-dependent activity with cyclic substrate, this ratio decreased approxiAMP, 1 mM (O-O), or cyclic GMP, 25 PM mately threefold.

(O---O), as substrate. CDR, 1 pg, was added to assays of dependent activity. See text footnote 2 for abbreviations.

II -01

0

01

I

I

02

03

04

I

I

II

G

05

06

07

ki

CDR and Cyclic Nucleotide

Substrate

Activation of the Ca2+-dependent phosphodiesterase by microgram quantities of

CAMP (mM1

FIG. 2. Inhibition of Ca2+-dependent cyclic GMP hydrolysis by cyclic AMP. Ca2+-dependent phosphodie&erase activities were determined with 1 @g of added Caz+-dependent regulator (CDR) at 3.3 6.7 (O-O), 10.1 (M-W and 20.1 FM (0 -O), (0-O) 3H-labeled cyclic GMP as substrate at 5 mM MgCl+ Assays were conducted with 0.34 pg of purified enzyme/incubation tube.

FIG. 3. Inhibition of Caz+-dependent cyclic AMP hydrolysis by cyclic GMP. Caz+-dependent phosphodiesterase activities were determined with 1 pg of added Ca2+-dependent regulator (CDR) at 0.1 0.2 (O-o), 0.33 (m-m), and 0.67 mM (0 -•), *H-labeled cyclic AMP as substrate at 5 (0 -Cl) mM MgCl,. Assays were conducted with 1.7 pg of purified enzyme/incubation tube.

306

BROSTROM AND WOLFF TABLE II

RELATIVE

tions of cyclic AMP and cyclic GMP were tested on the activation of the phosphodiesterase at a series of CDR concentrations and type Cyclic nuRatio of rates of (Fig. 6). Substrate concentration exerted moderate influences on the CDR cleotide conhydrolysis centration (cyclic sensitivity of the enzyme. Half-maximal GMPkyclic (PM) activation with 250 (uM cyclic GMP as subAMP) strate occurred at 25 ng of CDR as opposed 2.5 4.1 to 60 ng with 25 PM cyclic GMP. Half25 1.1 maximal activation occurred at 80 ng of 2.5 4.3 CDR with 250 FM cyclic AMP as substrate 25 1.7 and at 200 ng with 25 ,UM cyclic AMP as 2.5 4.7 substrate. 25

RATES OF HYDROLYSIS OF CYCLIC AND CYCLIC AMP”

Preparation

Homogenate Extract Purified enzyme

GMP

1.3

a Fresh whole brain homogenates were prepared in 50 mM Tris-chloride buffer as described in the text. Extracts represent the supernatant fraction obtained when the homogenate was clarified by centrifugation at 18,000 g for 35 min. Ca*+-dependent activity (nmol mg-* mine*) was determined at 0.2 mM free Ca*+ with 1 pg of added Caz+-dependent regulator in the assay.

CDR at 5 mM MgClz resulted in a sixfold increase of enzyme activity as compared to samples without added CDR. The apparent K, of the CDR-dependent phosphodiesterase for cyclic AMP decreased with increasing quantities of CDR in the assay (Fig. 4). K, values ranged from 4 mM at 50 ng of CDR to 0.18 mM at 10 fig of CDR. Maximal activities obtained at each concentration of CDR, however, were constant at 370 nmol (mg-’ min-9. The apparent K, for cyclic AMP for the CDR-independent (basal) activity was identical to that observed for the CDR-dependent enzyme at 10 pg of CDR. A comparable study was conducted with cyclic GMP as substrate and similar results were obtained (Fig. 5). Apparent K, values of the CDRdependent phosphodiesterase for cyclic GMP ranged from 200 PM at 10 ng of CDR to 8 PM at 1 pg of CDR, while maximal velocities were constant at 120 nmol mg-’ min-l. The apparent K, of the CDR-independent activity was also 8 PM. The data from Figs. 4 and 5 were replotted as the reciprocal of velocity versus the reciprocal of [CDR] to determine the effect of substrate on the activation of the enzyme by CDR. Complex nonlinear plots were obtained which were not readily interpretable. The effects of two concentra-

CDR and Magnesium

Ion

The MgZf requirement of the phosphodiesterase activity was examined initially with saturating amounts of CDR. Activity was not apparent in controls incubated with EDTA (5 mM). As MgClz concentrations were adjusted to 0.5 mM, the enzyme exhibited rapid increases in both Ca2+-independent and Ca2+-dependent activity with 25 pM cyclic AMP as substrate (Fig.

FIG. 4. Influence of CDR on the cyclic AMP concentration dependence of the Ca2+-dependent phosphodiesterase. Cal+-dependent activities were measured at 0.05 (AA, 0.10 (m---ml, 0.25 (m-a), ).O (0-O) and 10 pg (O-O) of CDRAncubation tube. These values were corrected for the Cae+-independent activity of controls incubated with EGTA. A control curve (A-Al representing this Cal+independent activity (no added CDR) is also plotted. Assays were conducted with 3.4 pg of enzyme and at 5 mM MgCl,. See text footnote 2 for abbreviation.

CALCIUM-DEPENDENT

PHOSPHODIESTERASE

307

Mg2+ from 500 to 8 PM with 25 PM cyclic AMP as substrate (Fig. 8). Variation of CDR from 50 ng to 1 pg decreased the apparent K, for Mg2+ from 2000 to 16 I.LM

FIG. 5. Influence of Caz+-dependent regulator (CDR) on the cyclic GMP concentration dependence of the Cae+-dependent phosphodiesterase. Caz+-dependent phosphodiesterase activities were measured at 0.01 (A-A), 0.025 (m---W, 0.05 (U-O), 0.10 (0-O) and 1 pg (O-O) of CDR/incubation tube and were corrected for Ca2+independent activity (A-A) as in Fig. 4. Assays were conducted with 0.68 pg enzyme and at 5 mM MgCl,.

7A). At MgCl, concentrations above 0.5 mM, Caz+-independent activity increased, Ca2+-dependent activity decreased, and the sum of the two activities remained constant. The apparent conversion of a portion of the Ca2+-dependent form to the independent form was further emphasized when the data were expressed in doublereciprocal format (Fig. 7B). A linear relationship was observed between Mg2+ concentration and total phosphodiesterase activity whereas complex relationships appeared to exist between Mg’+, Ca2+-independent activity, and Ca2+-dependent activity. The apparent conversion of the Ca2+-dependent to the Ca2+-independent phosphodiesterase activity with variation of Mg2+ was less pronounced with 25 PM cyclic GMP as substrate. The effect of CDR on the apparent K, of the phosphodiesterase for Mg2+ was examined by analyzing changes in total enzyme activity with variation of Mg2+ concentration. Variation of CDR in the assay from 75 ng to :I pg reduced the apparent K, for

FIG. 6. Effects of substrate on the interaction of Ca*+-dependent regulator (CDR) with the CDR-dependent phosphodiesterase. Assays were conducted with from 0.01 to 10 Fg of CDRlincubation tube at n ) and 25 PM (O--O) cyclic GMP and at 250 (rn0) and 25 PM (O-O) cyclic AMP. Phos250 (Clphodiesterase, 0.85 pg, was added to each incubation tube. MgCl, was present at 5 mM.

FIG. 7. MgZ+ concentration dependence of the phosphodiesterase. The phosphodiesterase activity of 1.7 pg of enzyme/incubation tube was assayed with 25 ELM cyclic AMP as substrate at varying concentrations of MgCl,. Ca*+-independent activity 0) represents the basal activity of the enco-zyme without Ca”+-dependent regulator (CDR) or Ca*+. Total phosphodiesterase activity (A-A) includes both the independent activity and the increment in activity observed with the addition of 3 Fg of CDR and 0.2 mM free Ca’+. Ca2+-dependent activity 0) was calculated as total activity minus incodependent activity.

308

BROSTROM AND WOLFF

cyclic nucleotide and one-third as great at ,UM cyclic nucleotide. In alternate experiments, buffer systems other than imidazole were tested for effects on the ratio of velocities (cyclic GMP/cyclic AMP) at 2.5 PM substrate. Only minor changes were observed with N - Tris(hydroxymethy1) methyl-Zaminoethane sulfonic acid (Tes), Tris, phosphate, pglycerophosphate, and N-2-hydroxyethyl piperazine-N’S-ethane sulfonic acid (Hepes) buffers at pH 7.5. An extract of yeast containing a variety of cofactors did not affect the ratio nor did boiled extracts of fresh rat brain. 250

DISCUSSION 1

I I

1 2

3

I 4

RECIPROCAL

5

6

Mq’+(mM)

Fro. 8. CaP+-dependent regulator (CDR) influence on the Mge+ concentration dependence of cyclic AMP hydrolysis by the phosphodiesterase. The total phosphodiesterase activity of 1.7 pg of enzyme/ incubation tube was measured with 25 w cyclic AMP as substrate at0.75 (A-A), 0.10(A-A), 0.15 (O-01, 0.30 (m-m), 1 (O-O), and 3 pg (0-O) of CDR. MgCl, was varied from 0.15 to 10 mhr.

with 25 PM cyclic GMP as substrate (Fig. 9). Attempts to express the Ca’+-dependent component of the total phosphodiesterase activity in a similar double-reciprocal format provided complex nonlinear plots, such as those discussed earlier (Fig. 7B). Replotting the Ca’+-dependent activity as the reciprocal of activity versus the reciprocal of [CDR] at various concentrations of Mg2+ provided complex concave curves that were difficult to interpret (data not illustrated). Increasing Mg2+ concentrations appeared to lower the apparent K, of the enzyme for CDR, but the apparent K, could not be determined. The relationships between CDR and Mg2+ at various concentrations of cyclic AMP and cyclic GMP are presented in Table III. With either substrate, total phosphodiesterase activity increased as CDR was elevated at constant Mg2+ or as Mg2+ was increased at constant CDR. Enzymic velocities with cyclic GMP as substrate were approximately fourfold greater than with cyclic AMP as substrate at 2.5 FM

The three factors known to interact with and modulate the activity of the Ca2+-dependent phosphodiesterase are the cyclic nucleotide substrate, Me+, and Ca2+ complexed with the Ca2+-binding protein, CDR (Ca”+*CDR). In this report the association of each of the three factors with the enzyme has been characterized kinetically and determined to be facilitated by the two alternate factors. A partially purified en-

1

i FIG. 9. Ca2+-dependent regulator (CDR) influence on Mg*+ concentration dependence of cyclic GMP hydrolysis by the phosphodiesterase. The total phosphodiesterase activity of 1.7 pg of enzyme/incubation tube was measured with 25 /.LM cyclic GMP as substrate at 0.05 (A-Al, 0.075 (o-o), 0.15 (m-m), 0.30 (O-O), and 1 fig of CDR. MgCl, was varied from 0.15 to 10 (0 -0) mx

CALCIUM-DEPENDENT TABLE

III

INFLUENCE OF MgZ+ AND CDR ON THE RELATIVE RATES OF CYCLIC AMP Nucleotide (N-f)

CDRb (ng)

Nanomoles

of nucleotide

hydrolyzed

AND CYCLIC GMP HYDROLYSIS~ per milligram

Cyclic AMP Magnesium chloride (rnbf) 0.2

2.5

309

PHOSPHODIESTERASE

0 15 75 1000

1.8 2.8 4.6 6.4

1.0 1.9 3.8 5.0 6.6

per minute

, Cyclic GMP Magnesium chloride (rnM) 5.0 2.4 5.5 6.6 7.3

0.2

1.0

5.0

3.2 7.0 7.0 25.4

4.7 7.4 22.1 26.6

4.5 16.3 27.4 30.1

25

0 15 75 1000

11 17 42 53

14 25 43 53

18 40 52 55

8 22 57 75

11 35 61 78

14 65 72 90

250

0 15 75 1000

63 138 223 252

78 174 237 265

97 240 279 295

23 80 85 113

29 80 94 109

51 109 107 112

’ Values represent total phosphodiesterase enzyme/incubation tube. b CDR, Ca2+-dependent regulator.

activity

zyme, identical physically and kinetically to the Ca2+-dependent phosphodiesterase of brain extract, was utilized in conducting these experiments. Although only one form of phosphodiesterase was demonstrated in the preparation by a variety of physical and kinetic criteria, that form possessed some basal activity as assayed with the addition of EGTA without added CDR. Several lines of evidence provide support for concluding that this Ca’+-independent phosphodiesterase activity is associated with or derived from enzyme which is normally dependent on the presence of Ca2+.C!DR. First, a constant ratio of dependent to independent activity was observed as the phosphodiesterase was denatured to successively greater extents at high temperature. When such studies were performed with CDR added during the heating stage, both activities were denatured in parallel at lower temperatures. Second, the respective apparent K, values for cyclic AMP and cyclic GMP were identical for the independent activity and for maximally activated dependent activity. Third, as Mg2+ concentrations were in-

at each condition.

Assays

contained

0.85 pg of

creased in the incubation, an apparent conversion of dependent activity to independent activity occurred (Fig. 7). Although independent activity accounts for X5-20% of the total phosphodiesterase activity observed, the significance of this contribution is unclear. The enzyme may exist as a mixture of two interconvertible forms or, alternately, the independent form may arise from spontaneous, irreversible activation of the dependent form. Activation of the phosphodiesterase by the Ca2+-dependent regulator has been suggested by a number of workers to involve two steps (6, 12, 18). The first involves an interaction of Ca*+ with CDR to form an active complex: Ca*+ + CDR $ Ca*+.C!DR.

VI

This complex then associates with and activates the enzyme (PDE, phosphodiesterase) : Ca*+CDR + PDE. InactiveCa*+*CDR.PDE a&w. El Incubation increasing

of the phosphodiesterase with amounts of CDR leads to the

310

BROSTROM

AND

WOLFF

activation of the enzyme at lower Ca2+ number of tissue preparations (9, 19,26); it concentrations, presumably through mass also represents a major form of cyclic AMP action effects (see Eq. [l]; (12). phosphodiesterase from several sources inThe present experiments were con- cluding brain (26-28). Neither Ca2+*CDR ducted at free Ca2+ concentrations (0.2 nor Mg2+ was observed to change the submM) such that almost all of the CDR added strate specificity of the Ca2+-dependent was present in the incubation as the phosphodiesterase. Activation of the enCa’+CDR complex. Ca’+*CDR was found zyme with these substances promoted parto decrease the respective apparent K, valallel variations in kinetic behavior toues of the enzyme for cyclic AMP and wards both cyclic AMP and cyclic GMP. cyclic GMP more than 20-fold without The threefold higher relative velocity occhanges of V (Figs. 4 and 5). On the other curring with high concentrations of cyclic hand, these substrates exerted only moderAMP as opposed to cyclic GMP as subate effects on the association of Ca2+CDR strate reflected the inherently greater V of with the phosphodiesterase (Fig. 6). High the enzyme with cyclic AMP as substrate. concentrations of cyclic GMP promoted the The fourfold higher relative velocity with association more effectively than low con- 2.5 pM cyclic GMP as opposed to cyclic centrations of cyclic GMP or equimolar con- AMP as substrate (Table III) was a refleccentrations of cyclic AMP. Similar effects tion of both the higher V with cyclic AMP were noted previously with a phosphodiesand the much lower apparent K, for cyclic terase of C-6 glioma cells, and it was GMP. From a knowledge of the relative V concluded that they arose as an expression and the apparent K, of the maximally of different degrees of substrate saturation activated enzyme for each cyclic nucleotide of the enzyme (12). These overall findings (cyclic AMP, 180 PM; cyclic GMP 8 PM), are generally consistent with and extend one can calculate, utilizing the Michaelisthe observations of Kakiuchi et al. (9, 19) Menten equation, that at all concentrabut do not support the conclusions of Wicktions below 0.5 PM, concentrations of cyclic son et al. (20). GMP equimolar to cyclic AMP will be hyAn inverse relationship was found to drolyzed seven times more rapidly than exist between the Ca2+*CDR complex and cyclic AMP. Since both cyclic nucleotides the Mg2+ concentration dependence of the are thought to occur at concentrations in phosphodiesterase. Ca2+CDR greatly de- brain which are considerably below the creased the apparent K, of the phosphodirespective apparent K, values determined esterase for Mg2+ while V remained con- for the enzyme in vitro, each nucleotide stant; similar results were obtained with would be removed by a first-order process. either cyclic AMP or cyclic GMP as sub- The sevenfold higher first-order rate constrates (Figs. 8 and 9). Alternately, the stant observed for the hydrolysis of cyclic sensitivity of the enzyme to low amounts GMP would support the argument that the of Ca’+*CDR was increased at higher con- Ca2+-dependent phosphodiesterase funccentrations of Mg2+. It is difficult to evalutions physiologically as a cyclic GMP phosate this finding in the context of the regulaphodiesterase. This conclusion, however, tion of the phosphodiesterase by CDR. In- may be premature in the absence of detinitracellular Mg2+ is thought to be present tive information regarding free cyclic nuat free concentrations of 0.5-1.0 mM (21- cleotide concentration ranges occurring at 23). Variation of intracellular Mg2+ (24,25) the intracellular site(s) of the phosphodiescould be construed as a potential mecha- terase. Also the possibility cannot be ruled nism by which the sensitivity of the phos- out that additional unknown factors may phodiesterase to Ca’+.CDR (and hence to exist in viva which alter enzyme speciof the Ca2+-defluctuation of free Ca2+) could change ficity. The relationship while total CDR remained constant. pendent phosphodiesterase to other forms The Ca2+-dependent phosphodiesterase of cyclic nucleotide phosphodiesterase in represents the predominant portion of the brain and other tissues (27, 29-32) recyclic GMP phosphodiesterase activity in a mains to be clarified.

CALCIUM-DEPENDENT ACKNOWLEDGMENTS The authors wish to express their appreciation to Mr. Kenneth Feiler for his excellent technical assistante in the course of this work. REFERENCES 1. BRADHAM, L. S. (1972) Biochim. Biophys. Acta 276, 434-443. 2. VON HUNCIEN, K., AND ROBERTS, S. (1973) Eur. J. Biochem. 36, 391-401. 3. JOHNSON, R. A., AND SUTHERLAND, E. (1973) J. Biol. Chem. 248, 5114-5121. 4. 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. 5. TEO, T. S., AND WANG, J. H. (1973) J. Biol. Chem. 248, 5950-5955. 6. LIN, Y. M., LIU, Y. P., AND CHEUNG, W. Y. (1974) J. Biol. Chem. 249, 4943-4954. 7. WOLFF, D. J., AND BROSTROM, C. 0. (1974)Arch. Biochem. Biophys. 163, 349-358. 8. BROSTROM, C. O., HUANG, Y. C., BRECKENRIDGE, B. McL., AND WOLFF, D. J. (1975) Proc.

311

PHOSPHODIESTERASE

AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 17. CUATRECASAS, P. (1970) J. Biol. Chem. 245, 3059-3065. 18. WANG, J. H., TEO, T. S., Ho, H. C., AND STEVENS, F. C. (1975) in Advances in Cyclic Nucleotide Research (Drummond, G. I., Greengard, P., and Robison, G. A., eds.), Vol. 5, pp. 179-194, Raven Press, New York. 19. KAKIUCHI, S., YAMAZAKI, R., TESHIMA, Y., UENISHI, K., AND MIYAMOTO, E. (1975) in A’dvances in Cyclic Nucleotide Research (Drummond, G. I., Greengard, P., and Robison, G. A., eds.), Vol. 5, pp. 163-178, Raven Press, New York. 20. WICKSON, R. D., BOUDREAU, R. J., AND DRUMMOND, G. I. (1975)Biochemistry 14,669-675. 21. GUNTHER, T. (1967) 2. Nuturforsch. 22b, 149154. 22. BUNN, H. E., RANSIL, B. J., AND CHAO, A. (1971) J. Biol. Chem. 246, 5273-5279. 23. VELOSO, D., GUYNN, R. W., OSKARSSON, M., AND VEECH, R. L. (1973) J. Biol. Chem. 248,48114819. 24. ROSE, I. A. (1968)Proc. Nut. Acad. Sci. USA 61, 1079-1086.

Nat. Acad. Sci. USA 72, 64-68. S., YAMAZAKI, R., TESHIMA, Y., AND K. (1973)Proc. N&l. Acad. Sci. USA 70, 3526-3530. 10. STEINER, A. L., PAGLIARA, A. S., CHASE, L. R., AND KIPNIS, D. M. (1972) J. Biol. Chem. 247, 1114-1~120.

25. SCARPA, A. (1974) Biochemistry 13,2789-2794. 26. KAKIUCHI, S., YAMAZAKI, R., TESHIMA, Y., UENISHI, K., AND MIYAMOTO, E. (1975) Biochem. J. 146, 109-120. 27. STRADA, S. J., UZUNOV, P., AND WEISS, B. (1974) J. Neurochem. 23, 1097-1103. 28. UZUNOV, P., SHEIN, H. M., AND WEISS, B., (1974) Neuropharmacology 13,377-391.

11. GOLDBERO,

29. THOMPSON,

9. KAKIUCHI, UENISHI,

12. 13. 14.

15.

N. D.,

O’DEA,

R. F.,

AND HADDOX,

M. K. (1973) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 3, pp. 155-223, Raven Press, New York. BROSTROM, C. O., AND WOLFF, D. J. (1974)Arch. Biochem. Biophys. 165, 715-727. WOLFF, D. J., AND SIEGEL, F. L. (1972) J. Biol. Chem 247, 4180-4185. HUANG, Y. S., AND KEMP, R. G. (1971) Biochemistry 10, 2278-2283. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427.

16. LOWRY, 0. H., ROSEBROUGH,

N. J., FARR, A. L.,

J. W.,

AND APPLEMAN,

M. M. (1971)

Biochemistry 10, 311-316. 30. CHEUNG, W. Y. (1970) in Role of Cyclic AMP in Cell Function (Greengard, P., and Costa, E., eds.), Vol. 3, pp. 51-65, Raven Press, New York. 31. APPLEMAN, M. M., THOMPSON, W. J., AND RusSELL, T. R. (1973) in Advances in Cyclic Nu-

cleotide Research (Greengard, son, G. A. eds.), Vol. 3, pp. Press, New York. 32. HARDMAN, J. C., AND O’MALLEY, (1974) Methods in Enzymology, 205-273, Academic Press, New

P., and Robi65-98, Raven B. W. 0. (eds.) Vol. 38, pp. York.