Phosphatidylinositol specific isoenzymes of phospholipase D from Catharanthus roseus. Purification and characterization

plan cience ELSEVIER SCIENCE IRELAND

Plant Science 97 (1994) 143-151

Phosphatidylinositol specific isoenzymes of phospholipase D from C a t h a r a n t h u s roseus. Purification and characterization Andrea Becher, Josef B. Wissing, Claudia Wylegalla, Karl G. Wagner* Gesellschaft fiir Biotechnologische Forschung, Ag. Enzymologie, Mascheroder Weg 1, D-38124 Braunschweig, Germany (Received 5 October 1993; revision received 14 December 1993; accepted 10 January 1994)

Abstract Microsomal membranes from suspension cultured Catharanthus roseus ceils were found to contain phospholipase D activity (EC 3.1.4.4) towards phosphatidylinositol (PI). After extraction with buffer containing Triton X-100, two isoenzymes with apparent molecular weights of 50 000 and 125 000 were partially purified. The enzymes accepted only PI as substrate and transphosphatidylation activity could not be detected. Whereas several phospholipids were slightly inhibitory, phosphatidyl glycerol had stimulatory properties. The enzymes required divalent cations for activity, Ca 2+ and Mg 2÷ were equally effective. In the presence of deoxycholate and Ca 2÷, Kmvalues for PI were found to be in the range 20-40/zM. Although the smaller protein had a slightly higher temperature stability and a slightly lower pI value, both isoenzymes revealed equal enzymic properties. This is the first report on a plant PI-specific phospholipase D. Key words: Catharanthus roseus; Microsomal membranes; Phospholipases; Suspension cultured plant cells

1. Introduction Phospholipases of type D (PLD) cleave phospholipids between the glycerol-bonded phosphate and the hydrophilic head group [1-3]. Whereas the data on phosphatidylinositol (PI)-

* Corresponding author. Abbreviations: Chaps, 3-[(3-cholamidopropyl)dimethylamino]l-propanesulfonate; CL, cardiolipin; DG, diacylglycerol; HEPES, N-2-hydroxyethylpiperazine-N'-2-et hanesulfonic acid; lns-lP, inositol-l-phosphate; [lns-2-3H]PI, phosphatidyl[2-3H]inositol; Mes, 2-(N-morpholino)ethanesulfonic acid; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol, PIP, phosphatidylinositol monophosphate; PLD, phospholipase D; PS, phosphatidylserine.

specific enzymes are scant, phosphatidylcholine (PC)-specific activities have been described from both animal and plant tissues. The PC-specific enzymes from both kingdoms were shown to possess phosphatidyltransferase activity [1-3]. In the case of the animal cells, interest in PLDcatalysed degradation of PC was mainly focused on the role of the cleavage products in signal transduction [4,5]. On the other hand, PC-specific enzymes from plants were highly purified [6-8] and applied for the preparation of various phospholipids exploiting their phosphatidyltransferase activity [9-12l. Much less is known on the role and properties of enzymes with PI specificity. Whereas evidence of their existence has been reported from animal

0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0168-9452(94)03806-K

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cells [13,14], in plant cells, PI-specific activity was unknown up to now [1-3], although an enzyme with broad specificity was described recently which displayed phosphatidyltransferase and hydrolase activity towards PC and PI [15]. The present work describes the partial purification and properties of two isoenzymes of PI-specific phospholipase D that were extracted from the microsomal fraction of suspension cultured Catharanthus roseus cells. 2. Materials and methods 2.1. Plant cells Catharanthus roseus suspension cultured cells

were grown in Murashige-Skoog medium with 3% sucrose, 5.4 #M c~-naphthylacetic acid, and 0.5 #M kinetin in the dark at 27°C and subcultured every 2 weeks by transferring 3 g cells into 80 ml of new medium. 2.2. Enzyme assays

Activities were determined with [Ins-2-3H]PI (33 Bq/nmol) as substrate at 30°C. Unlabelled PI (from soybean, 99%), dissolved in chloroform, was added into a reaction vessel and dried under a stream of nitrogen; water containing the deoxycholate was added, to give a 250/zM PI and 5 mM deoxycholate concentration and the mixture was sonified four times with a Branson sonifier (40 W, 10 s) interrupted by cooling in ice (1 min). Labelled PI, dissolved in toluene/ethanol (1:1), was added into a separate reaction vessel and dried under a stream of nitrogen; thereafter the solution of unlabelled PI with deoxycholate was added and the mixture was vortexed for 30 min. The enzyme (10 /zl, 0.3-1.4 /zg protein) was added to 50 /~1 assay buffer (100 mM HEPES, 20 mM CaC12, 50 mM NaC1, pH 7.5) and incubated for 5 min at 30°C; the reaction was started by the addition of PI (40 tzl). After 20 min the reaction was stopped by addition of 370 #1 chloroform/methanol (1:2) containing 1% concentrated HC1, 120/zl 2 M KC1, and 120 #1 chloroform. After vortexing the phases were separated by centrifugation for 1 min at maximum speed in an Eppendorf 3200 centrifuge. The lower phase was discarded, the aqueous upper phase was washed with 500 /zl chloroform and

A. Becher et al./Plant Sci. 97 (1994) 143-151

again centrifuged. Then, 200/zl of the upper phase were added to 3 ml Quicksafe A (Zinsser Analytic) and counted in a BF-Betascint 5000 (Berthold). To discriminate between phospholipase D and C activity, aliquots of the water phase were analysed by anion-exchange HPLC as described previously [16]. The label corresponding to inositol and inositol-l-P, respectively, was obtained from the total amount of radioactivity determined by liquid scintillation counting, multiplied by the fractions of label obtained by HPLC. Ins-1P phosphatase activity was tested at the conditions described above for the phospholipase D assay omitting PI and adding [2-3H]Ins-lP (570 Bq). Extraction was performed as described above and the products of the aqueous phase were separated on small Dowex anion exchange columns [16]. 2.3. Enzyme purification

All buffers contained 0.1 mM phenylmethylsulfonyl fluoride; with the exception of buffer A, all buffers contained 15 mM mercaptoethanol and 10% (v/v) glycerol. In addition, buffer A contained 25 mM Tris/Mes (pH 6.8), 300 mM sucrose, 10 mM KC1, 1 mM MgCI 2, and 0.1% BSA; buffer B contained 40 mM Tris-HCl (pH 7.5), 50 mM NaC1, 5 mM MgCI 2, 1 mM EDTA, and 1% Triton X-100; buffer C was as buffer B but contained only 0.1% Triton X-100; buffer D was as buffer C but contained 1 M NaCI; buffer E was as buffer C but contained 100 mM NaC1. The cells (400 g) were harvested on the 7th or 8th day of the growth cycle by filtration, suspended in 1200 ml ice-cold buffer A and homogenized with 6 pulses of 30 s duration (interrupted by 2 min cooling in ice) with an Ultra Turrax homogenizer (Janke and Kunkel). The suspension was filtered through Micracloth (Calbiochem) and centrifuged for 10 min at 800 x g. The supernatant was centrifuged at 28 000 x g for 1 h (Sorvall RC2-B, GSA-rotor). The pellets (microsomal fraction) were extracted with 200 ml buffer B; 60-ml fractions were treated with 30 strokes (maximum speed) in a PotterElvehjem homogenizer. The suspension was centrifuged at 48 000 x g for 60 min and the supernatant stored frozen at -70°C. For Q-Sepharose (XK 26/20, Pharmacia) chro-

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A. Becher et al./Plant Sci. 97 (1994) 143-151

matography, the extract of 400 g cells was applied to a 50-ml column (2.6 x 9.5 cm, flow rate 2 ml per min), which had been equilibrated with buffer C, and was eluted with the following NaC1 gradient: 100 ml buffer C, linear increase from buffer C to 50% buffer D (600 ml), then linear increase to 100% buffer D (100 ml), and 200 ml buffer L. The fractions containing activity were stored at -70°C. To exchange buffer D for buffer C, the fractions were applied to a 390-ml Sephadex G-25 (XK 50/30, Pharmacia) column (5 x 20 cm, flow rate 2 ml per min) which was equilibrated and eluted with buffer C. To concentrate, the fractions containing activity were pooled, applied to a 8-ml QSepharose (HR 10/10, Pharmacia) column (1 x 10 cm, flow rate 1 ml per min, equilibrated with buffer C), and eluted with buffer C/buffer D (1:1). The total activity was collected in 7 ml and stored at -70oc. Preparative gel filtration was performed on a 500-ml Sephacryl S 200 (HR, Pharmacia) column (2.6 x 94 cm, flow rate 1 ml per min) with buffer E (Fig. 1). Analytical gel filtration was performed with the same column as well as with a 26-ml Ultrogel TSK G-3000 SW column (Pharmacia) (0.75 x 60 cm) and a 100-ml Superose 6 (HR

16/50, Pharmacia) column (1.6 x 50 cm) using buffer E and Dextran Blue, thyroglobin, aldolase, ferritin, catalase, transferrin, alcohol dehydrogenase, BSA, chymotrypsinogen A and ribonuclease A (Pharmacia) as reference proteins. Isoelectric focusing was performed according to Persson and Overholm [17] with Immobiline pH 4 - 7 (Pharmacia); the re-swelling buffer contained 4% Triton X-100, 1.5% Chaps, 20% glycerol, 0.5% Ampholine pK 4-6.5 (Pharmacia), and 10 mM DTT. After development, the gel was washed four times (5 min) with 0.05% Triton X-100, 50 mM NaC1, 1 mM EDTA, 15 mM mercaptoethanol and cut into 2-mm strips which were eluted with 100/~1 assay buffer for 2 h at 4°C by shaking; 60/A were added to 40 #1 containing the substrate and assayed for phospholipase activity. Protein was determined according to Bradford [18]. [Ins-2-3H]phosphatidylinositol and [2-3H1myoinositol 1-phosphate were purchased from Amersham Buchler, unlabelled phospholipids were from Sigma Chemie, Triton X-100 f r o m Pierce Europe, and sodium deoxycholate from Serva Feinbiochemika. 3. Results 3.1. Activity assay and enzyme purification

I i i i E E o

=E

0.4

120

8°l

40

m

0.3

oE 0.2 /

\\

/

0.1 ~

)

0.0 150

200

250 Elution v o l u m e

300

350

ml

Fig. 1. Sephacryl S-200chromatographyof phospholipase D. The microsomalmembrane fraction, obtained from 240 g suspension culturedcells, was extracted,purified on Q-Sepharose, desalted on SephadexG-25, and concentratedon Q-Sepharose as describedin Section2. Sevenmillilitreswere applied on the Sephacryl column; 5-ml fractions were collected and the column was run with buffer E.

Phospholipase activity was determined at pH 7.5 in 50 mM HEPES buffer. In the absence of surfactant, a pH optimum between 6 and 7.5 was found, while in the presence of 2 mM deoxycholate, maximum activity occurred between pH 6 and 7 [19]. Deoxycholate is precipitated at slightly acidic pH and this pH range depended on the nature and concentration of ions present. Therefore, the pH of the assay was chosen to 7.5, although activity was slightly lower than at optimum pH. The substrate concentration was chosen as 100 /~M; under the assay conditions, the Km value of PI was about 37 /zM (described below). This ensured that under the assay conditions (20 min), the enzymic reaction was linear with time and with the amount of enzyme protein (up to 0.5 #g for the 50 kDa enzyme and up to 1.4/~g' for the 125 kDa enzyme) [1911 Purification started with the microsomal fraction obtained from suspension cultured Catharan-

146

A. Becher et aL / P l a n t Sci. 97 (1994) 143-151

thus roseus cells of the late log phase (day 7 or 8). Separate experiments showed that 85-90% of the enzyme activity and about 33% of the proteins were extracted from the microsomal membranes with buffer B which contained 1% Triton X-100. The extracted activity was rather stable; both storing at 4°C for 24 h and freezing and thawing did not exhibit any reduction. Separation on Q-Sepharose showed that about 30% of the protein and 15% of phospholipase activity were in the flow-through fraction (buffer C, 50 mM NaCI); the bound enzyme was eluted between 80 and 180 mM NaC1 in a single peak (not shown). Exchange of the buffer on a Sephadex G25 column and subsequent concentration on QSepharose resulted in an increase in the total activity of about 33%. The subsequent gel filtration (Fig. 1) led to two activity peaks resulting from proteins with obviously different sizes. The small third peak on the front could be an aggregation artifact eluting with the exclusion volume although this has not been thoroughly studied. Table 1 shows the data for the purification of the isoenzymes from 1400 g wet cells, although the chromatographic steps were performed with aliquots (400 g cells). Starting with the microsomal membranes, the purification factor for the individual isoenzymes was about 70 and the yield about 50% (about 5/~mol min -l from 1400 g wet cells). In the case of both fractions, SDS gel elec-

trophoretic separations and staining with silver nitrate showed several protein bands [19]; in order to obtain homogenous proteins, further purification steps would be required. The lipid content of the purified fractions has not been determined; however, from analyses in the case of PA kinase [20,21], a membrane-bound enzyme obtained from the same cells, one may conclude that the lipid content should be negligible after applying the three chromatographic steps. 3.2. Substrate and region specificity of phospholipase D Although C. roseus microsomal membranes contain PI-specific phospholipase activities of both D and C type [22], the conditions applied for the extraction and chromatographic purification resulted in the separation of the D-activity only, which was obviously also more stable than the Cactivity (data not shown). The activity peak of the first chromatographic step (Q-Sepharose, not shown) and the two peaks of the final gel filtration step (Fig. 1) were found to contain only D-activity. This was shown by separating the labelled products of hydrolysis from the aqueous phase by HPLC [16]; all fractions containing activity from the two peaks in Fig. 1 were analysed and exhibited only inositol as enzymic product, whereas inositol-l-phosphate (Ins-lP) was not formed. Furthermore, the main fractions were analysed for

Table 1 Purification of phospholipase D isoenzymes Step

Total protein (mg)

Total activity (nmol min -I)

Specific activity (nmol min -1 mg -I)

Degree of purification (-fold)

Membrane extract Q-Sepharose SephadexG-25Q-Sepharose Sephacryl S-200 Peak 1 Peak 2

1340 166 132 19 20.4

6414 5491 7280 1415 3561

4.8 33.1 55.2 74.5 174.6

1 6.9 11.5 71 a 67 a

The data are related to 1400 g (wet weight) C. roseus cells as starting material. The PLD activities were determined in the presence of 1 mM deoxycholate. aAssuming that the distribution of the activities of two isoenzymes was the same in the membrane extract as found in Sephacryl S200 chromatography (28% peak 1 and 72% peak 2), specific activities of the isoenzymes in the membrane extract were calculated as 1.05 (peak 1 enzyme) and 2.6 nmol min -I mg -l (peak 2 enzyme); this results in the indicated degrees of purification for the individual isoenzymes.

147

A. Becher et al./Plant Sci. 97 (1994) 143-151

phosphatase activity using labelled Ins-lP as described in Section 2; however, this activity could not be detected. The substrate specificity of both isoenzymes was tested with unlabelled PC, PE, PG, PS, and PI under standard assay conditions (100/zM or 250 pM lipid substrate). The formation of PA, visible after separation of the chloroform/methanol extract by TLC and staining with iodine vapour [23,24], was only detected with PI. All the other phospholipids did not reveal any cleavage product. Furthermore, phosphatidyl[N-methyl-aH]choline used as substrate (0.5 pCi) could not be cleaved by both isoenzymes, as evidenced by the standard assay and counting the labelled aqueous cleavage product. The monophosphorylated PI (PIP) was also not a substrate. [32p]PIP was prepared by using PI kinase as described [25], separated by preparative TLC, eluted from the silica gel by chloroform/methanol (l:l) and used in the standard assay (330 Bq). Transphosphatidylation was tested under standard assay conditions in the presence of unlabelled PI and 500 /~M choline, ethanolamine and glycerol, respectively. In this case the lipid products analysed by TLC only revealed PA and no indication of PC, PE or PG. Similar experiments were performed in the presence of 200, 500 or 1000 #M [3H]inositol and unlabelled PI (or unlabelled PA); however, labelled PI could not be extracted. Obviously, the present isoenzymes did not possess any transphosphatidylation activity.

zyme. These experiments were repeated twice and showed that the difference in the pI values of the two phospholipase D isoenzymes was significant. The temperature optimum (20 min reaction) was at 42°C in the case of both isoenzymes. When enzyme aliquots were incubated for 20 min at different temperatures, frozen and assayed at 30°C after thawing, the 50 kDa isoenzyme did not exhibit any reduction in activity at pre-incubation temperatures up to 56°C, whereas the 125 kDa enzyme showed reduction in activity at preincubation temperatures above 30°C. Obviously

800

A .=- ~ ~ - - - B - - __

Size exclusion chromatography on Sephacryl S200 showed two main fractions (Fig. 1). The total activity of the smaller protein was about twice that of the larger isoenzyme. Their apparent molecular weights determined by analytical gel filtration on Sephacryl S-200 (Section 2) were found to be 126 000 and 47 000. The smaller protein was also tested with Ultrogel TSK G-300 which gave a value of 50 000, whereas filtration on Superose 6 resulted in a value of 125 000 for the larger protein. Isoelectric focusing in the presence of Triton and Chaps followed by extraction and assaying the phospholipase activity resulted in a pI of 4.75 for the 50 kDa protein and 4.95 for the 125 kDa isoen-

Mg2+

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400

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20

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40

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3.3. Properties of the phospholipase D isoenzymes

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600

Mg2+

B

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200

5

10

15

20

25

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Fig. 2. Dependence of PLD activity (50 kDa isoenzyme) on divalent cations in the presence of 2 mM deoxycholate (A) and without surfactant (in the presence of 100 pM PG) (B). The pH values were 7.5 (A) and 6.7 (B). The data are average values from two (A) or three (B) determinations; SD were below 4- 10%.

A. Becher et al./Plant Sci. 97 (1994) 143-151

148

the larger protein denatured more quickly than the smaller one.

A

30

3.4. Influence of divalent cations Fig. 2A shows that phospholipase D is dependent on the presence of divalent cations with Ca slightly more active than Mg, although with both ions the same plateau was obtained (2 mM deoxycholate). When the enzyme was tested in the presence of 1 mM deoxycholate (not shown), the higher activation by Ca compared with Mg in the lower concentration range was more obvious. The activity was nearly zero without the addition of divalent cations, although from the enzyme preparations (buffer E, 5 mM MgCI2) small amounts of Mg were introduced into the assay mixture (50 #M and 5 #M in the case of the 125 kDa and 50 kDa isoenzyme, respectively). Mn ions also showed a slight activation at 2 mM, but higher concentrations were inhibitory; Co and Cu ions, however, revealed only a strong inhibition. Although the data of Fig. 2 were obtained with the 50 kDa isoenzyme, the 125 kDa enzyme showed the same behaviour with no significant differences. The results of Fig. 2A also show that the standard assay conditions with 10 mM CaCI 2 were near to optimum. To exclude specific surfactant-divalent cation interactions, with the 50 kDa isoenzyme, the cations were tested (at pH 6.7) in the absence of deoxycholate (Fig. 2B) but in the presence of 100 #M PG as lipid activator (see below). The data show that in the absence of surfactant, the activity was much lower at the lower ion concentrations, although at higher concentrations similar maxima were obtained. In the presence of 10 mM CaC12, NaCI did not affect the activity up to 100 mM; at higher concentrations (300 mM NaCI) there was a reduction by 35% (data not shown). Apparent Km values of PI were determined in the presence of 1 and 2 mM deoxycholate and at varying Ca 2+ concentrations. Hyperbolic curvature or linear Lineweaver-Burk plots were only obtained, however, in the presence of low Triton concentrations (added with the enzyme solutions). While at 0.1% Triton, a sigmoidal curvature was obtained, at 0.05% Triton and lower, the curvature was hyperbolic. Under these conditions, a Km

E t... E 20 ¸

10

0 0.0

0.5

1.0 1.5 Triton X-100 mM

2.0

2.5

3.0

600

=~

450

=

300 q

"~

150

1

2

3

4

Deoxycholate mM

Fig. 3. Influenceof surfactants on the PLD activity.(A) Inhibition by Triton X-100 in the presence of 2 mM deoxycholate (125 kDa isoenzyme);(B) influence of deoxycholate(50 kDa isoenzyme).The data are average values from two determinations; SD were below ±7%.

value for PI of 37 #M was determined at 12 mM Ca 2÷ for both isoenzymes (at 1 and 2 mM deoxycholate); determinations at lower Ca 2÷ concentrations led to lower Km values (23 #M at 6 mM and 20 #M at 3.6 mM Ca 2+, at 2 mM deoxycholate) and slightly higher Vmax values.

3.5. Influence of surfactants and phospholipids Fig. 3 shows the influence of surfactants; Triton X-100 which was used for extraction and purification of the enzymes revealed a quite different behaviour from that of deoxycholate which was used in the standard activity assay. Triton, tested from

A. Becher et al,/Plant Sci. 97 (1994) 143-151

0.001 to 2.8 raM, reduced the activity to a residual level of about 20%. It was shown that this effect was reversible, as removal of Triton by gel filtration restored the activity. The two isoenzyme fractions gave comparable results both in the case of deoxycholate and Triton, indicating that the two isoenzymes did not differ in their affinities towards the surfactants tested. The experiment with deoxycholate was performed with 100 /~M PI and 160 #M Triton X-100 which was introduced by the enzyme solution; an experiment performed with 200 #M PI (not shown) gave very similar results. Fig. 3B shows a complicated dependency on the deoxycholate concentration. At low concentrations, activity was reduced to a minimum followed, however, by an intermediary maximum at 2 mM and a further reduction at higher concentrations. In order to elucidate the complicated behaviour, turbidity of the assay solutions of Fig. 3B (omitting the enzyme protein) was measured by recording light absorption at 600 nm. The mixture of 100 #M PI and 160 #M Triton X-100 (assay conditions) displayed a rather high value of light scattering obviously due to bilayer aggregate formation and incorporation of Triton molecules into these bilayers (critical micelle concentration of Triton X-100 is about 0.29 mM). With increasing deoxycholate concentration, light scattering increased slightly (maximum at 0.75 mM) indicating the formation of larger aggregates. The maximum of enzyme activity at 1.5-2.0 mM deoxycholate, however, was correlated with a drop in light scattering to nearly zero; obviously only at the higher deoxycholate/ phospholipid ratios were deoxycholate micelles, which are known to be of small size, formed, tolerating phospholipid molecules without large disturbances [26]. At higher deoxycholate concentrations, the reduction in enzyme activity (Fig. 3B) was correlated with a slight increase in light scattering and also visible gel formation. Unfortunately, nothing is known up to now on mixed micelle formation of deoxycholate with PI, although that with PC has been studied [26]. The effect of phospholipids on the activity of phospholipase D was tested in the presence of 100 #M PI and 2 mM deoxycholate (Fig. 4). Whereas PA was only slightly inhibitory at rather high (un-

149

250

200

~,-~

--~--

~ ~ - - ~-~, PG

/ / /

o~ 150

/ /

<

/

100 ~

-

-

~ --

50

PC PE Ct.

0 0.0

0.1

0.2

03

0.4

0.5

0.6

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Fig. 4. Influenceof phospholipids.PLD activity(50 kDa isoenzyme)was testedin the presenceof 2 mM deoxycholateand 100 #M PI. The data are averagevaluesfromthree determinations; SD werebelow ± 7%. v, PG; O, PA; t , PC; A, PE; t'l, CL (cardiolipin).

physiological) concentrations, PC, PE and also cardiolipin reduced the activity significantly at lower concentrations. Interestingly, PG stimulated the activity markedly at rather low concentrations. This stimulation was obviously specific and not the consequence of the negative charge of the head group, otherwise PA and cardiolipin would also have been stimulatory. It was further tested whether the second product of hydrolysis, inositol, had any influence; however, inositol did not reveal any effect up to a concentration of 500 #M. 4. Discussion

The data presented show that the phospholipase purified from microsomal membranes of Catharanthus roseus suspension cultured cells is a type D enzyme with a strict substrate specificity for PI and thus represents a novel enzymatic activity from plants. It differs from the known plant phospholipase D enzymes [1-3] which showed a broad substrate specificity, although with highest activities towards PC, and possessed transphosphatidylation activity, a property not displayed by the present enzyme. Most of the known enzymes were reported not to transform PI [1,2]. More recently, however, a partially purified enzyme from cotton

150

A. Becher et al./Plant Sci. 97 (1994) 143-151

seeds was reported to accept PI both in the transphosphatidylation reaction and the formation of PA, although PC was a better substrate [15]. Interestingly, a crude enzyme preparation from cauliflower florets was also reported to accept PI in a transphosphatidylation reaction to produce two isomers of bis(phosphatidyl)inositol [271. D-Specific hydrolysis of PI is also not common in the animal kingdom; only recently were activities detected in human neutrophils during cell activation [13] and in canine kidney cells [14], although the activity has not been purified up to

lower pI value of the smaller protein, all the other properties of these isoenzymes proved to be identical. As the suspension cultured cells also possessed phospholipase D activity in the soluble fraction with the same substrate specificity as the enzymes presented [19], it is an interesting question how the two isoenzymes are interrelated and how the membrane-bound enzymes and soluble forms are related. Studies are currently being performed in this direction.

nOW.

This work has been supported by the Fonds der Chemischen Industrie.

Activation by Ca 2÷ ions is a common property of plant phospholipase D enzymes with broad specificity [1]; several activities displayed an absolute requirement for Ca 2÷ which could not be replaced by Mg 2÷. This is in contrast to the present PI-specific enzyme which was also dependent on the presence of divalent cations, however, Ca z+ and Mg 2+ were equally effective. The specific stimulation of the present enzyme by PG is rather interesting, as other anionic phospholipids such as PA and cardiolipin were inhibitory. This is in contrast to plant enzymes with broad specificity which were reported to be activated by anionic phospholipids such as PA and PIP 2 [1,3,28]. The influence of surfactants has not been studied thoroughly in the present work. Deoxycholate showed a complex behaviour, i.e. it was inhibitory at low and high concentrations with a maximum of stimulation around 2 mM, while Triton X-100 was only inhibitory. With the known plant phospholipase D enzymes, stimulation by both deoxycholate and Triton X-100 was reported [1]. The present work revealed two isoenzymes which were separated by gel filtration. The apparent molecular weights were determined as 50 000 and 125 000. As about 15% of phospholipase activity was in the flow-through fraction of the first chromatographic step; a further isoenzyme cannot be excluded. On the other hand, this fraction could also consist of phospholipase C activity, as the cleavage specificity of the flow-through fraction could not be determined. With the exception of a slightly higher temperature stability and a slightly

5. Acknowledgement

6. References 1 M. Heller, Phospholipase D. Adv. Lipid Res., 16 (1978) 267-326. 2 T. Galliard, Degradation of acyl lipids: hydrolytic and oxidative enzymes, in: P.K. Stumpf (Ed.), The Biochemistry of Plants (Lipids: Structure and Function), Vol. 4, Academic Press, London, 1980, pp. 85-115. 3 M. Waite, The Phospholipases, Plenum Press, New York, 1987. 4 M.M. Billah, J.C. Anthes and T.J. Mullmann, Receptorcoupled phospholipase D: regulation and functional significance. Biochem. Soc. Trans., 19 (1991) 324-329. 5 M. Liscovitch, Signal-dependent activation of phosphatidylcholine hydrolysis: role of phospholipase D. Biochem. Soc. Trans., 19 (1991) 402-407. 6 W. Witt, G. Yelenosky and R.T. Mayer, Purification of phospholipase D from citrus callus tissue. Arch. Biochem. Biophys., 259 (1987) 164-170. 7 M.H. Lee, Phospholipase D of rice bran. 1. Purification and characterization. Plant Sci., 59 (1989) 25-33. 8 R. Lambreeht and R. Ulbrich-Hofmann, A facile purification procedure of phospholipase D from cabbage and its characterization. Biol. Chem. Hoppe-Seyler, 373 (1992) 81-88. 9 H. Eibl and S. Kovatchev, Preparation of phospholipids and their analogs by phospholipase D. Methods Enzymol., 72 (1981) 632-639. 10 S.Y. Lee, N. Hibi, T. Yamane and S. Shimizu, Phosphatidylglycerol synthesis by phospholipase D in a microporous membrane bioreactor. J. Ferment. Technol., 63 (1985) 37-44. 11 L.R. Juneja, N. Hibi, N. Inagaki, T. Yamane and S. Shimizu, Comparative study on conversion of phosphatidylcholine to phosphatidylglycerol by cabbage phospholipase D in micelle and emulsion systems. Enzyme Microb. Technol., 9 (1987) 350-354. 12 L.R. Juneja, T. Kazuoka, N. Goto, T. Yamane and S.

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