BIOCHIMICA ET BIOPHYSICA ACTA 257

BBA 12212

M E T A B O L I S M OF P H O S P H O L I P I D S

VII. ON T H E LIPID REQUIREMENT

FOR PHOSPHATIDIC ACID PHOSPHATASE ACTIVITY

R. COLEMAN AND G. H I 3 B S C H E R

Department of Medical Biochemistry and Pharmacology, The Medical School, University of Birmingham, Birmingham (Great Britain)

(Received November 26th, 1962)

SUMMARY

I. The t reatment of phosphatidic acid phosphatase (L-a-phosphatidate phos- phohydrolase, EC 3.1.3-4) with a variety of organic solvents causes a reduction of enzymic activity.

2. Under specified conditions, the addition of lipid preparations brings about an almost complete reactivation of phosphatidic acid phosphatase preparations which have been solvent-deactivated.

3. The results are discussed in relation to the function of membrane-bound enzymes.

INTRODUCTION

In a previous paper of this series 1, attempts have been described to purify and solu- bilise phosphatidic acid phosphatase (L-a-phosphatidate phosphohydrolase, EC 3.1.3.4) from the microsomal fraction of pig kidney using autolysis, hydrolytic en- zymes, detergents, higher alcohols and repeated freezing and thawing. In the course of purification of this enzyme, relatively more lipid than protein was retained and the possibility that the enzyme was a lipoprotein could not be excluded. The present work is concerned with the role of lipid in the action of phosphatidic acid phosphatase.

MATERIALS AND METHODS

The assay of phosphatidic acid phosphatase and most of the materials used have been described 1.

Preparation of purified enzyme

Phosphatidic acid phosphatase was purified as previously described from pig kidney 1. This involved preparation of the microsomal fraction (Stage I), autolysis of the microsomal fraction and treatment with RNAase (Stage II), and extraction with n-butanol followed by dialysis of the extracted material (Stage III). The final dialysed preparation was concentrated at 4 ° by ultra-filtration 2 without any altera- tion in specific activity or recovery (Stage IV). It was stored at 4 ° .

B~ochim. Biophys. Acta, 73 (1963) 257 -266

258 R. COLEMAN, G. HfJBSCHER

Preparation of solvent powders of the enzyme

All extract ions were performed at - -2o ° in a freezing mixture. Fil trations and fur ther t rea tment were done at 2 ° as quickly as possible. All solutions were gassed with nitrogen before and during extraction. Fil tration and drying were performed under a nitrogen hood to minimise oxidations.

An aliquot of the enzyme preparat ion (Stage II, 20 mg of protein/ml, in o.oi M Tris buffer (pH 7-4)) was added slowly with constant stirring to 12 vol. of solvent. After mixing, the material stood for I h and was then filtered under suction. The cake was immediately resuspended in the original volume of solvent and stood for I h after which it was again filtered. The cake was partially dried in a s t ream of nitrogen and then placed in an evacuated desiccator over solid K O H overnight at 2 °. Three solvents were used in these experiments: (a) acetone; (b) ace tone-methanol diethyl ether (7 : I : 2, v/v); (c) e thanol -d ie thyl ether (3 : I, v/v).

Preparation of pig-kidney acetone powders

Pig-kidney acetone powder was prepared from minced whole kidney by homo- genisation in IO vol. of acetone, taking the same precautions as mentioned in the above paragraph. After filtration, the cake was dried under a s t ream of nitrogen and the powder stored over concentrated sulphuric acid in a vacuum desiccator at 2 °.

Solvent extractions of acetone powder

Aliquots of this powder were further t reated by addition of IOO vol. of solvent (w/v). The mixture was homogenised in a blendor for 2 min and left for I h at - -20 °. I t was filtered and dried over concentra ted sulphuric acid in a vacuum desiccator. The solvents used in these experiments were: (a) e thanol-die thyl ether (3 : i , v/v) and (b) light petroleum (4o-6o°)-n-butanol (7 : 3, v/v).

Extraction of the enzyme by repeated passage down a column of solvent

The method used is similar to tha t of MORRISON, CRAWFORD AND STOTZ ~ who described the deactivat ion of a succina te-cytochrome c reductase preparat ion by repeated passage of the preparat ion down a column of solvent, the extracted aqueous phase being collected at the base of the column. All operations were performed in a cold room at a temperature of 4 °. The column of solvent was in a 5o-ml buret te measuring 65 × I cm. Between 5.0 and 7.5 ml of the enzyme solution (either Stage I or Stage IV) suspended in o.o15 M Tris buffer (pH 8.0) (between 5 and IO mg of protein per ml) were delivered down a column of 50 ml of solvent as a very fine spray from the tip of a Pasteur pipette held just below the surface of the solvent. The fine spray fell through the solvent and collected as a separate phase at the base of the column from where it could be collected for recycling. There was no appreciable change in the volume of the aqueous phase during extraction.

Use of ester determination as an estimation of the lipid content in enzyme preparations

The measurements of carboxylic ester bonds 4 was thought to be the most suitable criterion for judging the relative amounts of lipid in enzyme preparations,

Biochim. Biophys. Acta, 73 (1963) 257 -266

L I P I D R E Q U I R E M E N T OF P H O S P H A T I D I C ACID P H O S P H A T A S E 259

since the nature of the lipid was unknown. The only modification introduced was that the acid-denatured protein of the enzyme preparation was removed by centri- fugation lust prior to measurement of the absorbancy.

Preparation of lipids for reactivation experiments

Two types of lipid were prepared. Type A was the lipid obtained in the solvent phase in the course of the extraction of the enzyme preparation. Type B was pre- pared essentially by the method of ROUSER et al), from pig kidney. Minced pig kidney was extracted 3 times with IO vol. of chloroform-methanol (2 : I, v/v) and the combined extracts were taken to dryness under reduced pressure in a stream of nitrogen at 35-4 o°. The residue was taken up in I /5oth of the original volume of the combined extracts using the same solvent. The solvent was removed in the same way and the residue dried overnight in a desiccator over KOH. The desiccator had been flushed with nitrogen prior to evacuation.

Aqueous suspensions of lipids used in the reactivation experiments

The lipids (dissolved in ether) were layered onto distilled water or a buffer containing O.Ol 5 M Tris-o.oo5 M EDTA (pH 7.4). The ether was evaporated under reduced pressure at 35-4 °0 , with constant agitation to produce an aqueous emulsion which, if necessary, was further dispersed by the aid of a Pot ter -Elvejhem homo- geniser. The dispersed Type B-lipid was then dialysed vs. IOO vol. of o.o15 M Tris- o.ooi M EDTA (pH 8.0) at 4 °. The buffer was changed twice during 24 h.

Lipid-enzyme ratios during reactivation

In the reactivation experiments, the amount of Type A-lipid added to the solvent-extracted enzyme preparation was equal to the amount of lipid present in the enzyme preparation prior to extraction. When Type B-lipid was used, double this amount was added.

E X P E R I M E N T S A N D R E S U L T S

Lipid content and stability of the enzyme

The purified phosphatidic acid phosphatase preparation (Stage IV) always contained significant amounts of lipid as determined by the carboxylic acid ester linkages. The ratio of/zmoles of earboxylic acid ester per mg of protein varied widely from preparation to preparation, a ratio of 0. 7 being an average value.

The microsomal fraction from pig kidney (Stage I) could be frozen and thawed at least 9 times without loss of phosphatidic acid phosphatase activity 1. In contrast, freezing and thawing of the purified dialysed enzyme preparation (Stage I I I ) brought about an almost complete loss of enzymic activity (see Table I). The remaining en- zymic activi ty was completely sedimented, together with most of the lipid, by cen- trifugation at 3 600 ooo g. min. Before freezing and thawing, only a third to a half of the enzymic activi ty was sedimented by centrifugation at 3 600 ooo g- rain.

When a fractionation of the Stage-I I I enzyme preparation was a t tempted with

Biochim. Biophys. Acta, 73 (1963) 257-266

260 R. COLEMAN, G. HUBSCHER

T A B L E I

THE EFFECT OF FREEZING ON PURIFIED PHOSPHATIDIC ACID PHOSPHATASE

The preparation (Stage III) had a specific activity of 2. 7 and was suspended in 0.o 3 IV1 Tris buffer (pH 7.7) containing 1.8 mg of protein/ml. The enzyme had been frozen and thawed twice during the course of a few hours. After thawing, the enzyme was centrifuged at 3 6oo ooo g. rain. giving a sediment and a supernatant liquid. The lipid content was measured by determining the

carboxylic acid ester content.

Total enzyme Total protein Total lipid Preparation units

(%) (%) (%)

Enzyme before freezing ioo ioo ioo Enzyme after freezing approx. 8 ioo ioo Sediment approx. 7 67 81 Supe rna tan t o 36 6

ammonium sulphate, a turbidity was obtained at 45% saturation. After centrifuga- tion at 14o ooog.min. , the previously turbid material was now part ly floating as a thin film on the surface and part ly adhering to the sides of the centrifuge tube. The enzymic activity was associated, however, with the floating film, the precipitate on the sides of the centrifuge tube, and the remaining aqueous phase. Thus no purification was achieved, even when the concentration of ammonium sulphate was increased stepwise above 45 % saturation. But it is noteworthy that a floating film or precipitations on the sides of the centrifuge tube developed upon addition of ammonium sulphate rather than the usual sedimentable material.

Enzymic activity of powders produced by organic solvents

These and the following experiments were an a t tempt to extract lipid from enzyme preparations under conditions in which a minimum of protein denaturation might be expected.

T A B L E I I

ENZYMIC ACTIVITY" AND LIPID CONTENT OF SOLVENT POWDERS

The enzyme used (Stage-II preparat ion) had a specific act ivi ty of 1.3 2 and contained o.6 5 #moles of carboxylic acid ester bonds per mg of protein. The relative specific act ivi ty was expressed as uni t s of enzyme recovered relative to the recovery of protein. The relative ratio of l ipid/protein is calculated f rom the to ta l /zmoles of carboxylic acid ester recovered relative to the recovery of

protein. For details of prepara t ion of powders see METHODS.

Total To~al Total Relative Relative Preparation enzyme ¢Jrotein lipid specific ratio

units • lipid[ (%) (%) (%) activity protein

Enzyme (Stage II) IOO IOO Ioo i .ooo i ,ooo Powder after t r ea tmen t wi th

acetone 53 7 ° 65 o.758 o.928 Powder after t r ea tmen t wi th

ace tone-methano l -d ie thy l ether (7 : I : 2) I8 55 31 °.327 °.563

Powder after t r ea tmen t wi th e thanol -d ie thyl ether (3 : i) 8 51 21 o.157 o.412

B i o c h i m . B i o p h y s . A c t a , 73 (1963) 257-266

LIPID REQUIREMENT OF PHOSPHATIDIC ACID PHOSPHATASE 201

The solvent powders used in initial experiments were prepared from a Stage-II preparation. They were suspended in o.o2 M maleate buffer (pH 6.o) (I : 2oo, w/v), left for 3 h at o ° with frequent dispersion using a Pot te r -Elvehjem homogeniser. These suspensions were then assayed at pH 6.o for phosphatidic acid phosphatase, carboxylic acid ester linkages and protein content. The results shown in Table I I indicate that all solvents used produced a depression in enzymic activity which was more marked with solvent mixtures of increased extraction power. The specific act ivi ty and lipid-protein ratios are more accurate guides than the recovery figures for protein and ester content of the enzyme preparations, since the latter are subject to losses due to filtration and resuspension. These losses will have no influence on the relative ratios, however. The relative rate of loss brought about by the action of the solvents was of the order: enzymic activity > lipid > protein. Thus the loss in enzymic activity was more closely correlated with the loss of lipid than with that of protein. This trend became even more apparent in the experiments summarised in Table I I I . There was virtually no loss of protein but corresponding losses of enzymic

T A B L E I I I

E N Z Y M I C A C T I V I T Y A N D L I P I D C O N T E N T O F S O L V E N T P O W D E R S P R E P A R E D

F R O M A C E T O N E P O ~ V D E R

The acetone powder was prepared f rom total pig-kidney homogenate and had a specific act ivi ty of o.34 o and contained 0.27/~mole of carboxylic acid ester bonds per mg of protein. The light pe t ro l eum-n -bu t ano l powder and the e thano l -e the r powder were prepared f rom this acetone powder as described under METHODS. The relative specific act ivi ty and the relative ratio of lipid•

protein were calculated as indicated in Table n .

Preparation

Total Relative Total Total Relative enzyme protein lipid specific ratio units lipid[ (%) (%) (%) activity protein

Acetone powder ioo ioo ioo i .ooo i .ooo Light pe t ro l eum-n-bu tano l 73 io i 64 0.730 0.640 E thano l -d ie thy l ether powder 67 lO6 61 o.670 o.61o

act ivi ty and lipid. In these experiments, the enzyme had been suspended in 5 ° vol. (w/v) of 0.02 M Tris buffer (pH 8.0) prior to assay of enzymic activity at pH 6.0.

I t can be argued that the enzyme is being extracted into the solvent, but if so, there should be a loss of protein in the solvent powder. Furthermore, no enzymic act ivi ty was detected when the solvent phase obtained after extraction was taken almost to dryness under reduced pressure and the remainder emulsified in water.

At tempts to reactivate the enzyme by adding to it aqueous suspensions of either the lipid extracted from the enzyme itself (Type A) or the total lipids from pig kidney (Type B) are shown in Table IV. I t can be seen that these lipids produced only slight if any reactivations.

Effect of repeated passage down a column of solvent and reactivation of the enzyme preparation by lipids

Treatment of microsomal preparations from pig kidney (Stage I) with solvents employing the repeated passage down a column of solvent resulted in a deactivation

B i o c h i m . B i o p h y s . Ac ta , 73 (1963) 257-266

2 6 2 R. C O L E M A N , G. H / J B S C H E R

T A B L E I V

THE EFFECT OF ADDED LIPIDS TO SOLVENT-EXTRACTED ENZYME POWDERS

The acetone and acetone-methanol-ether powders were obtained from a Stage-ll enzyme pre- paration as shown in Table II and the light petroleum-n-butanol powder was prepared as indicated in Table III. The enzyme preparations used in Expts. i and 2 were suspended in 0.02 M maleate buffer (pH 6.0) and the enzyme in Expt. 3 in 0.2 IV[ Tris (pH 8.o). All pre-incubations were done

a t O °,

Time of pre- Relative rate Specific Expt. Additions incubation (k) of reaction acivity

I A c e t o n e p o w d e r o i o o i . o i A c e t o n e p o w d e r + a c e t o n e e x t r a c t 3 lO3 - - A c e t o n e p o w d e r + a c e t o n e e x t r a c t 24 lO3 - -

2 A c e t o n e - m e t h a n o l - e t h e r p o w d e r o i o o 0 .43 A c e t o n e - m e t h a n o l - e t h e r p o w d e r + a c e t o n e -

m e t h a n o l - e t h e r e x t r a c t 3 113 - - A c e t o n e - m e t h a n o l - e t h e r p o w d e r + a c e t o n e -

m e t h a n o l - e t h e r e x t r a c t 24 112 - - A c e t o n e - m e t h a n o l - e t h e r e x t r a c t o o - -

3 L i g h t p e t r o l e u m - n - b u t a n o l p o w d e r o IOO 0 .28 L i g h t p e t r o l e u m - n - b u t a n o l p o w d e r + T y p e - B

l i p i d o lO 5 - - T y p e - B l i p i d o o - -

of phosphatidic acid phosphatase (see Table V). The degree of deactivation of this enzyme depended on the type(s) of solvent used and in general, less polar solvents alone were not as effective as mixtures of a less with a more polar solvent. I t is in- teresting that the Stage-IV preparation could also be deactivated: the purification procedure includes t reatment with n-butanol in an aqueous one-phase system which might have already influenced the lipid composition of the enzyme.

T A B L E V

DEACTIVATION OF PHOSPHATIDIC ACID PHOSPHATASE BY REPEATED PASSAGE DOWN A COLUMN OF SOLVENT

In Expts. I and 2 the enzyme preparation used was pig-kidney microsomal fraction (Stage I) w i t h a spec i f i c a c t i v i t y o f I . I . I n E x p t . 3 a S t a g e - I V p r e p a r a t i o n w i t h a spec i f i c a c t i v i t y o f 2. 7

w a s u s e d . F u r t h e r d e t a i l s a r e d e s c r i b e d u n d e r METHODS.

Recovery of No. of enzymic

Expt. Solvent used transfers activity (%)

i N o n e - - i o o I s o o c t a n e 4 73 I s o o c t a n e - c h l o r o f o r m (9 : i ) 4 68 I s o o c t a n e - n - b u t a n o l (9 : I) 4 36

2 N o n e - - IOO L i g h t p e t r o l e u m ( 4 0 - 6 0 °) i o 7 ° L i g h t p e t r o l e u m ( 4 o - 6 o °) s a t u r a t e d w i t h

m e t h a n o l i o 54 3 N o n e - - - i o o

L i g h t p e t r o l e u m ( 4 o - 6 o ° ) - n - b u t a n o l (7 :3 ) 7 57 L i g h t p e t r o l e u m ( 4 o - 6 o ° ) - n - b u t a n o l (7 :3) i o 4 °

B i o c h i m . B i o p h y s . A c t a , 73 (1963) 257 - 2 6 6

LIPID REQUIREMENT OF PHOSPHATIDIC ACID PHOSPHATASE 203

I n t h e fo l l owing e x p e r i m e n t s , a S t a g e - I V p r e p a r a t i o n was d e a c t i v a t e d b y pa s -

s age t h r o u g h l i g h t p e t r o l e u m ( 4 o - 6 o ° ) - n - b u t a n o l ( 7 : 3 , v / v ) . T h e r e s u l t s q u o t e d

in T a b l e V I i n d i c a t e t h a t t h e e x t r a c t e d e n z y m e w a s v e r y u n s t a b l e w h e n s t o r e d a t

4 ° ( t e s t s 4 a n d 7). T h i s m a y h a v e b e e n d u e to t h e p r e s e n c e of t h e o r g a n i c s o l v e n t

d i s s o l v e d in t h e a q u e o u s phase . T h e e n z y m i c a c t i v i t y c o u l d b e s t a b i l i s e d b y e i t h e r

d i a l y s i s or b y a d d i t i o n o f l ip id ( T y p e B). T h i s l i p id p r e p a r a t i o n d i d Not in i t s e l f

possess e n z y m i c a c t i v i t y a n d d i d n o t a c t i v a t e t h e o r i g i n a l e n z y m e p r e p a r a t i o n

TABLE VI

REACTIVATION OF SOLVENT-DEACTIVATED PHOSPHATIDIC ACID PHOSPHATASE

BY TOTAL LIPIDS FROM PIG K I D N E Y ( T Y P E - B LIPID)

The original enzyme was a Stage-IV preparat ion with a specific act ivi ty of 1.95. The enzyme was extracted by IO transfers through light petroleum (4o-6o°)-n-butanol (7 : 3, v/v) using 7.5 ml of enzyme for 5 ° ml of column volume. In tests 11-15 the deactivated enzyme was dialysed for

24 h vs. 35 ° vol. of o.o15 1Vi Tris-o.ooi M EDTA (pH 8.o) a t 4 °.

Recovery of Test Enzyme preparation Lipid Conditions of reactivation enzymic

added activity (%)

i Original enzyme - - - lOO 2 Original enzyme + Lipid added just prior to assay 96 3 None + - - o 4 Ext rac ted enzyme -- - - 54 5 Ext rac ted enzyme + Lipid added just prior to assay 64 6 Ext rac ted enzyme -}- Enzyme and lipid incubated for

2 h a t o ° prior to assay 76 7 Ext rac ted enzyme kept 24 h a t o ° -- - - 13 8 Ext rac ted enzyme kept 24 h at o ° + Lipid added just prior to assay 14 9 Ext rac ted enzyme kept 24 h at o ° + Enzyme and lipid incubated for

2 h at o ° prior to assay 23 io Ext rac ted enzyme kept 24 h a t o ° + Enzyme and lipid incubated for

24 h at o ° prior to assay 72 I I Ext rac ted and dialysed enzyme -- - - 54 12 Ext rac ted and dialysed enzyme + Lipid added just prior to assay 61 13 Ext rac ted and dialysed enzyme + Enzyme and lipid incubated for

2 h a t o ° prior to assay 64 14 Ext rac ted and dialysed enzyme + Lipid added to the enzyme before

dialysis 78 15 Ext rac ted and dialysed enzyme + Treated as in tes t 14 followed by

storage for 96 h at o ° 93

( t e s t s 2 a n d 3). W h i l e t h e r e a c t i v a t i o n s r e c o r d e d in t e s t s 4 - 6 a n d 7 - 1 0 m i g h t b e

e x p l a i n e d b y m e r e r e m o v a l o f t h e o r g a n i c s o l v e n t f r o m t h e a q u e o u s p h a s e b y t h e

l ip id , t h i s is u n l i k e l y to b e t h e e x p l a n a t i o n for t h e r e a c t i v a t i o n s r e c o r d e d in t e s t s 11 -15 .

D i a l y s i s for 24 h vs. 35 ° vol . o f b u f f e r wil l r e d u c e t h e o r g a n i c s o l v e n t s in t h e e n z y m e p r e p a r a t i o n t o a v e r y low va lue . I f t h e o r g a n i c s o l v e n t w a s i n h i b i t o r y , i t s r e m o v a l

b y d i a l y s i s s h o u l d h a v e r e s t o r e d t h e e n z y m i c a c t i v i t y to a v a l u e close to t h e o r i g i n a l

one . I n f ac t , t h e a c t i v i t y a f t e r d i a ly s i s w a s u n c h a n g e d ( t e s t s 4 a n d I I ) a n d i t re-

q u i r e d t h e a d d i t i o n o f l i p id to r e a c t i v a t e t h e e n z y m e p r e p a r a t i o n ( t e s t s 11 -15) .

F r o m t h e v a r i o u s deg r ee s o f r e a c t i v a t i o n o b s e r v e d in t e s t s 12 -15 , i t w o u l d a p p e a r t h a t t h e r e w a s i n i t i a l l y a f a s t r a t e o f r e a c t i v a t i o n fo l lowed b y a r e l a t i v e l y s low one.

Biochim. Biophys. Acta, 73 (1963) 257 -266

264 R. COLEMAN, G. HUBSCHER

TABLE VII

R E A C T I V A T I O N O F S O L V E N T - D E A C T I V A T E D P H O S P H A T I D I C A C I D P H O S P H A T A S E

B Y A D D I T I O N O F T H E L I P I D E X T R A C T E D W I T H T H E S O L V E N T ( T Y P E - A L I P I D )

The enzyme preparation used in this experiment, the solvent-extraction of the preparation and other experimental conditions were the same as those described in Table VI.

Recovery o] Test Enzyme preparation Lipid Conditions of recombination enzymic added activity

(%)

I Original enzyme -- - - ioo 2 None + - - o 3 Extracted enzyme -- - - 53.5 4 Extracted enzyme + Enzyme and lipid kept for 2 h at

o ° prior to assay 61.5 5 Extracted enzyme kept for 24 h - - 7-7

at o ° 6 Extracted enzyme kept for 2 4 h

a t o ° + Enzyme was stored for 2 4 h a r D ° in the presence of lipid prior to assay 56.0

+ Lipid added to the enzyme before dialysis for 24 h 77.5

7 Extracted and dialysed enzyme

Thus after o, 2, 24 and 12o h, the enzymic activity was 61, 64, 78 and 93% respec- tively of that observed before deactivation with the organic solvent.

I t seemed important to see whether the total lipids (Type B) could be replaced by the lipid extracted from the enzyme preparation (Type A). From the results shown in Table VII it may be concluded that the reactivations observed with the Type-A lipid were very similar to those observed with the Type-B lipid.

DISCUSSION

T h e presence o f s igni f icant a m o u n t s o f l ip id in pur i f ied p h o s p h a t i d i c ac id p h o s p h a t a s e

p r e p a r a t i o n s , t he a l m o s t c o m p l e t e i n a c t i v a t i o n o f th is p r e p a r a t i o n a f t e r f reez ing

a n d t h a w i n g , a n d t h e f loa t ing o b s e r v e d d u r i n g i ts f r a c t i o n a t i o n w i t h a m m o n i u m

s u l p h a t e sugges t ed t h a t t h e a c t i v e f o r m of t h e e n z y m e m a y be a l ipopro te in . Th is was f u r t h e r s u p p o r t e d b y resul t s o b t a i n e d w i t h s o l v e n t - e x t r a c t e d p repa ra t i ons .

T h e t r e a t m e n t of c rude or p a r t i a l l y pur i f ied p h o s p h a t i d i c ac id p h o s p h a t a s e w i t h o rgan ic so lven t s u n d e r a v a r i e t y o f e x p e r i m e n t a l cond i t i ons caused in all cases a

r e d u c t i o n in ac t i v i t y . I n some of these d e a c t i v a t e d p repa ra t i ons , t he a c t i v i t y cou ld

be p a r t i a l l y or a l m o s t c o m p l e t e l y r e s to red b y t h e a d d i t i o n of l ipids. S e v e r a l f ac to rs m a y p l a y a p a r t in t he d e a c t i v a t i o n p h e n o m e n a desc r ibed in t h e

e x p e r i m e n t a l sect ion. T h e d e a c t i v a t i o n m a y be due to an unspeci f ic d e n a t u r a t i o n o f p ro t e in b y d e h y d r a t i o n , to t he i n h i b i t o r y ac t ion o f t he so lven t on t h e e n z y m e , to t h e r e m o v a l o f specific or s t r u c t u r a l lipid(s) f rom the e n z y m e (assuming t h a t t h e e n z y m e is a l ipopro te in) and f inal ly to t he r e m o v a l of s o m e as y e t u n k n o w n c o f a c t o r

wh ich is soluble in o rgan ic so lvents . W h i l s t some p ro t e in d e n a t u r a t i o n m a y h a v e o c c u r r e d in t he p r e p a r a t i o n of t he so lven t powders , i t is un l ike ly t h a t d e n a t u r a t i o n

h a d t a k e n p lace in t h e c o l u m n - e x t r a c t i o n p rocedure , s ince an a lmos t c o m p l e t e re- a c t i v a t i o n could be d e m o n s t r a t e d . I t is n o t e w o r t h y t h a t IMAI AND SATO 6 a n d SCANU

Biochim. Biophys. Acta, 73 (1963) 257-266

LIPID REQUIREMENT OF PHOSPHATIDiC ACID PHOSPHATASE 265

AND HUGHES ~ who used similar techniques for the preparation of their solvent powders reported a reactivation of certain enzymes when lipid was added to the powder preparations. Since the solvents themselves did not appear to be responsible for the reduced activity of the enzyme preparation after solvent extraction (as shown by the dialysis experiment quoted in Table VI), the remaining two possi- bilities are: a specific lipid (or a non-lipid cofactor with lipid-like solubilities) is re- quired for opt imum activity or, lipid in general is required as a structural factor for the secondary or tert iary structure of the enzyme.

The results presented here do not allow an unequivocal interpretation of the observed phenomena but strongly point to the possibility that a lipid plays an im- por tant role in the reaction mechanism of phosphatidic acid phosphatase, This en- zyme is part of a lipid-protein membrane system (the endoplasmic reticulum) and is also participating in the biosynthesis of some of the lipids which make up this membrane system. Thus it is likely that the lipid is, at least partially, responsible for maintaining functional properties of the membrane system, in addition to its generally recognised structural role.

Phosphatidic acid phosphatase is not the first example of an enzyme which requires lipid for opt imum activity. In addition to the enzymic activities quoted above 6,~ the activity of some oxidative enzymes of mitochondria can be decreased by extraction with solvents s-10 and by repeated precipitations from detergent solu- tions n. Some of these types of preparations can be reactivated by solubilised lipids TM. Recently, a reactivation of fl-hydroxy butyric dehydrogenase (EC 1.1.1.3o ) has been reported in which pure synthetic phosphatidyl choline has been used la. The presence of sulfhydryl groups was also required. This was the first demonstration of a reactiva- tion of an enzyme by a specific phospholipid and the requirement for phosphatidyl choline was an absolute one. I t is thought that phosphatidyl choline combines with the enzyme providing a hydrophobic region in which the sulfhydryl group(s) of the enzyme may react 14.

Phosphatidic acid phosphatasel, 15-19 catalyses one in a series of reactions which lead to the formation of triglycerides and of lipid precursors in phospholipid biosyn- thesis. The other members of this multi-enzyme system are: lauryl CoA synthe- tase 2°-24, glycerophosphate acyl transferase(s)25, 2s diglyceride acyl transferase27, 28, monoglyceride acyl transferase ~9-sl, and possibly also monoglyceride kinase s*. In each of the reactions catalysed by these enzymes, at least one of the participating substrates is only sparingly soluble in water due to its lipidic nature, and all of the enzymes mentioned above occur predominantly in particulate subcellular structures. In some casesl,16-1s, 2~ a solubilisation of the enzyme from the subcellular structures has been at tempted, but convincing evidence for a true solubilisation has so far not been presented. Furthermore, the preparation of a submitochondrial particle has been described which had, with respect to triglyceride biosynthesis, the same en- zymic activities as the original mitochondrial preparationSS: it synthesised tri- glycerides using either a-glycerophosphate, phosphatidic acid, or diglyceride as precursors. Thus it seems that there are certain similarities regarding the physical nature of the enzymes making up this multi-enzyme system.

In contrast, glycero kinase s4-s6 which utilises water-soluble substrates is present either in the cell sap or, if present in subcellular structures, can be readily extracted. In summary, it would seem that the early stage in the biosynthesis of glycerides is

Biochim. Biophys. Acta, 73 (1963) 257-266

266 R. C O L E M A N , G. H O B S C H E R

catalysed by enzymes which are soluble or can be readily solubilised. When the intermediate in this multi-enzyme system becomes a larger molecule with lipidic properties, the enzymes from this point on are in particulate subcellular structures to which they seem to be firmly bound.

ACKNOWLEDGEMENTS

We thank Professor A. C. FRAZER for his encouraging interest in this work and Miss WYATT for skilled assistance. The work was supported by a grant from the Medical Research Council to one of us (R. C.).

R E F E R E N C E S

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