rac-glycerol 1:2-cyclic phosphate 2-phosphodiesterase, a new

Biochem. J. (1978) 173, 579-589Printed in Great Britain

rac-Glycerol 1:2-Cyclic Phosphate 2-Phosphodiesterase, a New SolublePhosphodiesterase of Mammalian Tissues

By NEVILLE CLARKE and REX M. C. DAWSONDepartment ofBiochemistry, A.R.C. Institute ofAnimal Physiology,

Babraham, Cambridge CB2 4AT, U.K.

(Received 24 November 1977)

1. A soluble phosphodiesterase is present in mammalian tissues which rapidly hydrolysesenantiomorphs of rac-glycerol 1:2-cyclic phosphate, producing rac-glycerol 1-phosphate.2. The enzyme has been purified up to 1700-fold by a combination of acetone precipitationand chromatography on DEAE-Sephadex A-50, Sephadex G-150 and hydroxyapatite.3. The Km with glycerol cyclic phosphate as substrate is 7.2mM, and the pH optimumbroad (6.9-7.5). The molecular weight (by gel filtration) of the enzyme is approx. 35 500.4. The phosphodiesterase has no requirement for Ca2+ or Mg2+, but is stimulated byreducing agents (cysteine, dithiothreitol) and Fe2+. 5. The purified phosphodiesterasepreparation also hydrolysed 3':5'-cyclic AMP, producing 5'-AMP exclusively, and2':3'-cyclic AMP, forming 3'-AMP and 2'-AMP in the ratio 7:3. Bis-(p-nitrophenyl)phosphate was slowly hydrolysed, but other phosphodiesters tested were not attacked.6. The phosphodiesterase is inhibited by theophylline and o-phenanthroline. It is inhibitedby Pi and by a variety of phosphomonoesters, of which certain aromatic primaryphosphates are particularly effective.

Both glycerol 1:2-cyclic phosphate and glycerolphosphate can be formed through the action ofglycerophosphinicocholine diesterase (EC 3.1.4.2),one of the enzymes involved in the turnover ofphospholipids (Clarke & Dawson, 1976). In furtherexperiments (N. Clarke & R. M. C. Dawson,unpublished work), small amounts of glycerol1:2-cyclic phosphate have been detected in mam-malian tissues. We show in the present work thattissues also contain phosphodiesterases that hydrolyseglycerol cyclic phosphate, producing glycerol phos-phate. In kidney, one of these phosphodiesterases isassociated with the particulate fraction of tissuehomogenates. It is strongly inhibited by EDTAand has a pH optimum around 8.9. Anotherphosphodiesterase is a low-molecular-weight solubleenzyme uninhibited by EDTA and with a pHoptimum near neutrality. In the present paper wereport the purification of this latter enzyme and anexamination of its characteristics. It has not beenpossible to equate the enzyme with- any phospho-diesterase hitherto described. In all tissues, includingspleen, the rate at which extracts hydrolyse glycerolcyclic phosphate is appreciably higher than the rateof hydrolysis of the p-nitrophenyl ester of thymidine3'-phosphate, a substrate for soluble phospho-diesterase II (EC 3.1.4.18). In addition, activitytowards this latter substrate disappears on puri-fication of the present enzyme.

Vol. 173

Experimental

Substrates

sn-Glycerol 2:3-cyclic phosphate. This was pre-pared by the base-catalysed methanolysis of eggphosphatidylcholine (Maruo & Benson, 1959;Brockerhoff, 1963). Phosphatidylcholine (36mg of P)was dissolved in 10.2ml of chloroform, and 6.8mlof ethanolic 0.3 M-KOH was added and the mixtureincubated for 50min at 18°C. K+ cations wereremoved by adding 10ml of 0.21 M-NH4CIO4 inmethanol/water (43:7, v/v), the insoluble KCO04being separated by centrifugation at 0°C. The glycerolcyclic phosphate was recovered from the supernatantby preparative paper ionophoresis at pH 3.6 (Dawson& Clarke, 1972). It had a mobility towards theanode slightly faster than Pi (Mp1 1.04), whereasglycerophosphocholine, the other phosphorus-con-taining product of the methanolysis, was isoelectric.

rac-Glycerol 1:2-cyclic phosphate. This was ob-tained from butyrylglycerol cyclic phosphate pre-pared as described by Clarke & Dawson (1976). Onpaper ionophoresis at pH 3.6 the butyrylglycerolcyclic phosphate gave a single phosphorus-containingspot (Mp, 0.79). On paper chromatography inethanol/5 M-NH3 (2: 1, v/v) the compound hadRF 0.83. It (0.8g of P) was dissolved in 24ml of aq.18M-NH3, and after hydrolysis for 2h at 18°C thereaction mixture was evaporated to dryness in vacuo

579

N. CLARKE AND R. M. C. DAWSON

at 65°C. The residue was solubilized in 48ml of dryNH3 in methanol (5 % saturation) and the ammoniumsalt of glycerol cyclic phosphate precipitated at 18°Cwith lOvol. of acetone. This solubilization andreprecipitation were repeated twice. The final pro-duct was dissolved in 60ml of 5 %-satd. NH3 inmethanol and treated with an equal volume of diethylether. A small precipitate containing glycerolphosphate was removed by centrifugation at 2000gand discarded. The supernatant solution wasevaporated to dryness below 50°C and the residue(ammonium glycerol cyclic phosphate) was stored inchloroform/methanol (1:3, v/v) at -28°C. The yieldwas 60% (based on phosphorus content) and noappreciable decomposition occurred during 8 months.On paper ionophoresis at pH 3.6 the product gave asingle phosphorus-containing spot (Mp. 1.03), andthis homogeneity was also found after paperchromatography in ethanol/S M-NH3 (RF 0.68). Acidhydrolysis (60min, 0.05 M-HCI, 40°C) completelydecomposed the product into glycerol phosphate.This eliminated the possibility of contamination withglycerol 1:3-cyclic phosphate, which is much morestable under acid conditions (Khorana et al., 1957).

Glycerol cyclic [32P]phosphate. The starting pointof this synthesis was [32P]phosphatidylcholine or[32P]phosphatidylethanolamine, which were pre-pared biosynthetically from Saccharomyces cere-visiae (Hazlewood & Dawson, 1975). These weredeacylated with alkali to give glycerophospho-'base'derivatives (Dawson et al., 1962), which were thenhydrolysed in 1 M-HCl (15min, 100°C) to releaseglycerophosphoric acid. The glycero[32P]phosphoricacid was then converted into its cyclic derivative asdescribed above.

Butyrylglycerol phosphate. Butyrylglycerol cyclicphosphate (see above) was dissolved in 0.05M-HCIat a concentration of 0.5mg of P/ml and heated at100°C for 3 min. After neutralization with 0.1 M-NH3,the hydrolysate was loaded on a column (10cm x 1 cm)of Dowex IX (OH- form). Elution of the columnwith 0.O5M-(NH4)2SO4 produced an initial phos-phorus-containing peak identified as glycerol phos-phate. This was followed by the major product,which proved on analysis (Clarke et al., 1976) tocontain glycerol, butyric acid and phosphorus inequimolecular ratios. Although this latter product islikely to consist of a mixture of positional isomersof butyrylglycerol phosphate, it nevertheless gavea single spot on paper ionophoresis at pH 3.6(Mp1 0.73) and on paper chromatography in ethanol/SM-NH3 (RF 0.62).

Enzyme assay

Method 1. The usual complete reaction mixturecontained, in a total volume of 0.18 ml, a final

concentration of the following reagents: ammoniumrac-glycerol 1: 2-cyclic phosphate (11 mm), glycyl-glycine/NaOH buffer, pH 7.5 (70mM), MgCI2 (IOmM),dithiothreitol (15mM), ammonium ascorbate (29mM),FeCI2 (0.3mM) and alkaline phosphatase (0.8 unit)(calf intestinal; grade I; Boehringer, Mannheim,Germany). The phosphodiesterase (0.025-SOug ofprotein) was added in SO,ul of 0.1 M-glycine/NaOHbuffer, pH 8.5, a medium in which the enzyme provedto be relatively stable on storage. With the purifiedenzyme ofhigh specific activity, consistent results wereonly obtained if it was supplemented with a solutionof bovine serum albumin dialysed against water toact as a protein carrier.

Incubation was for 30min at 38°C togetherwith independent enzyme and reagent controls.The reaction was stopped by adding lOml of asolution containing 10% (w/v) HCI04, 0.36%(w/v) ammonium molybdate and 0.0045% (w/v)aminonaphtholsulphonic acid reagent (Bartlett,1959). After centrifugation at 2000g for 5min toremove precipitated protein, the Pi released wasdetermined by heating for 10min at 100°C andmeasuring the A830 of the solution.When cyclic AMP substrates were used, some de-

composition occurred during the colour develop-ment. Consequently, with 3': 5'-cyclic AMP, heatingwas shortened to 5min and the aminonaphthol-sulphonic acid reagent was increased to 0.009%(w/v). With 2': 3'-cyclic AMP, the HCI04 concen-tration was decreased to 7.5% (w/v), the heatingtime to 2.5 min and the aminonaphtholsulphonic acidreagent increased to 0.0135% (w/v). The hydrolysisof the p-nitrophenyl ester of thymidine 3'-phosphateand of bis-(p-nitrophenyl) phosphate were measuredby determining p-nitrophenol liberation (Razzell,1961).Method 2. The incubation was as in method 1,

with the omission of alkaline phosphatase. At theend of the incubation 0.5ml of 0.1 M-glycylglycine/NaOH buffer (pH8.0) containing 15mM-MgCl2 wasadded and the mixture immediately heated at 100°Cfor 10min. After cooling to 18°C, 2.0 units of calfintestinal alkaline phosphatase in 0.2ml of 0.1 M-glycine/NaOH buffer (pH 9.5) were added and themixture was incubated for a further 20min at 37°C.Duplicate incubations were carried out for 40minto ensure that all the phosphomonoesters liberatedby the phosphodiesterase had been decomposed.The Pi released was determined as in method 1.Method 3. The incubation was as in method 1

with omission of alkaline phosphatase. The hydro-lysis was stopped by cooling in ice and adding 20,1of 8M-NH3. A portion was spotted on paper andthe reaction products were ionophoresed at pH3.6(in pyridine/acetic acid/water, 1:10:89, by vol.) at48V/cm. The cyclic phosphate ester substrates wereseparated from their phosphomonoester products

1978

580

rac-GLYCEROL 1:2-CYCLIC PHOSPHATE 2-PHOSPHODIESTERASE

by ionophoresis for the following times, by usingappropriate markers: glycerol cyclic phosphate andits butyryl derivative, 2.5h; 3':5'-cyclic AMP and2':3'-cyclic AMP, 6h. The phosphorus-containingspots were located and measured as previouslydescribed (Dawson et al., 1962). This method had tobe used with glycerol cyclic [32P]phosphate substrate.The glycerol [32P]phosphate spot separated byionophoresis was digested and the 32P assayed byCerenkov counting (Hazlewood & Dawson, 1975).

Enzyme purification

All operations were at 4°C or below. Protein was

determined by the method of Lowry et al. (1951)with serum albumin as standard. The cortex was takenfrom the kidneys of two young pigs and homogenizedbriefly in a Waring blender and then in a homogenizer(Aldridge et al., 1960) in 2.94vol. of 0.1M-glycine/NaOH buffer (pH 8.5) containing 20mM-2-mercapto-ethanol. The homogenate was centrifuged for 4hat 34000g and the clear supernatant collected.

Acetonie precipitation. To 200ml batches of super-natant was added ammonium ascorbate (0.15 Mfinal concentration) and acetone (38 %, v/v). Theprecipitated protein was centrifuged (34000g, 3 min)and discarded. Acetone was added to the supernatantto give a final concentration of 50% (v/v) and theprecipitated protein collected by centrifuging as

before. This 38-50 %-acetone fraction was solubilizedin 29ml of 0.1 M-glycine/NaOH buffer (pH 8.5)containing both ammonium ascorbate (0.1 5M) and20mM-2-mercaptoethanol, and an acetone-precipi-tated fraction collected between 42 and 50% (v/v).The 42-50%-acetone fraction was resolubilized anddialysed against 50mM-ethanolamine/HCl buffer(pH 8.5) overnight.DEAE-Sephadex chromatography. DEAE-Sepha-

dex A-50 (6.7g) was mixed with 200ml of 0.5M-ethanolamine/HCl buffer (pH 8.5) and heated at100°C for 20min. After cooling and centrifuging, thegel was washed well with 50mM-ethanolamine/HClbuffer (pH8.5). Half the gel was poured to form thebase of a column (6.2cm diam.), and half was mixedwith the dialysed 42-50 %-acetone fraction con-

taining 0.9g of protein, which was quickly adsorbed.This gel was poured on the column, which was theneluted with ethanolamine/HCI buffer (pH 8.5) byusing a concentration gradient of 50mM increasing to160mm over 2 litres and then remaining constant.Fractions of high specific activity eluted between150 and 160mm were pooled and concentrated byadsorbing on a smaller DEAE-Sephadex column(2.5g) and elution in 12ml of 3M-ethanolamine/HClbuffer (pH 8.5).

Gelfiltration on Sephadex G-150. The eluate fromthe DEAE-Sephadex column (see above) (78mg of

Vol. 173

protein in 12ml) was applied to a Sephadex G-150column (118cm x 2.4cm) and the proteins were elutedwith 0.1M-glycine/NaOH buffer (pH8.5). The en-zyme emerged as a peak after most of the otherproteins had appeared. The active fractions werepooled and dialysed overnight against 20mM-KH2PO4/K2HPO4 buffer (pH 6.8).

Hydroxyapatite. Bio-Gel HTP (3.3g) was slurriedwith 20mM-KH2PO4/K2HPO4 buffer (pH6.8) andhalf was poured to form a column (1.1cm diam.). Theenzyme recovered from the Sephadex G-150 columnwas mixed with the residual half of the Bio-Gel HTPslurry and the remainder of the column poured.Protein was displaced from the column by a stepwise(150ml) increase in the concentration of the phos-phate buffer, 20, 36, 120 and 240mm. To assay theenzyme it was necessary to remove the phosphateions from the fractions emerging from the column bydialysing against water. The protein with highestspecific activity was eluted at 36mm-phosphatealthough some enzyme remained adsorbed up to240mM.

Repetition ofpreceding three steps. By repeating theion-exchange chromatography, gel-filtration andhydroxyapatite procedures on smaller columns afurther purification of up to 3-fold could beachieved, but with poor recovery.

Identification of the products ofcyclic AMP hydrolysis

The hydrolysis was allowed to continue almost tocompletion and a portion of the incubation mixturespotted on paper. With 3': 5'-cyclic AMP as substratethe products were separated by anodic paperionophoresis (pH3.6, 40V/cm, 8h) followed bychromatography in ethanol/iM-ammonium acetate(7:3, v/v) in the same direction. Under these con-ditions the migration distances of standards were asfollows: 3'-AMP, 32cm; 5'-AMP, 28.5cm; 3':5'-cyclic AMP, 37cm. For 2':3'-cyclic AMP the pro-ducts were examined by two methods. (1) Paper iono-phoresis (pH 3.6, 40V/cm, 24h) followed by sprayingto detect phosphorus, which was then measured(Bartlett, 1959). In this time 2'-AMP had migrated86cm, 3'-AMP 90cm and 2':3'-cyclic AMP 96cm.(2) Paper chromatography for 48 h in a two-phasesystem (Carter, 1950). Spots were located by u.v.,cut out, eluted with water and the A260 was measured.The RF values were as follows: 2'-AMP, 0.69; 3'-AMP, 0.62; 2': 3'-cyclic AMP, 0.56; adenosine,0.49.

Reproducibility

Each type of experiment was carried out at leastthree times with minor variations in the experimentalprocedure and the results agreed in all essentialdetails.

581


Results

Glycerol cyclic phosphate phosphodiesterase in varioustissues

After the kidneys of a rat were homogenized, boththe supernatant and pellet fractions were capable ofhydrolysing glycerol cyclic phosphate (Table 1).Although the activity in the supernatant was notinhibited by EDTA, that in the pellet was totallysuppressed, suggesting insoluble and soluble phospho-diesterases of different character.The pH optimum of the insoluble enzyme was

alkaline (pH 8.9), whereas it is shown below that thepH optimum of the soluble enzyme is nearneutrality. In the tissues examined, the solublephosphodiesterase was much more active than theinsoluble enzyme (Table 1), and in the liver, whichshowed very active hydrolysis by the soluble enzyme,there was hardly detectable activity in the insolublefraction. Further studies were therefore concen-

trated on the soluble phosphodiesterase.A survey of the soluble phosphodiesterase in

various tissues of the rat active against glycerol cyclicphosphate is shown in Table 2. It is clear thatthe richest sources of the enzyme are the liver,

Table 1. Glycerol cyclic phosphate hydrolysis by soluble andinsolubleparts ofhomogenates oftissuesfrom various species

Tissues were homogenized in lOvol. of 0.05 M-glycylglycine buffer, pH7.5, containing 20mM-2-mercaptoethanol or in O.1M-glycine/NaOH buffer,pH8.5. The homogenate was centrifuged at lOOOOOgfor 90min, and the precipitate was washed with IOvol.of water and then recentrifuged at 100OOOg for60min. The soluble portion was dialysed overnightagainst the buffer, and then both fractions wereassayed for glycerol cyclic phosphate hydrolysis(method 1). The soluble part was assayed at pH7.5and the insoluble portion at pH8.9.

Hydrolysis (,umol/min per g wetwt. of tissue)

Animal TissueRat Kidney (whole)

LiverBrain (cerebral hemispheres)Intestinal mucosa

Mouse Kidney*Liver*

Guinea Kidney (whole)pig Liver

Brain (cerebral hemispheres)Sheep Brain (cerebral hemispheres)

Kidney (cortex)*Liver*

Pig Kidney (cortex)*Liver*

* Glycine/NaOH buffer used.

Soluble19.827.52.2

12.94.47.8

13.827.16.82.18.310.422.88.3

Insoluble1.00.050.110.50

1.20.030.120.02

kidney and intestinal mucosa. All of the othertissues sampled showed some activity, apart fromwhole blood. The relative distribution of the enzymeappears to be different from that of the phospho-diesterases attacking 2': 3'-cyclic AMP (EC 3.1.4.16)and 3': 5'-cyclic AMP (EC 3.1.4.17) (Table 2), whichas expected were highly enriched in the nervoussystem (Butcher & Sutherland, 1962; Olafson et al.,1969). Moreover, the highest activity towards thep-nitrophenyl ester of thymidine 3'-phosphate, asubstrate of soluble phosphodiesterase II (spleenexonuclease, EC 3.1.4.18), was present in the spleen(Table 2). On the other hand, the activity towardsbis-(p-nitrophenyl) phosphate seemed to bear a fairlyconstant relationship to the rate of glycerol cyclicphosphate breakdown throughout the varioustissues.To obtain a suitable bulk source of the soluble

enzyme for purification and to examine interspeciesvariability, activities were examined in the livers andkidneys of the mouse, rat, guinea pig, sheep and pig(Table 1). The liver usually had a higher activityper g wet weight than the kidney, apart from in thepig, where activity in the kidney was much higher thanthat in the liver and approaching that found in ratliver. The pig kidney cortex and medulla showed verysimilar activity, but, since the medullary materialshowed more activity towards phosphodiestersubstrates other than glycerol cyclic phosphate,kidney cortical tissue was chosen as a bulk source ofenzyme for purification.

Purification of enzyme

A combination of acetone precipitation andfractionation on successive columns of DEAE-Sephadex anion-exchanger, Sephadex G-150 mole-cular-filtration gel and hydroxyapatite resulted in apurification that in a number of preparations was400-500 times the activity in the original super-natant, with a yield of about 5% (Table 3). Repetitionof the column-fractionation stages resulted in amaximum purification of 1400-1700 times, in verylow yield. Nevertheless sufficient activity wasavailable to investigate those situations, e.g. substratespecificity, that required a high degree of purity.Sodium dodecyl sulphate/polyacrylamide-gel elec-

trophoresis of the purest enzyme preparations showeda major band corresponding to a mol.wt. of 36000and three smaller bands of lower molecular weight.Attempts to elute an active phosphodiesterase fromthe gel proved unsuccessful, so it was impossible toequate the enzyme with any particular band.

Products of the enzymic reaction

With the purified enzyme and glycerol cyclicphosphate substrate, the sole product detected by

1978

582


Table 2. Breakdown ofvarious phosphodiesters by the soluble phosphodiesterases ofrat tissuesThe tissues were homogenized in 9 vol. of0.1 M-glycine/NaOH buffer, pH 8.5, in a Potter-type machine. The homogenateswere centrifuged at 1000OOg for 40min and the supernatants collected. Incubation conditions were as described inthe Experimental section with substrates added at 11 mM concentration (method 1).

TissuLiver

Kidney (aKidney (IKidney (r

Small-intColon mlPancreasHeart verSalivary fiSkeletal nStomachSpleenLungBrain (cejEpididymTestisUterusOvaryBlood

Rate of breakdown Rate of breakSubstrate ... of glycerol

cyclic phosphate Bis-(p-nitrophenyl)e ((pmol/min per g) phosphate

24.8 2.724.6

Ldult) 13.0-18.8 (5) 5.113-day-old rat) 14.7 5.8iewborn rat) 3.2

2.3estine mucosa 16.1ucosa 7.4 4.0

11.0 3.4itricle 6.5 3.8gland 6.4 4.9nuscle 4.7 3.3mucosa 3.4

3.4 4.02.9 4.4

rebrum) 1.5 3.9lis 3.6

1.9 4.82.42.30

(down (%K of that for glycerol cyclic phosphate)

2': 3'-CyclicAMP

3.9

6.18.4

7.07.94.09.82.4

15.011.5

128

9.7

3': 5'-Cyclic p-Nitrophenyl ester ofAMP thymidine3'-phosphate

5.0 0.9

1.41.5

1.21.00.93.5

47.6

8.812.8

4.56.27.65.74.0

15.918.7

514

12.5

Table 3. Purification of soluble glycerol c

phosphodiesterasefrom pig kidneySpecific activity

Purification (pmol/min per

step mg of protein)(1) Original supernatant 0.188(2) 42-50%-acetone 1.34

precipitation(3) DEAE-Sephadex 3.42

A-50 column(4) Sephadex G-150 15.2

column(5) Hydroxyapatite 91

column(6) Step 3 repeat 90.5(7) Step 4 repeat 214(8) Step 5 repeat 322

paper ionophoresis was glycerol phorpreparations gave some Pi, presumabl3phosphomonoesterase activity. A so

glycerol phosphate produced by enzyrof sn-glycerol 1:2-cyclic phosphate bydiesterase was examined with glyceidehydrogenase (EC 1.1.1.8) (Baldwin1968), and this indicated that 98% w

3-phosphate. Synthetic rac-glycerol 1:phate gave a glycerol phosphate th,

Vol. 173

yclic phosphate 53 % sn-glycerol 3-phosphate when subjected to de-cortex gradation with glycerol phosphate dehydrogenase.Purifi- This glycerol phosphate reacted with lithiumcation Yield periodate at pH 7.5 producing 1 mol offormaldehyde/(fold) (%) mol of glycerol phosphate, whereas authentic glycerol

I 100 2-phosphate produced no formaldehyde under the7.1 22.7 same conditions. These results indicate that no

glycerol 2-phosphate had been produced during the18.1 5.2 enzymic hydrolysis of the glycerol cyclic phosphate

and that the phosphodiesterase exclusively acts on80.5 5.0 the cyclic phosphate ring at its point of attachment

485 4.4 to the C-2 position of the glycerol moiety. Whenrac-butyrylglycerol cyclic phosphate was hydrolysed

483 2.3 by the enzyme, butyrylglycerol phosphate was the1140 1.6 only product identified by paper ionophoresis.1710 0.12 When 3': 5'-cyclic AMP was used as substrate, it

was shown that 5'-AMP was the only product. With2': 3'-cyclic AMP, both 3'-AMP (69-72 %) and

sphate. Crude 2'-AMP (28-31 %) were formed. In additionaly by secondary experiments we found that the same products inolution of the a similar ratio were formed by a supernatant of amic hydrolysis rat intestinal-mucosa homogenate; Whitfield et al.the phospho- (1955) reported that the main product formed by

rol phosphate calf intestinal phosphodiesterase was 3'-AMP. On the& Cornatzer, other hand we found that rat brain particulatevas sn-glycerol fraction gave 2'-AMP almost exclusively, in agree-:2-cyclic phos- ment with the results of Drummond et al. (1962) forat assayed as bovine brain.

583


Molecular weight

The purified enzyme was applied with proteinstandards of known molecular weight (cytochrome c,ribonuclease, trypsin inhibitor, whale myoglobin,ovalbumin, bovine serum albumin, calf intestinealkaline phosphatase) to a column ofSephadex G- 1 50and eluted with 0.02M-NaOH/glycine buffer, pH 8.5.Enzymic activities and A280 were measured incollected fractions. Plots of molecular weight againstelution volume (Andrews, 1967) gave the phospho-diesterase an approximate mol.wt. of 35500.

General properties of enzyme

The purified enzyme (1400-fold) had a Km of7.2mMagainst rac-glycerol 1:2-cyclic phosphate whenassayed by method 2. A broad pH optimum of 7.4was recorded in 0.07M-imidazole/HCl buffers (method1) and 7.5 in 0.1 M-glycylglycine buffer (methods 2and 3). A slightly lower pH optimum (6.9) wasobserved in a series of 0.1 M-pfi'-dimethylglutaratebuffers (methods 2 and 3).Under the conditions of assay, the hydrolysis of

substrate was practically linear with time up to 30minand proportional to protein concentration up to0.075,ug of protein (1400-fold-purified enzyme) inthe standard assay. Assays carried out by method 1departed more from the ideal than methods 2 and 3,presumably because the Pi produced during thedetermination of phosphodiesterase activity withalkaline phosphatase acted as an inhibitor (see below).This inhibition was partially relieved by the Mg2+present in the assay medium.

Stimulatory action of reducing agents and Fe2+ ions

The partially purified enzyme was substantiallybut variably (61-150%) stimulated by the addition ofdithiothreitol, the effect being maximal at 10mm(Fig. 1). The stimulation of the highly purified(1400-fold) enzyme tended to be less, but on aging thestimulation by dithiothreitol increased. Cysteine and2-mercaptoethanol were almost as effective asdithiothreitol. Ascorbate produced a small stimu-lation of the reaction (Fig. 1) and when added withdithiothreitol there was an additive effect.

Fe2+ cations also produced an additional stimu-lation of the enzymic activity already stimulated byoptimal concentrations of dithiothreitol and ascorb-ate. An effect was observed at concentrations aslow as 5AM, but maximal stimulation occurred at0.3mM (155% of control); above this concentrationprecipitation of Fe(OH)2 was observed and theeffectiveness declined. In the absence of the otherreducing agents, the stimulatory effect of the Fe2+cations was markedly less. Fe3+ ions could besubstituted for Fe2+ without loss of activity; pre-

180

0160 0

140

120

0

100

20X

0 10 20 30[Reducing agentl (mM)

Fig. 1. Stimulation ofglycerol cyclic phosphate hydrolysisby various reducing agents

Incubation conditions were as described in theExperimental section (method 1): 1,ug of 50-fold-purified enzyme with no reducing agents addedhydrolysed 7% of the substrate (11 mM) in 30minincubation at 380C. Reducing agents: *, ascorbate;o, dithiothreitol; al, ascorbate plus dithiothreitol;A, 2-mercaptoethanol; A. cysteine; U, thioglycollate.

sumably these became reduced to the Fe2+ form by thedithiothreitol/ascorbate mixture. Mn2+, Ni2+, Co2+and Cu2+ cations failed to produce any stimulationof the reaction.

Substrate specificity

Table 4 shows the specificity of the phospho-diesterase when tested against a variety of phospho-diester substrates. rac-Glycerol 1: 2-cyclic phosphateand its butyryl ester were attacked most readily. Ithas been indicated under 'Products of the enzymicreaction' that the phosphodiesterase 'shows noabsolute specificity towards enantiomorphs ofglycerol cyclic phosphate; sn-glycerol 1:2-cyclicphosphate was readily hydrolysed to completion.

2': 3'-Cyclic AMP was hydrolysed at about one-tenth of the rate of glycerol cyclic phosphate, andthere was no significant change in the relative ratesof attack on these two substrates during a 1700-foldpurification. 3': 5'-Cyclic AMP was hydrolysed atapproximately the same rate as 2': 3'-cyclic AMPby the original kidney supernatant, but the activitytowards this substrate fell to about 50-70% of theoriginal during purification (Table 4). 3': 5'-CyclicGMP was attacked minimally by the phospho-diesterase, and inositol 1:2-cyclic phosphate not atall. The p-nitrophenyl ester of thymidine 3'-phos-

1978

584


Table 4. Substrate specificity ofglycerol cyclic phosphate phosphodiesteraseIncubation conditions were as described in the Experimental section (method 1) with ascorbate, dithiothreitol andFe2" added. Substrates were added atlI mM concentration: 15-20% of the glycerol cyclic phosphate was broken downin 30min with the various enzyme preparations used. Results are expressed as percentages of the rate of breakdown ofglycerol cyclic phosphate. Degradation of less than 0.5% of the activity towards glycerol cyclic phosphate was shownwith p-nitrophenyl ester of thymidine 5'-phosphate, and with inositol 1:2-cyclic phosphate, glycerophosphocholine,glycerophosphoethanolamine, glycerophosphoinositol, phosphatidylcholine, phosphatidylinositol, ATP, NADH,PP1 and ADP.

Pig kidney enzyme activity(% of rate of breakdown of glycerol cyclic phosphate)

SubstrateGlycerol cyclic phosphateButyrylglycerol cyclic phosphate2': 3'-Cyclic AMP

3': 5'-Cyclic AMP

Kidney supernatant Purified 16-fold100 100

9111.6 13

9.8

3': 5'-Cyclic GMPp-Nitrophenyl ester of thymidine 3'-phosphateBis-(p-nitrophenyl) phosphate

2.71.14.7

6.0

1.40.113.6

Purified 320-fold100581612.5 (purified 1700-fold)5.47.1 (purified 1700-fold)1.20.14.0

Table 5. Effect of various substances on the action ofpurified phosphodiesterase on different phosphodiester substratesSubstrate concentration was 11 mM unless otherwise stated. Incubation conditions were as described in theExperimental section (method 2, except that method 3 used for inhibition by P,). The enzyme concentration wasadjusted so that 15-20% of substrate was broken down in 30min.

Phosphodiester substrateGlycerol cyclic phosphate

3': 5'-Cyclic AMP

Glycerol cyclic phosphate

3': 5'-Cyclic AMP

2': 3'-Cyclic AMPBis-(p-nitrophenyl phosphate)Glycerol cyclic phosphate (7mM)

3': 5'-Cyclic AMP (7mM)2': 3'-Cyclic AMP (7 mM)Bis-(p-nitrophenyl) phosphate (7mM)

* (NH4)2Fe(SO4)2 present in assay.

Substance added andconcentration (mM)EDTA 0.05

1.010.01.0

10.0MgCl2 0.25

5101125

121212

Pi 1310101010

Purification of enzyme(fold)

1400140020

140020

14001400

20 (enzyme dialysed 11 days)1400

20 (enzyme dialysed 11 days)

202020202020

Activity(% of original activity)

101103

93,* 959094

104115102

1 108114

115, 113, 128, 1281141055934

17, 1212,25

1445

phate, a substrate of phosphodiesterase II, was slowlyattacked by the original kidney supernatant, but thisactivity was lost during purification. Bis-(p-nitro-phenyl) phosphate was hydrolysed by the enzymeat about 4% of the rate of glycerol cyclic phosphateand this activity ratio did not change duringpurification. All other phosphodiesters tested werehydrolysed at rates of less than a few per cent of thatof glycerol cyclic phosphate.

Vol. 173

Effect of bivalent metal ions and EDTA

Apart from Fe2+ ions the hydrolysis of glycerolcyclic phosphate was only minimally stimulatedby any other metal ions tested. Mg2+ stimulateda 20-fold purified enzyme, dialysed for 11 daysagainst 0.1 M-glycine buffer, pH 8.5, by 2% at 10mMand 8% at 12mm (assay method 3). With a purerenzyme (1700-fold purified) and in the absence of

585


added Fe2+ ions the stimulation was 4% at 0.25mMand 15% at 5mM (assay method 2) (Table 5). With3':5'-cyclic AMP as substrate the stimulation byMg2+ tended to be somewhat higher (Table 5).Ca2+ (10mM) did not stimulate glycerol cyclic

phosphate hydrolysis, nor did Co2+, Ni2+, Mn2+ orCu2+ when tested at 0.3mm. Zn2+ (1 mm) inhibitedthe hydrolysis by 18 %.EDTA had little effect on the basic phospho-

diesterase activity observed in the absence of addedFe2+ ions. With a highly purified enzyme preparation(1400-fold), addition of 0.05mM-EDTA gave anactivity that was 101 % of control, and I mm gave103%. With a less-pure enzyme preparation (20-fold purified) dialysed for 11 days. lOmM-EDTA gaveactivities that were 93-95% of the control (Table 5).

Inhibitors

Theophylline produced some inhibition of thephosphodiesterase acting against both glycerol cyclicphosphate and 3': 5'-cyclic AMP substrates, andthis was also true for o-phenanthroline (Fig. 2). Thecharacteristics of the inhibition produced bytheophylline and o-phenanthroline were similar forthe hydrolysis of both these substrates and quiteunlike that of a brain supernatant preparationhydrolysing 3': 5'-cyclic AMP (Fig. 2). It can becalculated from the results given in Tables 2 and 4that the present glycerol cyclic phosphate phospho-diesterase would contribute at most a few per cent ofthe 3': 5'-cyclic AMP breakdown catalysed by thebrain supernatant, and therefore it is likely thatalmost all would occur through the specific cyclicAMP phosphodiesterase (EC 3.1.4.17), which isenriched in brain tissue.The phosphodiesterase was markedly inhibited by

P1 (Table 5); 10mM lowered the activity againstglycerol cyclic phosphate and 2': 3'- and 3': 5'-cyclicAMP to a low value. In these experiments the enzymicactivity had to be assayed by method 3 because ofthe presence of large amounts of added P1 in theincubation medium. The inhibitory effect of Pi couldbe partially alleviated by the addition of 10mM-Mg2+,which presumably decreases the effective concen-tration of phosphate ions through the formation ofmagnesium phosphate. NaF (10mM) and iodoacetate(10mM) produced a small inhibition of the phospho-diesterase activity against glycerol cyclic phosphatewhen assayed by method 3 (with dithiothreitolomitted), amounting to 23-28% and 20-21 %respectively.The most potent of the inhibitors of the enzymic

hydrolysis of glycerol cyclic phosphate that wereexamined were certain phosphomonoesters. Althoughall phosphomonoesters tested produced some inhibi-tion of the reaction, there was wide variation in theireffectiveness (Table 6). Thus phenyl phosphate and

80

0 2.5 5.0 7.5 10.0ITheophyllinel (mM)

o 100 _(b)

C 80 /

60

40-

20~~~~~

20

0 0.5 1.0 1.5 2.0[o-Phenanthrolinel (mM)

Fig. 2. Comparison of the inhibition by theophylline (a)and o-phenanthroline (b) of the purified phosphodiesterasehydrolysing glycerol cyclic phosphate and 3': 5'-cyclicAMP, and a rat brain supernatant fraction hydrolysing

3': 5'-cyclic AMPThe substrates were used at a concentration of0.4mm. Phosphodiesterase activity was assayed bymethod 2 with the incubation medium containing0.3mM-Fe2+. Symbols: 0, glycerol cyclic phosphate,kidney phosphodiesterase (1500-fold-purified); *,3': 5'-cyclic AMP, kidney phosphodiesterase (1500-fold-purified); a, 3': 5'-cyclic AMP, rat brain super-natant fraction (Table 2).

x-naphthyl phosphate were highly inhibitory, phos-phatidic acid and dibutyryl glycerophosphate andAMP were intermediary and phosphocholine andglucose 6-phosphate were much less effective. Theinhibition produced by phenyl phosphate was testedfor competitiveness. Lineweaver-Burk (1934) plotsindicated that phenyl phosphate was competingvery effectively with the substrate for the activecentre (Fig. 3). Phenyl phosphate itself was nothydrolysed by the enzyme preparation. The phospho-diesterase was inhibited by its hydrolysis product,glycerol 1-phosphate, but the effectiveness of thisas an inhibitor was low compared with some of theother phosphomonoesters (Table 6). The hydrolysisof 3': 5'-cyclic AMP by the enzyme was also

1978

586


Table 6. Inhibition of glycerol cyclic phosphate phospho-diesterase by various phosphomonoesters

The activity was measured by using method 3(see the Experimental section) and glycerol cyclic[32P]phosphate. The phosphodiesterase had beenpurified 1500-fold. The glycerol 1-[32P]phosphateproduced was separated by ionophoresis and itsradioactivity determined.

Sul

InhibitorPhenyl phosphate

a-Naphthyl phosphate

Methyl phosphateEthyl phosphatePhosphatidic acidDibutyrylglycerol

phosphatePyridoxal phosphateCetyl phosphatep-Nitrophenyl

phosphate5'-AMP

2'-AMP3'-AMP5'-CMPGlycerol 1-phosphate

Glycerol 2-phosphateATPPhosphocholineGlucose 6-phosphate

cbstrate Inhibitor:oncn. concn.(mM) (mM)11 0.25

0.451.0

11 0.250.45

11 0.2511 0.2511 0.4511 0.45

11 0.2511 0.450.4 5.511 9.011 1.511 9.00.4 9.00.4 9.00.4 9.0

11 1.511 5.011 10.011 20.00.4 5.00.4 10.00.4 20.00.4 20.00.4 9.00.4 9.00.4 9.0

Inhibition

5868 (62)*81516434 (34)*3443 (49)*40

1429958848769075681916253441576640341828

* Inhibition with 3': 5'-cyclicAMP (11 mM) as substrate.

inhibited by phenyl phosphate, phosphatidic acidand methyl phosphate to an extent approximatelyequivalent to that observed with glycerol cyclicphosphate as substrate (Table 6).

Discussion

The phosphodiesterase studied and substantiallypurified in the present investigation appears to bedifferent from any in mammalian tissue previouslycharacterized. Many phosphodiesterases in suchtissues are membrane-bound, active at an alkaline pHand require added bivalent metal ions, usually Mg2+or Ca2+, for full activity. The present enzyme appearsto be substantially soluble, it has a neutral pHoptimum and is not inhibited by EDTA. These

Vol. 173

,. I,,30.40.50.60.7 0.80.9 1.0 1.1 1.2 1.3

I/ISI (mM-')Fig. 3. Lineweaver-Burk (1934) plots for the inhibition ofglycerol cyclic phosphate phosphodiesterase by phenyl

phosphateThe phosphodiesterase was determined by usingmethod 3 with glycerol cyclic [32P]phosphatesubstrate. Enzyme was 1500-fold purified, assayed ina reaction mixture of 90,ul total volume. Symbols:A, no inhibitor present; *,0.03 mM-phenyl phosphate;a, 0.2mM-phenyl phosphate.

properties are more equivalent to those displayed bylysosomal phosphodiesterase II (Flanagan&Zbarsky,1977), but the latter can readily attack the p-nitrophenyl ester of thymidine 3'-phosphate (Razzell,1961; Erecin'ska et al., 1969), a substrate not hydro-lysed by the present purified enzyme. In conformitywith this observation the highest activity displayedtowards the p-nitrophenyl ester of thymidine3'-phosphate in simple extracts of various rat tissueswas found in the spleen (Table 2), whereas theactivity towards glycerol cyclic phosphate was highestin the liver. The ratio between the rates of hydrolysisof the two substrates varied over 50-fold betweenthe two tissues, again indicating that the substratesare broken down by different enzymes.A soluble phosphodiesterase active at neutral

pH in the presence of EDTA has been purifiedfrom the tobacco plant (Shinshi et al., 1976).However, unlike the present enzyme this phospho-diesterase readily attacked the p-nitrophenyl esterof thymidine 3'-phosphate. Its preferred substrate isbis-(p-nitrophenyl) phosphate, a phosphodiester thatis attacked by our enzyme at about 4% of the rate ofglycerol cyclic phosphate. We have measured thehydrolysis of glycerol cyclic phosphate by extracts ofvarious plants, including tobacco, and found thatalthough this substrate was hydrolysed, bis-(p-nitrophenyl) phosphate was broken down at a fasteror equal rate.The ability of the present enzyme preparation to

attack enantiomorphs of glycerol cyclic phosphateis in marked contrast to D-myo-inositol 1: 2-cyclicphosphate 2-phosphodiesterase, which we have

587


shown is specific for the D-enantiomorph of thesubstrate (Dawson & Clarke, 1972). Although themaximum rate of hydrolysis occurs with glycerolcyclic phosphate and its butyryl ester, the enzymepreparation can also attack 2': 3'-cyclic AMP,3': 5'-cyclic AMP and bis-(p-nitrophenyl) phosphateat substantial rates. The evidence available suggeststhat these latter phosphodiesters are being hydrolysedby the same active centre that is responsible for theactivity towards glycerol cyclic phosphate. The ratiobetween the rates of hydrolysis of glycerol cyclicphosphate and 2': 3'-cyclic AMP or bis-(p-nitro-phenyl) phosphate does not change on purification,whereas the relative activity towards 3': 5'-cyclicAMP is decreased slightly after a 1700-foldpurification from the supernatant. This does notnecessarily imply that the present enzyme when purecannot hydrolyse 3': 5'-AMP; the most likelyexplanation is that the original supernatant containscytoplasmic 3': 5'-cyclic AMP phosphodiesterase(EC 3.1.4.17) and sufficient Mg2+ for this to showsome activity towards 3':5'-cyclic AMP under theconditions of the assay. In contrast with the presentenzyme, the main mammalian enzyme that hydrolyses2': 3'-cyclic AMP (EC 3.1.4.16) appears to be mem-brane-bound and of low activity in kidney tissue(Drummond et al., 1962), although soluble phospho-diesterases that hydrolyse this substrate have beenreported.The probability that the glycerol cyclic phosphate

phosphodiesterase also attacks 2': 3'- and 3': 5'-cyclicAMP and bis-(p-nitrophenyl) phosphate is alsosupported by the lack of any requirement for Mg2+ oreffect of EDTA when these substrates were beingattacked. In addition, their hydrolysis, like that ofglycerol cyclic phosphate, was markedly inhibitedby Pi ions and phosphomonoesters. The hydrolysisof 3': 5'-cyclic AMP also showed the same pHoptimum (7.3) and was inhibited by theophylline ando-phenanthroline in a manner similar to the hydro-lysis of glycerol cyclic phosphate and distinct fromthat of specific 3': 5'-cyclic AMP phosphodiesterase.In addition, the ratio between bis-(p-nitrophenyl)phosphate and glycerol cyclic phosphate hydrolysisis reasonably constant in the supernatants fromvarious tissues of the rat (Table 2), although it islow in the liver. In additional experiments (N. G.Clarke & R. M. C. Dawson, unpublished work) wehave found that in sheep tissues proportionallymuch more (2.5-3.0 times) of the bis-(p-nitrophenyl)phosphate is decomposed by the supernatant fractioncompared with the rat tissues, which could indicatethat more than one type of phosphodiesterase isinvolved in the hydrolysis of this substrate.The complex kinetics of 3': 5'-cyclic AMP hydro-

lysis bymany tissue extracts, including kidney (Jard &Bernard, 1970), has been ascribed to multiple formsoftheenzyme (e.g. high- and low-Km forms). However,

unlike the present enzyme, these forms all requirebivalent metal ions for activation and are probablymultiple molecular aggregates of 3': 5'-cyclic nucleo-tide phosphodiesterase (Pichard & Cheung, 1976).Buchnea (1973) quoted preliminary experiments

that suggested that glycerol 1: 3-cyclic phosphatecould act as a substrate for purified 3': 5'-cyclicnucleotide phosphodiesterase. In additional experi-ments we have tested the commercially purifiedenzyme (Boehringer, 0.15 unit/mg) from ox heart andfound that rac-glycerol 1: 2-cyclic phosphate washydrolysed at about 3% of the rate of 3': 5'-cyclicAMP cleavage, quite different from the phospho-diesterase studied in the present investigation.Morishima (1974) has isolated a purified phospho-diesterase from the larvae of the silkworm that canattack both 2': 3'- and 3':5'-cyclic AMP in thepresence of EDTA. The former substrate washydrolysed at a rate that was 10 times greater thanfor the latter, which contrasts markedly with thepresent enzyme.The activation of the enzyme by reducing agents

such as dithiothreitol, cysteine, ascorbic acid andFe2+ ions could be produced by the reduction ofdisulphide bonds in the enzyme structure, oralternatively by these changing the polymeric form ofthe enzyme. Although a number of dehydrogenasescontain or have iron intimately associated with them,the stimulation of the phosphodiesterase by Fe2+ions is more likely to be associated with theirreducing capacity. Thus compared with such enzymesthe phosphodiesterase is insensitive to o-phenan-throline inhibition, which would suggest that thephosphodiesterase activity has no absolute require-ment for iron. A few reports have occurred describingthe activation of EDTA-insensitive 3': 5'-cyclic AMPphosphodiesterases by Fe2+ or Fe3+ (Okabayashi &Ide, 1970; Brewin & Northcote, 1973).The marked competitive inhibition observed with

certain phosphomonoesters is likely to be due to thelatter having a higher affinity for the active centreof the enzyme than the substrate itself, althoughno phosphatase action occurs. That this affinitycan exist is perhaps indicated by the productinhibition of the activity by glycerol 1-phosphate,suggesting that the dissociation of the enzyme-product complex may be of significance in deter-mining the Km value.

It is not known whether glycerol cyclic phosphateis the main physiological substrate of the enzyme.Although this phosphodiester has so far only beenshown to be produced from glycerophosphocholineand glycerophosphoethanolamine through the actionof glycerophosphinicocholine diesterase (Clarke &Dawson, 1976), it is conceivable that other enzymesforming glycerol phosphate might also producesmall amounts of the cyclic derivative under certainconditions. In preliminary experiments (N. G.

1978

588

rac-GLYCEROL 1:2-CYCLIC PHOSPHATE 2-PHOSPHODIESTERASE 589

Clarke & R. M. C. Dawson, unpublished work) wehave detected low concentrations of glycerol cyclicphosphate in several mammalian tissues. The cyclicderivative can act as a precursor of tissue glycero-phospholipids and triacylglycerol (Buchnea, 1973),but presumably only after hydrolysis to glycerolphosphate.

References

Aldridge, W. N., Emery, R. C. & Street, B. W. (1960)Biochem. J. 77, 326-327

Andrews, P. (1967) Lab. Pract. 16, 851-856Baldwin, J. J. & Cornatzer, W. E. (1968) Biochim. Biophys.Acta 164, 195-204

Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-471Brewin, N. J. & Northcote, D. H. (1973) Biochim. Biophys.Acta 320, 104-122

Brockerhoff, H. (1963) J. Lipid Res. 4, 96-99Buchnea, D. (1973) Lipids 8, 289-294Butcher, R. W. & Sutherland, E. W. (1962) J. Biol. Chem.

237, 1244-1250Carter, C. E. (1950)J. Am. Chem. Soc. 72, 1466-1471Clarke, N. & Dawson, R. M. C. (1976) Biochem. J. 153,

745-747Clarke, N. G., Hazlewood, G. P. & Da-wson, R. M. C.

(1976) Chem. Phys. Lipids 17, 222-232Dawson, R. M. C. & Clarke, N. (1972) Biochem. J. 127,

113-118Dawson, R. M. C., Hemington, N. & Davenport, J. B.

(1962) Biochem. J. 84, 497-501

Drummond, G. I., Iyer, N. T. & Keith, J. (1962) J. Biol.Chem. 237, 3535-3539

Ereciniska, M., Sierakowska, H. & Shugar, D. (1969)Eur. J. Biochem. 11, 465-471

Flanagan, P. R. & Zbarsky, S. H. (1977) Biochim. Biophys.Acta 480, 204-218

Haziewood, G. P. & Dawson, R. M. C. (1975) J. Gen.Microbiol. 89, 163-174

Jard, S. & Bernard, M. (1970) Biochem. Biophys. Res.Commun. 41, 781-788

Khorana, H. G., Tener, G. M., Wright, R. S. & Moffatt,J. G. (1957) J. Am. Chem. Soc. 79, 430-436

Lineweaver, H. & Burk, D. (1934) J. Am. Chem. Soc. 56,658-666

Lowry, 0. H., Rosebrough, N. J., Farr, A. L. &Randall, R. J. (l951)J. Biol. Chem. 193,265-275

Maruo, B. & Benson, A. A. (1959) J. Biol. Chem. 234,254-256

Morishima, 1. (1974) Biochim. Biophys. Acta 370, 227-241Okabayashi, T. & Ide, M. (1970) Biochim. Biophys. Acta

220, 116-123Olafson, R. W., Drummond, G. I. & Lee, J. F. (1969)

Can. J. Biochem. 47, 961-966Pichard, A. L. & Cheung, W. Y. (1976)J. Bio. Chem. 251,

5726-5737Razzell, W. E. (1961)J. Biol. Chenm. 236, 3028-3030Shinshi, H., Miwa, M., Kato, K., Noguchi, M.,

Matsushima, T. & Sugimura, T. (1976) Biochemistry15, 2185-2190

Whitfield, P. R., Heppel, L. A. & Markham, R. (1955)Biochem. J. 60, 15-19

Vol. 173

rac-glycerol 1:2-cyclic phosphate 2-phosphodiesterase, a new

Documents