Soluble and particulate forms of phosphoinositide phosphodiesterase in ox brain

324

BIOCHIMICA

ET BIOPHYSICA

ACTA

BBA 56074

SOLUBLE

AND PARTICULATE

PHOSPHO~I~ST~RASE

K. M. W. XEOUGH department (Received

OF PHOSPH~INOSITID~

and W. THOMPSON

of Biochmnistry, University February

FORMS

IN OX BRAIN

of Toronto, Toronto 5, Ontario (Canada)

Sth, 1972)

SUMMARY I. Phosphoinositide phosphodiesterase was recovered in both supernatant and well-washed particulate fractions of homogenates of beef brain. z. The membrane-bound enzyme was solubilized with z M KC1 and both supernatant and KCI-extracted enzymes were partially purified by ammonium sulphate fractionation and adsorption to and desorption from calcium phosphate gels. 3. Both enzymes could hydrolyse tri-, di- and monophosphoinositides and in each case the products were identified as diglycerides and inositol phosphates. 4. The optimum pH for monophosphoinositide hydrolysis was 5.S, that for diphosphoinositide 6.0, and for triphosphoinositide, 7.2-7.8, with both supernatant and KCl-extracted enzymes. Hydrolysis of all three lipids was stimulated by cetvltrimethylammonium bromide, when added in suitable molar proportions, characteristic for each substrate. Ca2+ was a better activator of mo~lophosplloinositide hydrolysis than the cationic detergent. Calculation of apparent k’, values indicated that, with both enzymes, there were no marked differences in afhnity for any of the inositide substrates. Heat denaturation of both enzyme fractions was almost identical with respect to polyphosphoinositide hydrolysis but with nlo~lopl~ospl~oi~os~tideas substrate the supernatant enzj’me appeared to be slightly more labile than the KClextracted enzyme.

INTRODUCTION

It was shown recently1 that brain tissues in vitro have a high capacity for splitting phosphoinositides into diglycerides and inositol phosphates. With triphosphoinositide as substrate, homogenates of brains from r-day-old rats showed low activity but activity increased rapidly in older rats corresponding to the age period prior to and during myelination. Substantial enzymatic activity was located both in the supernatant fraction and in the well-washed particulate fraction of brain homogenates from rats at all ages. This report describes a further investigation of both particulate and supernatant enzyme activities. The partic rlate fraction has been solubilized by

PHOSPHOINOSITIDE

PHOSPHODIESTERASES

32.5

IN BRAIN

salt extraction and a limited degree of enzyme purification attained. Both enzymes have been found to hydrolyse monophosphoinositide, ~phosphoinosi~de and triphosphoinositide and some characteristics of the enzymes are described. METHODS

Preparation of brai% extracts Beef brain obtained fresh from the slaughterhouse was transported to the laboratory on ice. The tissue was homogenized in 4 vol. IO m&I Tris-HCl buffer, pH 7.2, at 4 “C in a Potter-Elvehjem homogenizer, then centrifuged for I h at IOOOOO xg. The pellet was resuspended in the buffer in a volume equal to that of the original homogenate and sedimented again. This was repeated twice. The washed pellet was suspended in z M KC1 in IO mM Tris-HCl, pH 7.2, and stirred for 14-16 11at 4 “C. Buffer was then added to dilute the KC1 to I M and the suspension centrifuged for I h at zoo ooo xg to yield a salt extract and a residual pellet. Samples of the original homogenate, high-speed supernatant, washes, salt extract and residual pellet were then dialysed extensively at 4 “C against frequent changes of IO mM Tris-HCl, pH 7.2. Any precipitate which developed on dialysis was centrifuged down and discarded. The dialysed preparations were stored at -15 “C. Purification of szlpernatan,t and KU-sohble fractions Substantial enzyme activity was found in the supernatant fraction of the brain homogenate and in the salt extract, derived from the particulate fraction. The enzymes in these fractions were partially purified as follows: fractions precipitating between 25 and 55 y0 saturated ammonium sulphate were prepared by adding saturated ammonium sulphate solution in IOO mM Tris-HCI, pH 7.2, at room temperature. The 25-55 y0 cut was dissolved in IO mM Tris-NC1 buffer, pH 7.2, and dialysed overnight at 4 “C against the same buffer. The dialysed fractions were subjected to ten cycles of freezing at -70 “C and thawing at 37 “C and the small precipitates that formed were centrifuged down and discarded. The enzymes were adsorbed on calcium phosphate gel (1.4 g of gel per g of protein) for 30 min at 4 “C and then the gel-protein complex was extracted for 4 h with a solutions containing 0.8 M ammonium sulphate and 0.25 M sodium acetate in 25 mM Tris-HCI buffer, pH 7.2. The supernatant obtained after centrifuging this suspension was adjusted to 70% saturation with respect to ammonium sulphate and after 30 min a precipitate was centrifuged down. The precipitate was dissolved in IO mM Tris-HCl, pH 7.2 and dialysed overnight against the same buffer at 4 “C and then stored at -15 “C. Enzyme assays Hydrolysis of triphosphoinositide was assayed in 0.5 ml of the following mixture: I n&I triphosphoinositide, 1.2 mM cetylt~methylammo~um bromide, 50 mM 2-(N-morpholino)ethanesulphonic acid-50 mM N-z-hydroxymethyl piperazine-N-2ethanesulphonic acid buffer (adjusted to pH 7.2 with NaOH) and enzyme preparation (0.01-0.2 mg protein). Incubations were for IO min at 37 “C. Incubation conditions were the same for diphosphoinositide but with LO mM cetyltrimethylammonium bromide and the buffer was adjusted to pH 6.0. Monophosphoinositide (I mm) was hydrolysed in 50 mM 2-(N-morpholino)ethanesulphonic acid-50 mM N-z-hydroxyBiocBim. Biophys.

Acta,

270

(x972) 324-336

K. M. W. KEOUGH, W. THOMPSON

326

methyl piperazine-N-z-ethanesulphonic acid buffer, pH 5.8, usually in the presence of I mM CaCl, and with incubation times of 20-30 min. After incubation the tubes were placed in an ice bath and approximately IOO mg activated charcoal was added (except in the case of monophosphoinositide) followed by 0.2 ml of 5% bovine serum albumin and 1.3 ml IO”/~ perchloric acid. Phosphate analyses in the acid-soluble filtrates were performed as described previouslyl. Pi released from diphosphoinositide and triphosphoinositide was low, generally less than 5 “/Aof the total P released when crude brain extracts were assayed, and was negligible with more purified extracts. Pi release in monophosphoinositide assays was not detectable. The release of combined phosphate was used as an assay of enzyme activity in most experiments, but it was observed that the reaction rate decreased slightly with time. The incubation times, usually 10-30 min, depending on the substrate, were a compromise, chosen so as to give as little deviation from linearity as possible, but were long enough to allow release of an amount of P that could be conveniently measured. For identification of reaction products incubation mixtures were scaled up IO-fold and incubation times were increased to 30 min. The contents of each tube were divided into two portions, hydrolysis products as described chloroform-methanol (I : I, v/v). paper and the clear filtrate was

One portion was taken for analysis of previouslyl. To the other portion was The suspensions were filtered through dried in vacua. The lipid residue was

water-soluble added IO vol. defatted filter redissolved in

chloroform and applied to a shoit silicic acid column. The column was eluted with 20 ml chloroform followed by 20 ml methanol to give neutral lipid products and unhydrolysed substrates respectively. Inositol, acyl ester and glycerol assays were done as beforel. Tri- and diphosphoinositides were prepared as ammonium salts by a modification3 of the procedure of Hendrickson and Ballou4. Monophosphoinositide, prepared from pig liver, was purchased from Serdary Research Laboratories, London, Ontario. The monophosphoinositide was dissolved in chloroform-methanol (2: I, v/v) and shaken vigorously with 0.2 vol. 0.5 M HCl. The upper phase was removed and the lower phase was then shaken with chloroform-methanol-water (3:48:47, by vol.) containing I M ammonium acetate. This was repeated with “upper phase” solvent without the ammonium salt. The monophosphoinositide as ammonium salt ran as a single spot on thin-layer chromatography but in two preparations a slower moving spot with the mobility of lysophosphoinositide

was observed,

which accounted

for 4.6 and 7.00/d of the total

phosphate. The fatty acids of the substrates were analysed by gas-liquid chromatography+. Brain diphosphoinositide and triphosphoinositide were almost identical, containing 26-27% arachidonic acid and 36639% stearic acid as the main components. The pork liver monophosphoinositide was also rich in arachidonate (28.60/,) but had higher levels of stearate (50.4%), and lower levels of oleate, 8% as compared with 14-16% oleate in the polyphosphoinositides. RESULTS Sohble

and particdate enzyme actiaity Previous results1 had indicated that, with triphosphoinositide phosphodiesterase activity was associated with soluble and particulate

Biochim.

Biophys.

Acta,

270 (1972)

324-336

as substrate, fractions from

PHOSPHOINOSITIDE

PHOSPHODIESTERASE

327

IN BRAIN

sucrose homogenates of rat brain. As shown in Table I, a similar distribution was obtained with homogenates from beef brain, prepared in hypotonic buffer. A substantial proportion of the activity was found in the IOO ooo x g supernatant. Repeated washing of the IOOOOO xg pellet released decreasingly small amounts of enzyme (Washes x-3, Table I), leaving considerable activity in the washed pellets. It would be expected that any loosely-bound or trapped cytoplasmic enzyme would be released during this procedure and that the activity remaining represented a relatively firmly-attached membrane component. About half of the membrane-associated activity could be released by extraction of the pellets with 2 M KC1 solution (Table I). It is shown TABLE

I

PHOSPHOINOSITIDE

PHOSPHODIESTERASE

Fraction

Homogenate

ACTIVITY

XX SOLUBLE

AND

PARTICULATE

FRACTIONS

FROM

BEEF

Monofihosphoinositide

_.-Diphos$hoinositide

pnoles Ihydrolysedl g brain per h

~~t~v~t~

pmoles hydrolysed / g brain per h

% of recovered activity

flmo1e.s hydrolysedj g b9ais per k

zXkX$ activity

47.7

-

59.2

-

119.4

-

20.7

;::z

25.2

27.6 IO.5 3.3 0.7 57.9

XiZeved

BRAIN

Tri~hos~hoinositide

roooooXg

supernatant Wash I Wash 2 Wash 3 Washed pellet

7.9 I.4 0.9 27.2

2.4 1.6

46.8

9.6 3.0 0.7 52.8

pellet*

15.X

13.8

52.3 47.7

26.2

X3.5 9.9

7.7 5.7 3.5 56.9

6.0

99.3

% of recovered pellet activity

% of qecoueyea p&et ~t~v~ty

~~~~~~e~ p&et act~v~t~~ KC1 extract* KCl-extracted

45.9

57.6 53-7

56.7 43.3

32.2 24.6

5X.7 48.3

* These fractions were prepared from the washed ~ooooo x g pellet.

also that activity was exhibited with each of the three phosphoinositides as substrate and that distribution was similar in each case. Recoveries from the subfractions were consistently greater (rzo-IGO%) than the activity obtained with the initial homogenates. Possibly the accessibility or availability of the substrates to the enzymes is increased in the individual subfractions. Alternatively the procedures could result in the removal of an inhibitor. Because of unavoidable circumstances considerable time elapsed (1-1.5 h) between removal of the brain from the animals and preparation of the brain extracts. It was therefore possible that the multiple location of enzyme was the result of redistribution in the autolysing tissue. To check this possibility rat brains were processed in the same way without delay between decapitation and preparation of extracts. Table II indicates that enzyme activity was located in both soluble and particulate fractions, although the proportion of activity extracted into KC1 from the washed pellet was greater in rat than in beef brain. Partial pwi@xtion of enzymes Limited degrees of purification of phosphoinositide phosphodiesterases from different sources have been reported previously a~st7.In the present work numerous attempts were made to purify the particulate and soluble ox-brain fractions with only BaOGhim.

Bioghys.

/i&S,

270 (1972) 324-336

32s

Ii. M. W. KEOUGH,

TABLE

II

PHOSPHOINOSITIDE RAT

W. THOMPSOiY

PHOSPHODIESTERASE

ACTIVITY

IN SOLUBLE

AND

PARTICULATE-FRACTIONS

FROM

BRAIN

Fraction

0/Oof recovered activity

I00000 x g supernatant Wash I Wash 2 Wash 3 Washed pellet KC1 extract * * This fraction was obtained

DiPhosphoinositide

Triphosphoinositide

44 21 3

35 I8

0

:

31 I;,, qf pellet activity

32

89

90

from the washed IOOOOOx g pellet

marginally successful results. Table III lists the purification and recoveries of all activities against each of the three inositide substrates when the supernatant and KU-soluble brain fractions were subjected to the procedures described above. The supernatant enzyme was purified only s-10 fold over that of the original homogenate after three successive steps, with about 20% recovery of activity. A somewhat greater degree of purification was obtained with the KCl-soluble fraction, due to the fact that the enzyme when extracted from the particulate fraction with salt solution had a higher specific activity than the supernatant enzyme. Somewhat variable results were obtained with calcium phosphate gels. The recoveries and purification of monophosphoinositide phosphodiesterase after the gel treatment reported in Table III are poor, and may be due to the sensitivity of the hydrolytic reaction to Ca2+ (see Fig. 4). The data show also that recoveries of hydrolytic activity against each of monophosphoinositide, diphosphoinositide and triphosphoinositide were comparable in both enzyme preparations at other stages of purification. These too, were somewhat variable in repeated runs but it is probable that the variations are at least partly due to inherent limitations in the assay procedures rather than real differences in enzyme recoveries and purification. Identijcation qf hydrolysis products Large scale incubations were carried

out with enzyme

preparations,

obtained

by ammonium sulphate fractionation, and the water-soluble and lipid hydrolysis products characterized. The analyses are given in Table IV. The molar ratios of products are consistent with diglycerides being released in each case and stoichiometric amounts of water-soluble inositol mono-, di- and triphosphates from monophosphoinositide, diphosphoinositide and triphosphoinositide respectively. These indicate the presence of phosphodiesterase activity (phospholipase C) in both supernatant and KCl-soluble fractions. Characteristics of the enzymes Previous reports6v8 of pH optima of phosphoinositide phosphodiesterase activity in different brain extracts had shown that activity varied not only with Hf concentration but also with the type of buffer used. In those experiments different types of buffers were employed to cover the pH range 5.0-8.5 and the variations in activity were difficult to interpret. This has been avoided by using z-(N-morpholino)ethaneBiochim. Biophys.

Acta, 270 (1972) 324-336

RECOVERIES

4r.o

40.3

b

10.4

7.8

6.0

38.4 23.1

39.6 17.8

15.5

32.1

36.1

12.2

24.0

9.4 I 7.9

KC1 extract Ammonium sulphate fraction Freeze-thawed fraction Ca#Q)a gel fraction

I

I

35.5 56.8

25.0 19.1

29.2 37.7 15.1

47.4

which is set arbitrarily at I.

40.9 46.0

28.8

52.3

34.9

100

TPI

32.1

32.2

xoo

DPI

55.4

24.7

100

MPI

-_.. Recovery (%)

4x.2

22.3

13.3

I

Relative spec. act.* TPI MPI DPI

Abbreviations: MPI, monophosphoinositide; DPX, diphosphoinositide; TPI, triphosphoinositide. * Specific activity of fraction &moles substrate hydrolysed/min per mg protein) divided by specific activity of homogenate,

3

7.4

8.2

5.2

4.1

39.9

BRAIN

KCI-soluble enzyme

BEEF

Homogenate

FROM

z

$

gel

5.4

2.3

100

TPI

Fraction

PHOSPHODIESTERASES

fraction C+_OQh fraction

5.5

2.5

2.6

supernatant Ammonium sulphate fraction Freeze-thawed

f 43.9

100

100

I

I

Homogenate loooooxg 4.5‘3

DPI

MPI

TPI

MPI

DPl

Recovery ( y’)

Relative spec. act. *

OF PHOSPHOINOSITIDE

fE:

ta g’

AND

Supewkatant enzyme

PURIFICATION

PARTIAL

Fraction

III

TABLE

K. M. W. KEOUGH, W. THOMPSON

330 TABLE IV

PRODUCTSOF HYDROLYSISOF THE PHoSPHoINosITIDEs BY SUPERNATArTASD ENZYMES

FROM

BEEF

E+UyYiZe

Substrate

Acid-soluble Phosphate

Monophosphoinositide Diphosphoinositidc Triphosphoinositide *

K(‘l-SOLUULE

BRAIS

Supernatant KCI-soluble

1.00 I .oo

Supernatant KCI-soluble Supernatant KCl-soluble

$roducts*

I+d

fwoducts *

Glycevol

Inositol

--~- .4 cyl 1,ster I.30

0.g8

I.03

1.07

0.9-t

I.83

2.00

I.01

0.83

1 .CJX

2.00

I.Oj

I.04

I

3.00 3 .oo

1.75

I .02

J.o,j

I.0.j

1.1.5

I.C)8

ss

Molar ratios. Each value is the average of two separate assays.

sulphonic

acid-N-e-hydroxymethyl

piperazine-N-z-ethanesulphonic

acid buffers over

the pH range 5.0-8.2, with constant buffer ion species. The pH optima of the enzyme preparations, taken to the stage of ammonium sulphate precipitation, are shown in Fig. I. The cytoplasmic enzyme catalysed monophosphoinositide hydrolysis at optimum pH 5.8, diphosphoinositide at pH 6.0 and triphosphoinositide at pH 7.2. The KCl-soluble enzyme showed similar pH optima for each of the three inositides. While

a

b

0 5.0

6.0

7.0

6.0

PH Fig. I. pH-activity brain. Substrates:

5.0

6.0

7.0

6.0

PH

curves of (a) supernatant A, monophosphoinositide;

enzyme and (b) KCl-extracted enzyme from beef 0, diphosphoinositide; C, triphosphoinositide.

the pH curves exhibited fairly sharp optima with monophosphoinositide and diphosphoinositide, those for triphosphoinositide showed a broader range extending from approximately 7.0 to 7.8. It was reported6 previously that cetyltrimethylammonium bromide stimulated triphosphoinositide hydrolysis in extracts from acetone powders of brain and that the molar ratio of activator to substrate was critical for optimum activity. This was found to be the case also with the supernatant and KCl-soluble brain fractions. As shown in Fig. z with the KCl-soluble enzyme the optimum concentration of cetyltrimethylammonium bromide increased in a proportional manner with increasing triphosphoinositide concentration and maximum stimulation was obtained with an activator to B&him.

Biophys.

Acta,

270

(1972) 324-336

PHOSPHOINOSITIDE PHOSPHODIESTERASES

331

IN BRAIN

I5

IO

1

P 3 5

I.0

2.0

C~tyltrlmethylammonlum

3-O bromide (mm)

Fig. 2. The relation between substrate levels and optimum concentration of the activator, cetyltrimethylammonium bromide, in the hydrolysis of triphosphoinositide (TPI) by the KCl-extracted enzyme from beef brain. Concentration of triphosphoinositide (mM): 0, 0.4; 0, 0.8; A, 1.0; x, 1.2; A, 1.5.

substrate molar ratio of 1.6-1.8. With the supernatant enzyme the best molar ratios were 1.2-1.4. The data in Fig. 2 were prepared in a form to suit the equation, [S] = ([S] /v) V-K,, which is a linear interpolation of the standard Michaelis-Menten equation. When the data were plotted in linear form according to this equation (Fig. 3) using the conditions of optimum activator:substrate ratios, an “apparent Km” was derived for both soluble and KCl-extracted enzymes, which in each case was 1.6 mM. The term “K,” is used advisedly, and can only be considered as an approximation since the substrates were dispersed rather than in solution and the enzymatic reaction rate was not strictly linear with time. However, since all conditions of assay were maintained as constant as possible, the values do provide some means of comparison. Diphosphoinositide hydrolysis was also stimulated by cetyltrimethylammonium bromide, the most favourable ratios of activator to substrate being 1.6 and 1.0 with two different preparations of diphosphoinositide, and “apparent Km” values were derived in a similar manner. The hydrolysis of monophosphoinositide (I mM) was increased with cetyltrimethylammonium bromide at a concentration of 0.6 mM. However, this activator was less effective than Ca*+, which at I mM stimulated maximally Biochim.

Biophys.

Acta,

270 (1972) 324-336

332

K. M. W. KEOUGH, W. THOMPSON

3 E

0 I-

TPI (mM) )rmolcs hydrolyred /min

x IO’

--I*0

- 2.0 Fig. 3. Kinetic plot of the hydrolysis of triphosphoinositide (TPI) by ( l) supernatant enzyme and (0) KCl-extracted enzyme in the presence of cetyltrimethylammonium bromide. Molar ratios of substrate to activator were as described in text.

both the cytoplasmic and KCl-extracted enzymes (Fig. 4). Table V lists the “apparent K,” values for each of the three inositides determined under different assay conditions for each lipid. The “apparent Km” values were from 0.75 to 1.6 mM suggesting a slightly greater affinity for monophosphoinositide but with no appreciable differences between any of the substrates with either cytoplasmic or salt-extracted enzyme.

IO 6 6 4 2

I

2 Co”+

3

4

5

0.2

0.6

I.0

Cotyltrimethylammonium

(mM1

I.4 bromide (mM)

Fig. 4. Effect of (a) CaCl, and (b) cetyltrimethylammonium bromide on the hydrolysis phosphoinositide by ( l) supernatant enzyme and (0) KCl-extracted enzyme. Biochim.

Bzophys.

Acta,

270 (1972)

324-336

of mono-

PHOSPHOINOSITIDE TABLE

PHOSPHODIESTERASES

333

IN BRAIN

V

CHARACTERISTICS

OF SOLUBLE

AND PARTICULATE

FORMS OF PHOSPHOINOSITIDE

PHOSPHODIESTERASE

Substrate

Enzyme

pH optimum

‘Ii,,,’ (mM)

Maximum activation with I mM substrate

Monophosphoinositide

Supernatant

5.8

0.9

KCl-soluble

5.8

0.75

Supernatant

6.0

1.1

KCl-soluble

6.0

1.1

Supernatant

7.2

1.6

KCl-soluble

7.6

1.6

I mM Ca*+; 0.6 mM cetyltrimethylammonium bromide I mM Ca*+; 0.6 mM cetyltrimethylammonium bromide 1.0 or 1.6 mM cetyltrimethylammonium bromide r.o or 1.6 mM cetyltrimethylammonium bromide I .z-I .4 mM cetyltrimethylammonium bromide I .6-I .8 mM cetyltrimethylammonium bromide

Diphosphoinositide

Triphosphoinositide

Heat treatment

The effect of the three different ranging from 30 to to heat was similar 50 “C and 85-95s

heat treatment on the activities of both enzyme preparations with substrates was examined. Fractions were heated at temperatures 70 “C for 5 min and then cooled in ice and assayed. Susceptibility in all cases, in that there was 2+30% inactivation after 5 min at inactivation at 60 “C. Fig. 5 illustrates the effects of heating the

[:kbb

5 IO I5 min at 50°C

5 IO I5 min at SO’C

5 IO I5 min at 5O’c

Fig. 5. Effect of heat treatment on the ability of (0) supernatant enzyme and (0) KCl-extracted enzyme to hydrolyse (a) triphosphoinositide, (b) diphosphoinositide and (c) monophosphoinositide.

different enzyme preparations at 50 “C for varying periods of time. The inactivation curves with triphosphoinositide and diphosphoinositide as substrate were very similar with both enzymes. Some difference was observed with monophosphoinositide where the cytoplasmic enzyme fraction seemed rather more labile at 50 “C than the KClextracted enzyme (Fig. 5~). The significance of this can not be ascertained at present. It is possible that the different prior treatments of the fractions, (e.g. exposure to 2 M KCl), may be responsible for the apparent variation in heat labilities. DISCUSSION

The phosphoinositides are the only glycerophosphatides for which there is welldocumented evidence of hydrolysis by a phospholipase C in mammalian tissues. Such activity appears to be widely distributed. Extracts of acetone powders of beef brain were shown by Thompson and Dawsona to liberate diglycerides and inositol phosBiochim.

Biophys.

Acta,

270

(1972)324-336

Ii. 1w.W.

334 phates

from triphosphoinositide

and diphosphoinositide,

KEOUGH,

W.

but exhibited

THOMPSOIS

very

little

activity with monophosphoinositide. Monophosphoinositide phosphodiesterase activity was subsequently reported to be present in the soluble fraction of rat brains and in soluble and particulate fractions of guinea-pig braing. Soluble preparations of ox pancreaslO, pig thyroid11 and rat live? also hydrolyse monophosphoinositide and an enzyme partially purified from the cytoplasmic reported’ to hydrolyse monophosphoinositide, inositide. Keough

and Thompson’

recently

fraction of intestinal diphosphoinositide

found that phosphodiesterase

mucosa has been and triphosphocleavage

of tri-

phosphoinositide was a property of both soluble and particulate fractions from sucrose homogenates of rat brain. The membrane associated with this activity was not unequivocally identified, although there was some correlation between the distribution of triphosphoinositide phosphodiesterase and that of 5’-nucleotidase, a marker enzyme for the plasma membrane. The present work extends those observations inasmuch as substantial phosphodiesterase activity is shown to be present in both soluble and particle fractions of ox brain homogenates prepared in hypotonic buffers. The membrane-bound form of the enzyme can be solubilized by the procedure of KC1 extraction outlined above, a step which may be a useful starting point in further enzyme purification. Subfractionation gave greater recoveries of enzyme than was present in the original homogenate, so it is difficult to assess the relative distribution of soluble and particulate activities, but the results in Table I do suggest there is probably as much, if not more, membrane-bound than cytoplasmic activity in these homogenates. However it cannot be determined from the data what relation this distribution in vitro may bear to the situation i?z Go. The soluble and membranebound activities may be in equilibrium in the intact brain, but this may change and redistribution genizing the variance with phodiesterase small amount

may occur as a result of changes in the ionic environment etc. on homotissue. The findings of substantial membrane-bound activity are at those of Friedel et al.@ who reported that monophosphoinositide phoswas mostly in the supernatant of guinea-pig homogenates, although a of activity was associated with the synaptosomal fraction after osmotic

shock treatment, and Harwood and HawthorneI* who found that diphosphoinositide phosphodiesterase activity was essentially all in the cytoplasmic fraction of rat brain synaptosomes. However, in neither of these reports were optimum conditions for the hydrolysis of the substrates determined. Monophosphoinositide hydrolysis9 was assayed at pH 7.0 (compared with our pH optimum, 5.8) in the absence of added Ca2+, was measured in an unfortified buffer at pH 7.0, and diphosphoinositide breakdownI conditions which would indicate very low activities in our enzyme preparations. It is possible that, in unfortified assay media, membrane-bound activity may be particularly underestimated. Monophosphoinositide

phosphodiesterase, however, in non-neural tissues, pancreaslo, intestinal mucosa’, liveP, and thyroidll appears to be essentially a soluble enzyme, recovered in the supernatant fraction of homogenates of these tissues. In general, these have characteristics similar to the brain preparations, with a reported optimum pH in the range 5.2-6.0, and a requirement for Ca2+m.The finding of both membrane-bound and cytoplasmic activities in brain raises the possibility that the enzyme is adsorbed on or integrated in a membrane structure either specific to brain or present in greater amounts in brain than in non-neural tissues.

Biochim.

Biophys. Acta, 270 (1972) 324-336

PHOSPHOINOSITIDE

PHOSPHODIESTERASES

IN BRAIN

335

The question remains whether multiple forms of phosphoinositide phosphodiesterase exist, either different soluble and particulate forms, or possibly separate enzymes with different affinities for the three substrates. With each substrate and with both forms of enzyme the hydrolysis products unequivocally show phosphodiesterase activity splitting the lipids into diglycerides and inositol phosphates. Jungalwala et al.” and Dawson et al.13 have recently made the interesting finding that the soluble monophosphoinositide-splitting enzyme from pig thyroid released diglyceride and mainly a cyclic phosphorylinositol (cyclic inositol I : a-monophosphate) and a small amount of phosphorylinositol, at pH 5.3-5.5. As the pH of the incubation media was raised to 6.8 the proportion of phosphorylinositol liberated increased. Whether the soluble and particulate enzymes from brain also release the cyclic product has not been determined. The procedures used here for isolating and characterizing the watersoluble phosphates would not distinguish between phosphorylinositol and the cyclic derivative. It can be seen from Table III that the supernatant and KCl-extracted enzymes acting on any one substrate display similar behaviour throughout the different steps that yielded a limited degree of purification. Hydrolysis of each substrate has a characteristic optimum pH but the heat labilities of diphosphoinositide and triphosphoinositide phosphodiesterase from both sources were essentially the same. Some differences in heat-denaturation between soluble and salt-extracted enzyme with monophosphoinositide as substrate were observed, which could indicate that the enzymes were not identical, but the possibility that the results obtained could have been due to the different treatments of the two enzyme preparations cannot be disregarded. While the two enzyme preparations do exhibit certain similarities, the present data are based on properties of relatively crude extracts and the question of possible multiple forms of enzyme can only be resolved when much higher degrees of enzyme purification are attained. The apparent K,, values indicate that no high degree of specificity for any one of the three inositides is exhibited by enzymes from either fraction, Atherton and Hawthorne’ reported that the monophosphoinositide-spliting enzyme from intestinal mucosa had a K, of 2.8. IO-~ M for corn monophosphoinositide and 5. IO-~ M for brain monophosphoinositide. They attributed this difference to the degrees of unsaturation of the monophosphoinositide preparations and differences in the surface areas of the lipid dispersions. Similar considerations apply also to the dispersions of monophosphoinositide, disphosphoinositide and triphosphoinositide which, due to their different charge characteristics, in equimolar concentration undoubtedly present different surface areas to the enzyme. Both in the crude homogenates and in the partially purified soluble and salt-extracted preparations when reaction rates are compared under different optimum conditions for the three substrates, triphosphoinositide is hydrolysed about twice as rapidly as diphosphoinositide and 2.5-3.5 times more rapidly than monophosphoinositide. As yet there are no clear indications of the physiological significance of the inositide hydrolytic enzymes. Phosphodiesterase cleavage of the inositides would have the effect of reducing the concentration of acidic phospholipids at some localized membrane site, presumably changing the charge characteristics in the local environment and affecting the ion-binding capacity of the membrane. There have been reportsI that phosphoinositide phosphodiesterase in synaptosomal fractions of brain Biochim. Biophys. Ada. 270 (1972)

324-336

K. M. W. KEOUGH, W. THOMPSON

336

is activated by acetylcholine and speculation that this effect is a primary one related to ion flux and the depolarization of the membrane. However there is, as yet, no substantial evidence in support of this contention. The phosphodiesterase may be considered not only to remove lipid from a membrane but also simultaneously to generate

diglyceride and water-soluble inositol phosphates. The formation may have physiological implications not yet realized.

of these products

ACKNOWLEDGEMENTS

Mr G. MacDonald

gave skilful

by a grant from the Medical Research Graduate

technical

assistance.

Council of Canada.

The work was supported K.M.W.

K. held an Ontario

Fellowship.

REFERENCES I 2 3 4 5

6 7 8 g IO

II 12 13 14

K. M. W. Keough and W. Thompson, J. Neurochem., r7 (1970) I. P. Kemp, G. Hubscher and J. N. Hawthorne, B&hem. J., 79 (1961) 193. B. J. Holub, A. Kuksis and W. Thompson, J. Lipid Res., II (1970) 558. H. S. Hendrickson and C. E. Ballou, J. Biol. Chsm., 239 (1964) 1369. W. Thompson, Biochim. Biophys. Acta, 196 (1970) 162. W. Thompson and R. M. C. Dawson, Biochem. J., 91 (1964) 237. R. S. Atherton and J. N. Hawthorne, Eur. J. Biochem., 4 (1968) 68. W. Thompson, Can. J. Biochem., 45 (1967) 853. R. 0. Friedel, J. D. Brown and J. Durrell, J. Neurochem., 16 (1969) 37r. R. M. C. Dawson, Biochim. Biophys. Acta, 33 (1959) 68. F. B. Tunealwala. N. Freinkel and R. M. C. Dawson. Biochem. I., 121 (1071) IC) J, L. f&wood and J. N. Hawthorne, J. Neurochem., 16 (1969) ;377. R. M. C. Dawson, N. Freinkel, F. B. Jungalwala and N. Clarke, Biochem. J., 122 J. Durrell, J. T. Garland and R. 0. Friedel, Science, 165 (1969) 862.

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270 (1972) 324-336

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