Preparation and Characterization of Mouse Intestinal Phospholipase A T H O S OTTOLENGHI, Department of Physiology and Pharmacology,

Duke Medical Center, Durham, North Carolina 27710

intestine of albino mice infected with the tapeworm H y m e n o l e p i s nana (11).

ABSTRACT

Phospholipase has been prepared in a stable, partially purified form from the small intestine of mice infected with the tapeworm H y m e n o l e p i s nana. The enzyme(s) attacks diacylphosphatides with liberation of free fatty acids and a corresponding decrease in phospholipid phosphorus, without accumulation of lysophosphatides. The reactivity with various substrates is strongly influenced by their physical state, the presence of other finds, proteins or detergents. The phospholipids of some biomembranes (mitochondria, microsomes, red cell ghosts) are readily hydrolyzed under customary reaction conditions. The ensuing biochemical, morphological and functional alterations have been documented. In contrast, the diacylphospholipids of the cell membrane (intact erythrocytes, Pseudomonas aerunginosa) are not accessible to phospholipase action unless some alteration of the integrity of the cell is induced by physical or chemical means (hemolysis, polymyxin). The enzyme is proposed as a tool for the investigation of biomembranes and as a model for the study of phospholipase activity.

MATERIALS AND METHODS Animals

INTRODUCTION

In recent years a n u m b e r of animal tissues have been shown to contain enzymes capable of splitting phospholipids by hydrolysis of the fatty acid ester bonds. Various criteria and methods (positional specificity, substrate affinity, Ca++ requirement, pH optimum) have been used to characterize enzymes from different sources and resolve mixtures of these activities encountered in some tissues (1-3). The presence of phospholipases A and B in rodent intestine was first reported by Epstein and Shapiro (4) and has since been confirmed in many laboratories (5-9). Subbaiah and Ganguly have recently described the subcellular distribution of these enzymes in rat intestine (10). This report deals with the preparation and biochemical characterization of a stable, partially purified form of the phospholipase found in particularly high concentrations in the small

Adult male mice (Charles River, CD-1), average body weight 20g, were housed in groups of 20 per cage in quarters provided with temperature, humidity and light controls (7 A.M.-9 P.M.-7 A.M., light-darkness cycle). These animals were used for both induction of high intestinal phospholipase concentrations by worm infection and as sources of worm eggs to maintain a small colony of infected mice over the past 3 years. Routinely the mice were infected with H y m e n o l e p i s nana eggs 1 week after arrival, each animal receiving 3000-5000 eggs in 0.5 ml water by garage using a N1611 curved needle 18 GA x 1.5 (Biomed, Instrument Div., Popper & Sons, Inc., N.Y.). A high phospholipase content of the intestine, suitable for enzyme extraction and purification, is already present after 10-12 days and persists for at least 6 weeks (11). We have used most often 4-5 weeks infected mice for enzyme preparation as described in the results section. A slightly shorter time is optimal for the harvesting of eggs. For this purpose the lower third of the small intestine of six to eight mice infected 25-30 days earlier was removed, immersed in water in a Petri dish and cut longitudinally to disperse the contents by gentle shaking. The easily identifiable, white, thread-like worms were transferred with tweezers through three to four washes in water to remove gross contaminants, and finally collected in ca. 10 ml water. After transfer of the parasites and fluid to a small porcelain mortar, the eggs were freed by gentle grinding with a pestle followed by stirring for 10-15 min with the aid of a magnetic bar. Five microliters of the suspension were used for counting at 35x magnification and the concentration of the eggs was adjusted to 6000-10,000 eggs per milliliter. The yield from eight mice was sufficient for infecting 30 new animals by administering to each 0.5 ml of the continuously stirred suspension as described above. It is worth adding that harvesting of the parasites should not be postponed much later than the 30th day since the n u m b e r of worms to be

415

416

A. OTTOLENGHI

found declines considerably by the 5th week, probably as a consequence of self-cure. The latter process is accompanied by the development of i m m u n i t y to reinfection, and it is therefore important to avoid accidental transmission of the infection via bedding or nonsterilized drinking water bottles and cages, so that animals kept for weeks before infection do not become immune and give negative results. For the same reason, it is important to obtain mice from breeding colonies where Hymenolepis nana infection is totally absent. In our experience the source mentioned above has met this requirement over the past 3 years.

persions of Asolectin for use in the reactivation of beef heart mitochondrial enzymes were prepared and stored according to Fleischer and Fleischer (15).

Rat liver mitochondria and mierosomes:

These were obtained by the standard preparation methods in isotonic sucrose and were stored at -15 C in a sucrose volume corresponding to 50% of the liver weight. Lipid and protein fractions of the two preparations were obtained by extraction with aqueous acetone ammonia according to Fleischer and Fleischer (16). One volume of mitochondrial or microsomal suspension was mixed with 24 volumes of acetone-water-ammonia 22.5: 1.5: 0.004 v/v/v Su bstrates and the mixture separated at 800 x g for 10 The substrates of phospholipase, their rain. The pellets were then washed three times sources and preparation methods are listed with isotonic sucrose, sedimented at 20,000 x g for 10 min and resuspended in the same below. Phosphatidylcholine (PC): PC was prepared medium in one-half the original volume. These from egg yolk according to Ansell and Haw- preparations, containing 5.1 and 15.3 mg of thorne (12). The silicic acid column eluate in protein per milliliter, respectively, are here chloroform-methanol 7:3 was added with referred to as mitochondrial and microsomal 0.005% of the antioxidant 4-methyl-2-6-di-tert- protein. The corresponding acetone superbutyl phenol and brought to dryness in a rotary natants were evaporated under reduced presevaporator. After flushing with nitrogen the PC sure, resuspended in twice the original (mitowas resuspended in water to a final concen- chondrial or microsomal) volume of water and tration of 20/~mol/ml and stored frozen in 5-10 extracted according to Bligh and Dyer (17). rnl aliquots at -15 C. For use, the frozen Each chloroform extract was added with samples were thawed in lukewarm water. Ac- 0.005% of the antioxidant 4-methyl-2,6-di-tertcording to the particular experimental design, butyl phenol, brought to dryness under reduced the substrate was mixed directly with buffer or pressure, and the lipid residue was dispersed in dispersed by addition of Triton X-100 or one half the original volume of sucrose giving a sonicated in a Raytheon Model DF101 mag- suspension containing 15.3 and 6.3 gmol phosnetostrictive oscillator, 10 KC, for two succes- pholipid per milliliter mitochondrial and microsive 3 rain intervals. somal extract, respectively. All preparations Phosphatidylethanolamine (PE): PE was ob- were stored at -15 C and simply thawed before tained from egg yolk according to Ansell and use unless otherwise indicated. Hawthorne (13) and processed for storage and Red cellghosts: Washed human erythrocytes use as indicated above for PC. were lysed in 0.01 M Tris buffer pH 7.4 and Lysophosphatidylcholine (LPC): LPC was washed free of hemoglobin by repeated centriprepared from PC using snake venom according fugation at 14,000 x g for 10 vain in the same to Long and Penny (14). After extraction with medium. The cells were resuspended in eighthot alcohol, the phospholipid was dried at tenths of the original volume and incubated reduced pressure and redissolved in a small with phospholipase as specified in the results volume of water (60-80/~mol/ml). The solution section. was dialyzed overnight against distilled water Pseudomonas aeruginosa: The bacteria were and the concentration adjusted to 20/.tmol/ml, grown for 24 hr on Difco Nutrient Broth, for storage at -15 C. The clear solution resulting harvested by centrifugation, washed with water upon melting in lukewarm water was used three times and finally resuspended in water. routinely without further treatment. Cell concentration was adjusted so that, u p o n Soybean phosphatides (Asolectin): The mix- undergoing standard operations of incubation ture of phosphatides was obtained from a and further dilution, the control sample in commercial source (Associated Concentrates, water would give an optical density reading of Inc., Long Island, N.Y.). When used as a 0.13-0.16 at 500 X in a Coleman junior phosubstrate the material was dispersed in water (5 tometer. In the experiments reported here, the mg/ml) by homogenization in a motor driven cells were first incubated at room temperature Teflon-giass homogenizer. If needed, sonication with phospholipase and polymyxin; a 2 ml was carried out as indicated for PC. Microdis- aliquot was withdrawn at a chosen time and the LIPIDS, VOL. 8, NO. 7

MOUSE INTESTINAL PHOSPHOLIPASE initial OD reading was taken. Then 0.2 ml of 2 M NaC1 was added, rapidly mixed, and the cell's response was followed by short interval readings of the OD changes. Cells incubated without phospholipase served as controls. Enzyme Assays

Phospholipase activity: Reaction conditions varied to fit specific experimental goals. The following buffers were used at 0.1 M strength for the pH studies: acetate, MES, Tricine. Routinely 1 volume buffer was mixed with 0.5 volumes substrate in aqueous medium at 37 C, and the enzyme preparation was added in microliter amounts to start the reaction. The standard assay for lysophospholipase activity used 0. 1 M phosphate buffer pH 6.6 added with 0.5 volumes of 2 x 10 -2 M lysolecithin. Hydrolysis of the various substrates was determined by one of two basic procedures: (a) extraction of the unreacted phospholipid according to Bligh and Dyer (17) and estimation of the P content of the chloroform phase (Fiske and Subbarow [18] and Barlett [19]); (b) extraction and titration of the liberated fatty acids in heptane according to Dole (20). The first procedure was most useful when diacylphospholipids were studied, while the second was preferred when LPC was the substrate. Quantitative estimates involving both fatty acids and phospholipids were usually performed on Bligh and Dyer extracts. To obviate the interference with fatty acid titration normally encountered when diacylphospholipid are present, an aliquot of the chloroform phase was first dried, the lipid redissolved in 3-4 ml dry chloroform and loaded onto a small silicic acid column (at least 1 g/2.5 /~mol extracted phospholipid). The column was then washed with 3 volumes of dry chloroform, and the combined eluates dried under reduced pressure. The residue was dissolved in heptane and the fatty acids titrated with 0.01 N NaOH. This procedure, which works well with pure substrates, was controlled using known amounts of pure fatty acids. When very small quantities were involved, the fatty acid estimation method of Mahadevan et al. (21) was followed. Aliquots of the Bligh and Dyer extracts were also used for thin layer chromatography on silica plates (Eastman, Chromagram) following the two step procedure of Skipski and Barclay (22). Visualization of the phospholipid spots was achieved by spraying with dichlorofluorescein and viewing in UV light. Glycerylphospholycholine (GPC) was estimated by the ennaiodide method of Appleton et al. (23) adapted for PC and lysoPC preparations (24). The formation of giycerylphosphor-

417

ylethanolamine and GPC m red cell ghosts suspensions was detected by paper chromatography according to Dawson et al. (25). Oxidative enzymes o f beef heart mitochondria: Standard techniques were followed for the estimation of succinate-cytochrome reductase (26) of heavy beef heart mitochondria incubated at 30 C with mouse phospholipase. RESULTS Extraction and Purification of Mouse Phospholipase

The procedure developed and finally adopted in our laboratory for preparation of mouse intestinal phospholipase consists of the steps described below. The volumes and amounts given refer to a preparation run using eight mice; a larger number of animals can be processed with a proportional increase in fluid and reagents. Estimation of phospholipase B activity was used to follow the various stages of purification. Intestinal homogenate: Mice infected with Hymenolepis nana 4-5 weeks earlier are anesthetized with ether and killed by decapitation. The entire small intestine is dissected, cut in 4-5 segments and the content extruded by gentle pressure along the peritoneal surface. After weighing, the tissue is minced with scissors and homogenized in 19 volumes ice cold medium (8% sucrose, 1 x 10-2M Tris pH 7.4, 1 x 10-3M Mg, 1 x 10-3M dithiothreitol) with the aid of a motor driven Teflon-glass homogenizer. The homogenates are pooled (280 ml) and centrifuged at 8000 x g for 10 min. (For this and all the following centrifugation steps except dialysis and storage, we have found it advantageous to use a # 3 0 Spinco rotor and 38.5 ml tubes.) The supernatant (230 ml) contains 80-90% of the total homogenate activity. Precipitation with Protamine: The 8000 x g supernatant is added slowly, with stirring, with 0.25 volume of protamine sulfate (Calbiochem., 1% in water at room temperature) and the mixture is allowed to stand in the cold for 20 min prior to centrifugation (25,000 x g x 15 rain). The whitish pellets contain the bulk of the phospholipase activity. Extraction with Tris-Triton X-I O0: To each centrifuge tube are added in succession 5 ml 0.3M Tris pH 7.4 (containing 1 x 10-3M dithiotreitol) and 0.5 ml 1% Triton X-100. The pellet is first dispersed with the aid of a glass rod, and the suspension is diluted with 15 ml water containing 1 x 10-3M dithiothreitol and transferred to an ice cold beaker. Washing of the centrifuge tubes with an additional 20 ml of Tris-water medium aids to recover all the particulate material, and extraction is cornLIPIDS, VOL. 8, NO. 7

A. OTTOLENGHI

418

TABLE I Purification of Mouse Intestinal Phospholipase a Preparation 8000 x g supernatant Protamine-Triton X extract 40% Ammonium sulfate, ppt 60% Ammonium sulfate, ppt Calcium phosphate gel eluate--I Calcium phosphate gel eluates I + II

Total activity

%

Activity/mg protein

402,000 220,000

100 54

342 1123

213,000

53

1627

163,000

40

2634

60,000

15

6024

140,000

34

4740

aActivity values represent micromoles lysolecithin hydrolyzed per hour under standard assay conditions. p l e t e d by h o m o g e n i z a t i o n w i t h a few h a n d strokes in a Teflon-glass h o m o g e n i z e r . Centrif u g a t i o n at 2 5 , 0 0 0 x g f o r 15 m i n gives a lightly c o l o r e d s u p e r n a t a n t c o n t a i n i n g ca. 50% of the i n t e s t i n a l h o m o g e n a t e activity. T h e partial rec o v e r y at this step is due t o difficulties in solubilizing t h e e n z y m e f r o m t h e p r o t a m i n e precipitate rather than to inhibition by the d e t e r g e n t , w h i c h , at t h e a b o v e c o n c e n t r a t i o n s , does n o t a f f e c t t h e t o t a l p h o s p h o l i p a s e B activity.

Fractionation with ammonium sulfate I and H: T h e s u p e r n a t a n t o f the p r e c e d i n g step ( 1 4 0 ml) is m i x e d slowly, stirred w i t h 1 v o l u m e cold 80% s a t u r a t e d a m m o n i u m sulfate a n d allowed t o s t a n d in the cold for 15-20 min. T h e light p r e c i p i t a t e t h u s f o r m e d is s e p a r a t e d at 2 5 , 0 0 0 x g f o r 15 rain, a n d t h e slightly c l o u d y supern a t a n t is slowly a d d e d w i t h 0.5 v o l u m e cold s a t u r a t e d a m m o n i u m sulfate (final s a t u r a t i o n , 60%). A f t e r 3 0 rain in t h e cold, a well d e f i n e d p r e c i p i t a t e is p r e s e n t a n d is easily s e p a r a t e d b y c e n t r i f u g a t i o n f o r 15 m i n at 2 5 , 0 0 0 x g. Fractionation on calcium phosphate gel: T h e

pellets o f the p r e c e d i n g s t e p are dissolved in 4 0 ml cold 0. 1M Tris p H 7.4, 10-3M d i t h i o t h r e i t o l , a n d 12 ml c a l c i u m p h o s p h a t e gel s u s p e n s i o n ( 2 7 ) is added. A f t e r mixing, c e n t r i f u g a t i o n at 2 0 0 0 x g for 10 m i n s e p a r a t e s t h e gel, w h i c h is w a s h e d twice w i t h 60 ml 1% s o d i u m c h l o r i d e , 1 x 10-3M d i t h i o t h r e i t o l , a n d is finally e x t r a c t e d w i t h 20 ml ( a l t e r n a t i v e l y twice w i t h 10 m l a l i q u o t s ) 0.4M p o t a s s i u m p h o s p h a t e b u f f e r p H 7.4. Dialysis and storage: T h e gel eluate is dialy z e d o v e r n i g h t against two c h a n g e s of distilled w a t e r (8 liters), a n d t h e clear s o l u t i o n is s t o r e d f r o z e n at -15 C. The results o f o n e e x p e r i m e n t a l r u n are given in Table I. T h e p r o c e d u r e d e t a i l e d a b o v e gives p r e p a r a t i o n s averaging 1 m g p r o t e i n a n d 3 5 0 0 - 6 0 0 0 u n i t s l y s o p h o s p h o l i p a s e activity p e r milliliter; n o loss of activity in t h e f r o z e n s t a t e is d e t e c t a b l e over an e i g h t m o n t h period. Altern a t i v e l y , t h e dialyzed p r e p a r a t i o n can be freezedried a n d k e p t at -15 C as a p o w d e r . L y o p h y l i z a t i o n is a c c o m p a n i e d by a 50% loss of activity, b u t t h e p r e p a r a t i o n is stable a f t e r w a r d s over a

TABLE II Effect of Reaction Conditions on Phospholipase Activity Substrate a Phosphatidylethanolamine Phosphatidylcholine Asolectine Lysolecithin

None pH V pH V pH V pH V

5.2-5.9 95 0.0 6.2 45 5.2-7.3 418

Treatment or addition Sonication b Protein c Triton X-IO0 d 5.2 110 5.2 105 6.2 12

5.2-6.6 155 0.0 6.2 87 5.2-7.3 432

0.0 6.99 117 6.6 109

aSubstrate concentration: phosphatidylethanolamine 1.2 x 10-3M; phosphatidylcholine = 1.3 x 10-3M; asolectine = 1.1 x 10-3; lysolecithin = 1.4 x 10-3M. For each substrate the pH value or range indicated is that at which reaction velocity (V) is highest. V is given as millimicromoles of substrate hydrolyzed per hour by 1 ~g lyophilized enzyme. LIPIDS, VOL. 8, NO. 7

MOUSE INTESTINAL PHOSPHOLIPASE

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FIG. 1. Hydrolysis of mitochondrial phospholipids of rat liver by mouse phospliolipase. Hydrolysis rate expressed on abscissa as millimicromoles phospholipids hydrolyzed per hour by l #g lyophilized enzyme reacted with intact rat liver mitochondria (e s), their lipid extract (c-----~), and lipid extract added with mitochondrial protein (a----~). Phospholipid concentration in three series = 1.4; 1.6; and 1.6 #mol/ml, respectively; added protein = 1.01 mg/ml. prolonged period of time (more than 1 year in our experience). For testing with the various substrates the enzyme is best prepared in relatively concentrated solutions (0.5-1 mg protein/ml), since rapid loss of activity follows dilution in water at concentrations below 200 btg protein per milliliter. Addition of electrolytes or serum albumin is without effect, and only partial stabilization is achieved by the presence of SH groups (2 x 10-3M dithiothreitol). We have routinely used microliter volumes of freshly thawed material added to pre-equilibrated mixtures of buffers and substrates with good reproducibility of results. Further purification of the enzyme by gel filtration or ion exchange chromatography (Deae-Sephadex) is impractical, since little or no gain in specific activity is obtained by either procedure. Gel electrophoresis shows the presence of seven distinct bands (four major, three minor), two of which are associated with phospholipase activity recoverable from the sliced gel by extraction with glycerol buffer. Peak activity coincides with a band corresponding to 25% of the stained protein in the gel. Substrates

Mouse intestinal preparations readily hydrolyze the diacylphospholipids of mitochondria, microsomes, red cell ghosts and bacterial membranes, and attack with varying affinity the individual phospholipids reacted in the form of aqueous dispersions. The hydrolytic reaction

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FIG. 2. Hydrolysis of microsomal phospholipids of rat liver by mouse phospholipase. On abscissa hydrolysis rate expressed as millimicromoles phospholipid hydrolyzed per hour by 1 #g lyophilized enzyme reacted with: intact rat liver microsomes (: --); their lipid extract (c~-----o);lipid extract added with microsomal protein (A A). Phospholipid concentration in three series = 1.96; 2.39; and 2.39 #mol/ml, respectively. Added microsomal protein = 600 #g/ml. and the attendant biochemical characteristics (pH optima, apparent Vmax) are strongly influenced by the physical state of the substrates and the presence of nonlipid material (Figs. 1-5, Table II). Figure 1 shows the rather broad range of pH values at which the phospholipids of intact rat liver mitochondria are hydrolyzed. Separation of the lipid moiety by extraction with aqueous acetone-ammonia results in a considerable reduction of reaction rate and pH range with a shift of the optimum toward the neutral region. Addition of the protein residue to the lipid extract restores the pH curve to a range and height comparable to those seen with intact mitochondria (Fig. 1). Similar results are obtained with liver microsomes (Fig. 2). Recombination experiments of the type described above indicate that restoration of the protein to phospholipid ratio present in the intact organelles is not essential, since one-third of the equivalent protein is sufficient to produce a maximal effect on both hydrolysis rate and pH range. In this respect the mitochondrial protein is more effective than the corresponding microsomal preparation on a protein weight basis, even when microsomal phospholipids are the substrate. Partially purified phospholipid mixtures (Asolectin) and individual phospholipids behave LIPIDS, VOL. 8, NO. 7

A. OTTOLENGHI

420

TABLE III Stoichiometry of Reaction Products during Hydrolysis of Phospholipids by Mouse Phospholipase a Substrate

-APLP-P

+FA

FA/P

3.03 3.34 0.84 2.53

5.91 6.82 1.72 4.'/5

1.95 2.04 2.05 1.88

3.88 4.68

7.38 9.02

1.90 1.93

Egg lecithin Egg-phosp hatidylethano lamine Rat liver mitochondria

Reaction conditions or additions Sonicated, pH 5.3 Sonicated, pH 5.3 pH 6.99, Triton-X pH 6.23 Mitochon drial protein pH 6.23 pH 6.23

aValues in columns headed -APLP-P and +FA indicate, respectively, micromoles of phospholipid hydrolyzed and of fatty acid liberated under reaction conditions specified in last column. Table was compiled from experimental data obtained in a number of experiments using various enzymatic preparations of different specific activity. X - 1 0 0 has b e e n c o n f i r m e d i n a n u m b e r o f e x p e r i m e n t s similar to t h o s e s h o w n in Figure 4. Among the substrates tested, lysophosphat i d y l c h o l i n e shows t h e highest r e a c t i v i t y over a b r o a d p H range u n a f f e c t e d b y a d d i t i o n of p r o t e i n (Fig. 5), d e t e r g e n t or s o n i c a t i o n ; h e n c e it was selected as t h e s u b s t r a t e o f r e f e r e n c e d u r i n g the d e v e l o p m e n t o f t h e e n z y m e purific a t i o n p r o c e d u r e . O t h e r s u b s t r a t e s f o u n d susceptible to mouse phospholipase include p u r i f i e d p h o s p h a t i d i c acid a n d t h e p h o s p h o lipids o f bacterial m e m b r a n e vesicles (28-31).

similarly. A d d i t i o n o f p r o t e i n or d e t e r g e n t or s o n i c a t i o n o f the s u b s t r a t e c a n greatly i n f l u e n c e t h e rate of the h y d r o l y t i c r e a c t i o n , a l t h o u g h n o single e x p e r i m e n t a l variable i n d u c e s similar effects o n all s u b s t r a t e s t e s t e d , t h u s u n d e r l i n i n g t h e c o m p l e x i t y of t h e i n t e r a c t i o n s i n v o l v e d in initiation and enhancement of the enzymatic attack. F o r e x a m p l e , a d d i t i o n of m i t o c h o n d r i a l p r o t e i n a u g m e n t s b o t h r e a c t i o n rate a n d p H range for PE a n d A s o l e c t i n s u s p e n s i o n s (Fig. 3, Table I1) b u t has n o e f f e c t o n PC, w h i c h becomes susceptible to hydrolysis only after dispersion b y s o n i c a t i o n or b y a d d i t i o n o f T r i t o n - X (Fig. 4). These p r o c e d u r e s , b y contrast, r e d u c e t h e r e a c t i v i t y o f t h e o t h e r t w o s u b s t r a t e s as s h o w n in T a b l e II. T h e p r e s e n c e of t w o p H o p t i m a for PC a f t e r a d d i t i o n o f T r i t o n

Reaction

Products

Phospholipid hydrolysis by the mouse phosp h o l i p a s e results in l i b e r a t i o n o f f a t t y acids a n d a c o n c o m i t a n t decrease in p h o s p h o l i p i d P. As s h o w n in T a b l e III the m o l a r ratios of f a t t y acid t o P are q u i t e u n i f o r m u n d e r widely d i f f e r e n t experimental conditions and indicate that the

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3 4 5 6 7 8 pH. FIG. 3. Hydrolysis of phosphatidyl ethanolamine by mouse phospholipase. On abscissa reaction rate expressed as millimicromoles phosphatidylethanolamine hydrolyzed per hour by 1 ~g lyophilized enzyme reacted with 1.18 /~mol/ml substrate in aqueous dispersion (= =); after sonication (3 x 3' at 10 KC) (~----A); after addition of mitochondrial protein (335 #g/ml) (* x). LIPIDS, VOL. 8, NO. 7

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FIG. 4. Hydrolysis of phosphatidylcholine by mouse phospholipase. Hydrolysis rate expressed on abscissa as millimicromoles phosphatidylcholine hydrolyzed per hour by 1 ~g lyophilized enzyme reacted with phosphatidylcholine (1.33 umol/ml) in aqueous dispersion (~---~); after sonication (3 x 3' at 10 KC) (-" -'); after addition of mitochondrial protein (335 ~g/ml) (= --); after addition of triton X (8.3 #l/m1) (~--~).

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FIG. 5. Hydrolysis of lysophosphatidylcholine by mouse phospholipase. Hydrolytic rates are given as millimicromoles of lysophosphatidylcholine hydrolyzed per hour by 1 #g lyophilized enzyme reacted with lysophosphatidylcholine (1.44 #mol/ml) in aqueous dispersion (-- -'); or after addition of mitochondrial protein (500 #g/ml) ( ; ;). diacylphospholipids are hydrolyzed without accumulation of detectable amounts of lysophospholipids. The absence of a lysolecithin spot in thin layer chromatograms of reaction samples withdrawn at different stages of the hydrolytic reaction with PC supports this conclusion. Hydrolysis of PC and lysoPC is accompanied by the accumulation of acid soluble P and glycerylphosphorylcholine (GPC). Table IV shows the quantitative relationship between these products. Qualitative analysis by paper chromatography confirmed the presence of both GPC and its ethanolamine-containing analog, GPE in red cell ghosts incubated with mouse phospholipase. Attempts at separation of phospholipase A and B activities by the addition to PC of deoxycholate over a wide range of concentrations were unsuccessful due to a concurrent inhibition of the hydrolytic reaction as a whole. At present no indication that the two activities can be separated has been obtained, despite the variety of experimental conditions employed, including the multiple steps of the purification procedure as shown in Table V. on Biological

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91 8 0 -

Membranes

The mouse intestinal phospholipase was tested for its action on a number of biological membranes to ascertain its possible use as a tool in special experimental conditions, as is current

FIG. 6. Response to osmotic challenge of Pseudomonas aeruginosa cells after treatment with phospholipase, o-----e Control cells; ~ cells + polymyxin, 5 #g/ml; ~ cells + polymyxin, 10 ~g/ml; -- -- cells + polymyxin, 5 /~g/ml, + phospholipase, l 1 #g/ml;-" " cells + polymyxin, 10 /~g/ml, + phospholipase, 11 #g/ml. practice with other phospholipases (A1, A2, C, D). Intact bacterial cells, red blood cells and their ghosts and beef heart mitochondria were selected for this purpose, and the results are summarized below. Bacterial cells: Incubation of intact bacterial cells ( P s e u d o m o n a s a e r u g i n o s a ) with mouse phospholipase is without effect on bacterial phospholipids, although the latter are rapidly hydrolyzed when suspensions of cells disrupted by sonication are exposed to the enzyme. The apparent protection of the phospholipids from enzymatic attack in the i n t a c t cell can be removed by treatment with relatively low concentrations of Polymyxin, known to induce definite changes in bacterial membrane morphology (33). The two agents appear to exert cumulative effects with regard to alterations in cell function, since their combination causes a considerable impairment in the ability of the cells to react to osmotic challenge. Figure 6 (curve A), shows the characteristic response of normal cells to an increase in tonicity consisting of (a) a rapid decrease in cell size which is reflected in the sharp increase in optical density readings, followed by (b) a slow swelling process extending over 40 min and measured by the slow decline of the OD curve. While addition of polymyxin alone is without effect (curves B and C), combination with phospholipase causes lowering of the maximum OD value and acceleration of the swelling process in proportion to the amount of polymyxin (curves D and E). According to the evidence presented by Bernheim (34-36), these changes represent alterations in the permeability of bacterial LIPIDS, VOL. 8, NO. 7

422

A. OTTOLENGHI TABLE IV

TABLE V

Glycerylphosphorylcholine Formation from Phosphatidylcholine and Lysophosphatidylcholine Hydrolyzed by Mouse Phospholipase

Hydrolytic Activity of Mouse Intestinal Preparations on Diacyl- and Monacylphospholipids a

Reaction products b +FA or Substrate a -APLP-P ASP GPC Phosphatidylcholine 0.76 0.78 0.70 1.14 1.13 1.16 Lysophosphatidylcholine 1.48 1.55 2.55 2.92 4.11 4.22 4.05 aReaction conditions: (a) phosphatidylcholine (sonicated) 1.6 x 10-3M, acetate buffer pH 5.2 0.066M, enzyme 27 pg/ml; (b) lysophosphatidylcholine 6.6 x 10-3M, tricine buffer pH 6.6 0.066M, enzyme 8 pg/ml. bReaction products expressed as micromoles of hydrolyzed phospholipid (-APLP, for phosphatidylcholine; +FA for lysophosphatidylcholine and of released acid soluble P (ASP) and glycerylphospholycholine (GPC). m e m b r a n e s due to loss of selectivity to permeants. Work done in o t h e r laboratories (28-31) using m o u s e phospholipase preparations supplied by us has c o n f i r m e d the susceptability of bacterial m e m b r a n e phospholipids to attack by this e n z y m e . Red blood cells: Purified mouse phospholipase is w i t h o u t effect on the phospholipids of red cells f r o m various species (man, rat, sheep, pig) i n c u b a t e d in c o n d i t i o n s preventing hemolysis. It should be emphasized that crude intestinal h o m o g e n a t e s contain a lytic factor, which is effectively separated f r o m the phospholipase during the calcium p h o s p h a t e gel absorption step. E n z y m e preparations preceding this treatm e n t cause hemolysis and hydrolysis of RBC phospholipids, developing with apparently identical time courses in the initial stages of the reaction. Later preparations are inactive. In contrast with the lack of action on intact red cells, the mouse phospholipase readily hydrolyzes the phospholipids of red cell ghosts with liberation of free fatty acids and a corresponding decrease in phospholipid P. The h y d r o l y t i c process is a c c o m p a n i e d by a p r o f o u n d alteration of the ghost m o r p h o l o g y as revealed by freeze fracture electron micrographs. The normal pattern of distribution of particles on the internal fracture face of the ghosts consisting of small chains or clusters is shown in Figure 7A. A f t e r t r e a t m e n t with mouse phospholipase at different concentrations, areas of d e n u d a t i o n and f o r m a t i o n of large clusters are seen (B,C) indicating a rearr a n g e m e n t of the constitutive elements of the ghost m e m b r a n e . The m o r p h o l o g i c a l changes show some similarity to those observed by LIPIDS, VOL. 8, NO. 7

Preparation

PC

Homogenate 8000x gx 10' supernatant Tris-Triton extract from protamine, ppt 60% Ammonium sulfate, ppt Calcium phosphate get eluate

Substrate LysoPC PC]lysoPC

61

690

.088

81

841

.096

75

625

.120

97

1370

.071

267

3624

.078

aHydrolytic activity of various preparations of mouse intestinal mucosa reacted with phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) is given as micromoles hydrolyzed per hour per milligram protein. Values of last column are ratios of activities for two substrates. Weinstein and Wallach (37) after t r e a t m e n t of RBC ghosts with phospholipase C. Beef heart mitochondria: Rapid hydrolysis of the diacylphospholipids of these preparations occurs u p o n i n c u b a t i o n w i t h small concentrations of mouse phospholipase. Respirat o r y activity (succinate-cytochrome C reductase) is inhibited and can be restored by addition of microdispersed phospholipids. No a c c u m u l a t i o n of lysophospholipids can be detected and the necessity of multiple washes with albumin, as is the case w i t h snake v e n o m phospholipase, is obviated. DISCUSSI ON

The results r e p o r t e d here show the presence in the intestine of Hymenolepis nana-infected mice of an active phospholipase, capable of attacking all the major diacylphospholipids with liberation of free fatty acids and the corresponding g l y c e r y l p h o s p h o r y l fragments. In the following discussion the question of classification and o t h e r special aspects of the activity of the e n z y m e will be considered. Current t e r m i n o l o g y identifies the acylhydrolases acting on diacylphosphatides on the basis of their positional specificity as phospholipases A 1 and A2, and designates the e n z y m e s acting on m o n o a c y l p h o s p h a t i d e s (lysophospholipids) as lysophospholipases (E.C. 3.1.1.5.). This classification does n o t include all possible m o d e s o f e n z y m a t i c attack and McMurray and Magee (1) have recently p r o p o s e d the term "phospholipase B" ( h i t h e r t o used for the E.C. 3.1.1.5.) for " t h e e n z y m e which presumably hydrolyzes b o t h of the acyl ester linkages in diacylphosphatides s i m u l t a n e o u s l y . " The same

MOUSE INTESTINAL PHOSPHOLIPASE

423

FIG. 7. Action of mouse phospholipase on red cell ghosts. A = Control RBC ghosts; B,C = RBC ghosts incubated for 45 min with 120 and 360 gg phospholipase per milliliter, respectively, authors p o i n t out that the presence of several acylhydrolases in the same tissue often complicates the task of identifying single e n z y m a t i c activities. Such might be the case in the e x p e r i m e n t s r e p o r t e d here, in which a separation o f activities (diacyl- and monoacylhydrolases) did n o t o c c u r t h r o u g h o u t the sequence of

purification or after a d d i t i o n of d e o x y c h o l a t e . The negative results w i t h the latter p r o c e d u r e are particularly significant, since this is the usual m e t h o d for d e m o n s t r a t i n g in vitro the presence of the A 2 t y p e of e n z y m e . Differentiation on the basis of p H o p t i m a is i n f i r m e d by the d e p e n d e n c e of this p a r a m e t e r on such LIPIDS, VOL. 8, NO. 7

424

A. OTTOLENGHI

factors as s u b s t r a t e dispersion a n d t h e p r e s e n c e of o t h e r materials. T h e failure t o i d e n t i f y t w o e n z y m a t i c activities c a n be i n t e r p r e t e d for t h e time b e i n g as an i n d i r e c t i n d i c a t i o n of t h e p r e s e n c e in m o u s e i n t e s t i n e of a h y d r o l a s e a c t i n g o n b o t h acyl e s t e r b o n d s ( t y p e B of M c M u r r a y a n d Magee). This is, a d m i t t e d l y , a w o r k i n g h y p o t h e s i s t o be t e s t e d d i r e c t l y b y f u r t h e r studies. While t h e p r e s e n c e o f y e t a n o t h e r p h o s p h o lipase in the i n t e s t i n e is n o t surprising, in view of t h e m u l t i p l i c i t y o f e n z y m e s of this t y p e f o u n d in m a n y tissues, it is of c o n s i d e r a b l e i n t e r e s t t h a t s u c h h i g h levels s h o u l d develop following worm infection. An accompanying s h o r t c o m m u n i c a t i o n ( 1 1 ) details t h e actual q u a n t i t i e s , t h e time course a n d the d i s t r i b u t i o n of the e n z y m e in the i n t e s t i n a l tissue d u r i n g the various phases o f the i n f e c t i o n . I t suffices to p o i n t o u t here t h a t in t h e areas of h i g h e s t c o n c e n t r a t i o n , p h o s p h o l i p a s e activity can be readily s h o w n u s i n g as little as 2 0 - 5 0 / a g tissue, ca. 100 t i m e s less t h a n t h e a m o u n t n e e d e d w h e n o t h e r organs or n o r m a l i n t e s t i n e s are used. It seems r e a s o n a b l e t o i n f e r t h a t the p r o n o u n c e d increase in p h o s p h o l i p a s e activity has some y e t u n k n o w n r e l a t i o n s h i p to e i t h e r t h e local i n j u r y or the d e v e l o p m e n t of a p r o t e c t i v e b a r r i e r against the parasite. I n e i t h e r case, a n o v e l a n d u n s u s p e c t e d f u n c t i o n for the e n z y m e is suggested. The m o u s e i n t e s t i n a l p h o s p h o l i p a s e e x h i b i t s the wide v a r i a t i o n in p H o p t i m a a n d t h e d e p e n d e n c e on t h e p h y s i c a l s t a t e of t h e substrate t h a t has b e e n r e p o r t e d for a l m o s t all e n z y m e s of this class, irrespective of t h e i r sources ( 3 6 - 3 9 ) . As already p o i n t e d out, the c o n d i t i o n s t h a t m a x i m i z e t h e h y d r o l y t i c activity differ c o n s i d e r a b l y for the various s u b s t r a t e s a n d n o single e x p e r i m e n t a l variable applies equally t o all. O f t e n i n v e r s i o n of e f f e c t is seen in passing f r o m o n e s u b s t r a t e t o a n o t h e r ; this is true w i t h r e g a r d to b o t h t h e p h y s i c a l state of the s u b s t r a t e ( s o n i c a t i o n ) or t h e a d d i t i o n of dispersing m a t e r i a l ( d e t e r g e n t , p r o t e i n ) . T h e e f f e c t of p r o t e i n n o r m a l l y associated w i t h p h o s p h o l i p i d s in cell organelles is clearly s h o w n in the e x p e r i m e n t s u s i n g lipid e x t r a c t s f r o m m i t o c h o n d r i a a n d m i c r o s o m e s a n d some of t h e purified s u b s t r a t e s . The fact t h a t lipid m i x t u r e s are m o r e readily a t t a c k e d t h a n t h e i r p u r i f i e d c o m p o n e n t s i n d i c a t e s t h e i m p o r t a n c e o f multiple lipid-lipid a n d l i p i d - p r o t e i n i n t e r a c t i o n s . A n o t h e r aspect of the r e l a t i o n s h i p b e t w e e n e n z y m a t i c a t t a c k a n d the special s t a t e of t h e p h o s p h o l i p i d s is revealed in t h e e x p e r i m e n t s u s i n g e r y t h r o c y t e s a n d P s e u d o m o n a s aeruginosa. Our results are in a g r e e m e n t w i t h r e p o r t s , p u b l i s h e d d u r i n g the course of o u r w o r k LIPIDS, VOL. 8, NO. 7

( 4 1 - 4 4 ) , s h o w i n g the v i r t u a l inaccessibility of the m e m b r a n e p h o s p h o l i p i d s to a c y l h y d r o l a s e s as l o n g as t h e m e m b r a n e s t r u c t u r e is i n t a c t . H e m o l y s i s (or p r e t r e a t m e n t w i t h p o l y m i x i n in the case of the P s e u d o m o n a s cells) is sufficient to e x p o s e t h e p h o s p h o l i p i d s t o t h e a c t i o n of the e n z y m e . T h e m o r p h o l o g i c a l a l t e r a t i o n s of the RBC m e m b r a n e suggest a m o b i l i z a t i o n of some of its c o m p o n e n t s a f t e r h y d r o l y s i s of t h e p h o s p h o l i p i d s , as already s h o w n to be t h e case for t h e p h o s p h o l i p a s e C-treated ghosts ( 4 5 ) . It is of i n t e r e s t t h a t in t h e bacterial cells t h e b i o c h e m i c a l lesion is a c c o m p a n i e d b y a measurable a l t e r a t i o n in f u n c t i o n i n v o l v i n g loss of p e r m e a n t selectivity. O t h e r w o r k e r s ( 2 8 - 3 1 ) have u s e d o u r e n z y m e t o digest p h o s p h o l i p i d s o f m e m b r a n e vesicles of E. coli a n d have s h o w n a d i f f e r e n t i a l e f f e c t o n c a t a l y t i c activities associated w i t h t r a n s p o r t a n d t h e ability of t h e cells to r e t a i n t r a n s p o r t e d solute. T h e general s u s c e p t i b i l i t y o f p h o s p h o l i p i d s of cellular organelles a n d of e x p o s e d m e m b r a n e s to t h e a c t i o n of t h e e n z y m e is also confirmed by the effect on beef heart mitoc h o n d r i a . We have already r e p o r t e d elsewhere the a l t e r a t i o n s in n e r v e f u n c t i o n associated w i t h t r e a t m e n t o f t h e s q u i d giant a x o n w i t h p h o s p h o l i p a s e (46). In c o n c l u s i o n , a b r o a d s p e c t r u m of activity has b e e n s h o w n for this e n z y m e , w h i c h is n o w available in a stable, partially p u r i f i e d f o r m , t e c h n i c a l l y u s e f u l for f u r t h e r s t u d y as a m o d e l of p h o s p h o l i p a s e a c t i o n a n d as a tool f o r t h e i n v e s t i g a t i o n o f biological m e m b r a n e s . ACKNOWLEDGMENTS This work was supported in part by Grant AT(40-1)-3329 from the Atomic Energy Commission and Grant GB-29752X1 from the National Science Foundation. Technical assistance was provided by J.T. Rowland. R.E. Kohls (Norwich Pharmacal Co.) aided in establishing a colony of infected mice, and R.G. Kirk (Duke Medical Center) collaborated in the electron microscopy studies. REFERENCES 1. McMurray, W.C., and W.L. Magee, Ann. Rev. Biochem. 41:129 (1972). 2. Hanahan, D.J., "The Enzymes," Vol. V, Edited by P.D. Boyer, Academic Press, New York, 1971, p. 71. 3. Van Golde, L.M.G., R.N. McElhaney and L.L.M. Van Deenen, Biochim. Biophys. Acta 231:245 (1971). 4. Epstein, B., and B. Shapiro, Biochem. J. 71:615 (1959). 5. Marples, E.A., and R.H.S. Thompson, Ibid. 74:123 (1960). 6. Robertson, A.F., and W.E.M. Lands, Biochemistry 1:804 (1962). 7. Ottolengh.i, A., J. Lipid Res. 5:532 (1964).

MOUSE INTESTINAL PHOSPHOLIPASE 8. Gallai-Hatchard, J.J., and R.H.S. Thompson, Biochim. Biophys. Aeta 98:128 (1965). 9. Ottolenghi, A., Lipids 5:531 (1970). 10. Subbaiah, P.V., and J. Ganguly, Biochem. J. 118:233 (1970). 11. Ottolenghi, A., Lipids 8:426 (1973). 12. Ansell, G.B., and S.N. Hawthorne, "Phospholipids," Elsevier Publishing Co., Amsterdam, 1964, p. 87. 13. Ansell, G.B., and S.N. Hawthorne, Ibid., p. 99. 14. Long, G., and I.F. Penney, Biochem. J. 74:123 (1960). 15. Fleischer, S., and B. Fleischer, "Methods in Enzymology," Vol. X, Academic Press, New York, 1967, p. 423. 16. Fleischer, S., and B. Fleischer, Ibid., p. 412. 17. Bligh, E.G., and W.J. Dyer, Can. J. Biochem. Physiol. 37:911 (1959). 18. Fiske, C.H., and Y. Subbarow, J. Biol. Chem. 66:375 (1925). 19. Barlett, G.R., Ibid. 234:466 (1959). 20. Dole, V., J. Clin. Invest. 35:150 (1956). 21. Mahadevan, S., C.J. Dillard and A.L. Tappel, Analyt. Biochem. 27:387 (1969). 22. Skipski, V.P., and M. Barclay, "Methods in Enzymology," Vol. XIV, Academic Press, New York, 1966, p. 566. 23. Appleton, H.D., B.N. La Du, Jr., B.B. Levy, J.M. Steele and B.B. Brodie, J. Biol. Chem. 205:803 (1953). 24. Ottolenghi, A., and F. Bernheim, Rad. Res. 15:609 (1961). 25. Dawson, R.M.C., N. Hemington and J.B. Davenport, Biochem. J. 84:497 (1962). 26. Fleischer, S., and B. Fleischer, "Methods in Enzymology," Vol. X, Academic Press, New York, 1967, p. 427. 27. Wood, W.A., "Methods in Enzymology," Vol. II, Edited by S.T. Colowick and N.O. Kaplan, Academic Press, New York, 1955, p. 214. 28. Kaback, H.R., Ann. Rev. Biochem. 39:561 (1970). 29. Kaback, H.R., E.M. Barnes, Jr., and L.S. Milner, in "Recent Advances in Microbiology," Edited by A. Perez-Miravete and D. Pelaez, Liberia Inter-

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[ Revised m a n u s c r i p t r e c e i v e d A p r i l 5, 1 9 7 3 ]

LIPIDS, VOL. 8, NO. 7