Purification and Characterization of Membrane-Bound

Plant Physiol. (1 993) 102: 165-1 72

Purification and Characterization of Membrane-Bound lnositol Phospholipid-Specific Phospholipase C from Suspension-Cultured Rice (Oryza sativa 1.) Cells ldentification of a Regulatory Factor Kenji Yotsushima, Toshiaki Mitsui*, Toshiyuki Takaoka, Toshiro Hayakawa, and lkuo lgaue Department of Biosystem Science, Graduate School of Science and Technology (K.Y.), and Department of Applied Biological Chemistry, Faculty of Agriculture (T.M., T.T., T.H., I.I.), Niigata University, 2-lkarashi, Niigata 950-21, Japan

phospholipid breakdown is induced by light (Morse et al., 1987) and auxin (Ettlinger and Lehle, 1988) and that Ir3 stimulates Ca2+release from the store organelles (Drdbak and Ferguson, 1985; Reddy and Poovaiah, 1987; Schumaker and Sze, 1987; Ranjeva et al., 1988; Alexandre et al., 1990). This suggests that the inositol phospholipid turnover is involved in signal transduction of higher plants. Two different types of inositol phospholipid-specific PLC, soluble and membrane-bound enzymes, have been identified in severa1 plant cells (McMurry and Irvine, 1987; Melin et al., 1987; Pfaffmann et al., 1987; Tate et al., 1989; Kamada and Muto, 1991; Yotsushima et al., 1992). The membrane-bound PLC preferentially hydrolyzes PIPl and PIP over PI, whereas the soluble PLC is not able to hydrolyze PIP2 (McMurry and Irvine, 1987; Melin et al., 1987; Yotsushima et al., 1992). This suggests that the membrane-bound PLC is a key enzyme of the signal transduction system in higher plants. Previously, we reported the characterization of the soluble inositol phospholipid-specific PLC enzyme purified from suspension-cultured rice (Oryza sativa L.) cells (Yotsushima et al., 1992). In the present study, we have purified and characterized a membrane-bound PLC from rice cells and found a regulatory factor for the PIPz-hydrolyzingactivity of PLC.

A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. l h e apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5.1. l h e optimum pH for the enzyme activity was approximately 6.5, and the enzyme was activated by both CaZ+and SrZ+. The chemical and catalytic properties of the purified membranebound phospholipase C differed from those of the soluble enzyme reported previously (K. Yotsushima, K. Nakamura, 1. Mitsui, 1. lgaue [1992]Biosci Biotech Biochem 56: 1247-1251). In addition, we found a regulatory factor for the phosphatidylinositol-4,5bisphosphate (PIPJ hydrolyzing activity of phospholipase C from rice cells. l h e regulatory factor was dissociated from the catalytic subunit of phospholipase C during the purification. l h e regulatory factor was necessary to induce PIPz-hydrolyzing activity of both membrane-bound and -soluble phospholipase C; these purified enzymes had no activity alone. Because the plasma membranes isolated from rice cells could also act as a regulatory factor, the regulatory factor seems to be localized in the plasma membranes. Regulation of inositol phospholipid turnover in rice cells is discussed.

MATERIALS A N D METHODS

In mammalian cells, inositol phospholipid turnover is widely accepted as an important step in signal transduction across the plasma membrane via second messengers. Breakdown of phosphatidylinositols in response to externa1 stimuli is catalyzed by inositol phospholipid-specific PLC. The PLC cleaves PIP and PIPz to yield inositol phosphates and DAG. Ir3and DAG produced from PIPz are well known as secondmessenger molecules. IP3 promotes the release of Ca2+from interna1 stores to the cytoplasm (Bemdge and Irvine, 1984) and DAG serves as a cofactor for protein kinase C (Nishizuka, 1986). Recently, it has been reported that the activity of PLC is regulated by G proteins (Smrcka et al., 1991; Taylor et al., 1991) or tyrosine phosphorylation of the enzyme (Nishibe et al., 1990; Goldschmit-Clermont et al., 1991). In higher plants, it has been reported that the inositol

Materials

Malachite green was purchased from Kodak (Rochester, NY), and phospholipids and GTP-7-S were from Sigma. PI, ~-a-[myoinositol-2-~H(N)] (37-185 GBq/mmol), PIP[myoinosit01-2-~H(N)] (37-185 GBq/mmol), and PIPz [myoinositol-23H(N)](37-185 GBq/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). The suspensioncultured cells of rice (Oryza sativa L. cv Nipponkai) were prepared as described previously (Yotsushima et al., 1992). Abbreviations: DAG, diacylglycerol; GTP-y-S, guanosine 5 '-O-(3thiotriphosphate);IP, inositol-monophosphate;Ir2,inositol-1,4-bisphosphate; IP3, inositol-1,4,5-trisphosphate; PI, phosphatidylinositol; ,PIP, phosphatidylinositol-4-monophosphate;PP2, phosphatidylinositol-4,5-bisphosphate;PLC, phospholipase C.

* Corresponding author; fax 81-25-263-1569. 165

166

Yotsushima et al.

Enzyme and Protein Assays

A standard assay of PLC was essentially identical with the procedure described before (Yotsushima et al., 1992). The reaction mixture for the soluble enzyme consisted of 50 mM acetate buffer (pH 5.2), 0.1 mM CaC12, 0.08% (w/v) sodium deoxycholate, 1 mM PI, and enzyme in a final volume of 75 pL. In the case of the membrane-bound enzyme, 50 mM Trismalate-NaOH (pH 6.5) was used instead of 50 mM acetate buffer (pH 5.2) in the above reaction mixture. The enzyme reaction was started by adding substrate and was incubated for 30 min at 37OC. It was terminated with the addition of 0.1 mL of 4 N HC1/0.5% (w/v) CaC12 and 0.6 mL of CHC13:methanol(2:1, v/v) and immediately cooled on ice. The mixture was vortexed and then centrifuged at 20008 for 10 min to separate aqueous and organic phases. After centrifugation, 0.15 mL of the upper aqueous phase was mixed with 75 pL of 0.3 M sodium periodate and incubated for 1 h at 37OC to liberate phosphate from inositol phosphate. Seventy-five microliters of 2 M sodium sulfite and 1.5 mL of malachite green reagent (Itaya and Ui, 1966) were added to the mixture sequentially. After incubation for 10 min, 0.15 mL of 0.06% (w/v) Tween 20 was added, and the color developed was measured at 660 nm. A radioactive assay of PLC was as follows: The assay mixture consisted of 0.1 mM radioactive substrate (92.5 MBq/mmol; [3H]PI, [3H]PIP, or [3H]PIPz),50 mM Tris-maleate-NaOH (pH 6.5), 0.08% (w/v) sodium deoxycholate, 3 p~ Ca2+ (0.5 mM CaC12 plus 1 mM EGTA), and enzyme in a final volume of 0.1 mL. The radioactivity in the upper aqueous phase was determined by liquid scintillation counting (Aloka LSC-1050). Assay of PIP2 phosphatase was camed out using a reaction mixture consisting of 0.1 mM PIP2, 50 mM Tris-maleate-NaOH (pH 6.5), 0.08% (w/v) sodium deoxycholate, 3 FM Ca2+(0.5 mM CaC12 plus 1 mM EGTA), and enzyme in a final volume of 0.1 mL. After incubation for an appropriate time at 37OC, the reaction was terminated, and the aqueous phase was separated and collected as described above. The amount of liberated Pi in the aqueous phase was determined by the malachite green reagent. A control incubation in each enzyme assay was done without enzyme or with boiled enzyme. Protein was measured by the method of Lowry et al. (1951) with BSA as a standard.

Plant Physiol. Vol. 102, 1993

IP3 were determined by the above periodate oxidation-malachite green procedure using the standard curves of IP, Ir2, and Ir3. Preparation of Plasma Membranes

Rice plasma membranes were isolated by a slightly modified PEG/dextran aqueous two-phase method (Yoshida et al., 1986). The rice cells were homogenized with buffer 1 (50 mM Tris-HC1 [pH 7.51, 1 mM EDTA, 0.5 M mannitol) and centrifuged at 12,OOOg for 15 min at 4OC. The supernatant was centrifuged at 100,OOOg for 1 h at 4OC, and the pellet was suspended with buffer 2 (10 mM potassium phosphate [pH 7.81, 30 mM NaCI, 0.5 M mannitol). PEG and dextran T-500 dissolved in buffer 2 were added to the suspension to give a 5.8% (w/w) concentration of each polymer. The suspension was centrifuged at 800g for 15 min to facilitate phase settling. The upper phase was collected and diluted with 3 volumes of 20 ITLM Tris-HC1 (pH 7.5) and centrifuged at 100,OOOg for 1 h at 4OC to precipitate plasma membranes. Purification of Soluble PLC

The purification procedure for soluble PLC from the rice cells was described previously (Yotsushima et al., 1992). Purification of Membrane-Bound PLC

All purification steps were performed at O to 4OC. Step 1: Preparation of Crude Enzyme

Rice cells (1 kg fresh weight) were homogenized in an equal volume of 150 mM Tris-HC1 (pH 7.5), 0.3 M NaCl, and 0.1 mM PMSF using a Potter-Elvehjem-type Teflon homogenizer. After cell debris was removed by centrifugation at 12,0008 for 20 min, the supernatant was further centrifuged at 100,OOOg for 1 h. The pellet was washed with 150 mM Tris-HC1 (pH 7.5) and 0.1 mM PMSF to remove NaCl and recentrifuged at 100,OOOg for 1 h. The pellet was suspended with 10 mM Tris-HC1 (pH 7.5), 0.1 mM PMSF, and 1%(w/v) sodium cholate, sonicated at 80 W for 10 min in an ice bath, and then centrifuged at 100,OOOg for 1 h. The resulting supernatant was used as a crude enzyme. Step 2: DEAE-Cellulose Column Chromatography

Product Analysis

Products of hydrolysis of PIP2 by PLC were separated and characterized on a Dowex AG 1-X8 formate form column (1 mL) according to the methods of Bemdge et al. (1983). A sample of the aqueous phase described above was put on the column, and the column was eluted sequentially with 25 mL of H20, 10 mL of 5 mM disodium tetraborate/60 mM sodium formate, 10 mL of 0.1 M formic acid/0.2 M ammonium formate, 10 mL of 0.1 M formic acid/0.4 M ammonium formate, and 10 mL of 0.1 M formic acid/0.8 M ammonium formate. Authentic IP, Ir2,and IP3 were eluted in the 0.1 M formic acid/0.2 M ammonium formate, 0.1 M formic acid/0.4 M ammonium formate, and 0.1 M formic acid/0.8 M ammonium formate fractions, respectively. The amounts of IP, IP2, and

The crude enzyme was dialyzed against buffer A (10 mM Tris-HC1 [pH 6.51, 0.1 mM PMSF, and 0.2% [w/v] sodium cholate). The dialysate was applied to a DEAE-cellulose column (2.5 X 40 cm) equilibrated with buffer A. After the column was washed with 800 mL of buffer A, it was eluted with 1600 mL of a linear concentration gradient of O to 0.5 M NaCl in buffer A. The active fractions were pooled and concentrated by Amicon PM-10. Step 3: Sephadex C-700 Column Chromatography

The concentrated enzyme (4.5 mL) was applied to a Sephadex G-100 gel filtration column (2.5 X 70 cm) equilibrated with buffer A. The column was eluted with buffer A at a

167

Purification of Rice Membrane-Bound Phospholipase C

flow rate of 18 mL/h-'. The active fractions were pooled and concentrated by Amicon PM-10. Step 4: Chelate Cellulofine Column Chromatography The concentrated enzyme was dialyzed against buffer A and then applied to a Cu2+-linkedchelate cellulofine column (1.5 X 12 cm) equilibrated with buffer A. After the column was washed with 60 mL of buffer A, it was eluted with 120 mL of a linear gradient of O to 1M Gly in buffer A. The active fractions were pooled, dialyzed against buffer A, and concentrated by Amicon PM-10. Step 5: C3000 SW HPLC The enzyme sample (0.5 mL) obtained from the chelate cellulofine column was applied to a G3000 SW gel filtration column (7.5 X 600 mm; TOSOH TSK-GEL) equilibrated with buffer A and eluted with buffer A at a flow rate of 1 mL min-' using a Hitachi 638-30 HPLC system. The purified enzyme was stable at -2OOC for at least 1 month. IEF

The isoelectric point of the protein was determined using an electrofocusing column (Ampholine column LKB 8101). An enzyme sample in 20% (w/v) glycerol solution was poured into the center of a glycerol gradient (O-40%) containing 1%(v/v) Ampholine (pH 4 to 6). Electrofocusing was performed at 200 V for 20 min, at 400 V for 20 min, and at 800 V for 48 h, sequentially, at 4OC using 1% H3P04 (v/v) for the anode solution and 8% (w/v) NaOH for the cathode solution. After electrofocusing, the pH gradient was fractionated in 2-mL fractions, and the enzyme activity and the pH value in each fraction were measured. SDS-PACE

SDS-PAGE was performed according to the method of Laemmli (1970). Protein bands on an SDS gel were visualized using the silver staining kit (Bio-Rad). Myosin (205 kD), /3galactosidase (116 kD), phosphorylase b (97.5 kD), BSA (66 kD), egg albumin (45 kD), pepsin (34.7 kD), carbonic anhydrase (29 kD), trypsinogen (24 kD), and lysozyme (14.3 kD) were used as molecular mass standards. Regulatory Factor Preparation

A regulatory factor was prepared from the G3000 SW HPLC (fraction Nos. 10-15 in Fig. 3) as described in "Results." Reconstitution Experiments

Four methods were used to reconstitute the enzyme and the regulatory factor. With the first method, the purified membrane-bound PLC (0.19 pg) was preincubated with the heat-treated (at 100°C for 5 min) or untreated regulatory factor (0.34 Pg) in buffer A for 1 min at 37OC. With the second method, the membrane-bound enzyme (0.19 pg) was preincubated with the isolated plasma membranes (1.5 Pg) in buffer A for 1 min at 37OC. With the third method, the purified soluble PLC (6.7 pg) was preincubated with the heat-

treated (at 100°C for 5 min) or untreated regulatory factor (0.34 pg) in buffer A for 1 min at 37OC. With the fourth method, the soluble enzyme (6.7 pg) was preincubated with the isolated plasma membranes (1.5 pg) in buffer A for 1 min at 37OC. These reconstituted samples were subjected to the following PIP2 hydrolysis assay: The assay mixture consisted of 0.1 m~ PIP2, 50 mM Tris-maleate-NaOH (pH 6.5), 0.08% (w/v) sodium deoxycholate, 3 p~ Ca2+(prepared as a CaZ+-EGTA buffer), and the reconstituted enzyme sample in a final volume of 0.1 mL. The reaction was carried out at 37OC for 60 min. Inositol phosphate liberation was determined by the periodate oxidation-malachite green procedure. Neither the purified enzymes nor the regulatory factor had PIP2-hydrolyzing activities alone. An interna1 PIP2-hydrolzying activity of the isolated plasma membranes was determined by the same assay system. RESULTS Purification of Membrane-Bound PLC

A membrane-bound inositol phospholipid-specific PLC was solubilized from rice microsomal membranes with 1% (w/v) sodium cholate and subjected to DEAE-cellulose anion exchange, Sephadex G-100 gel filtration, chelate cellulofine, and G3000 SW gel filtration column chromatography. The separation profile of membrane-bound PLC on the G3000 SW gel filtration column is shown in Figure 1. The distribution of the PLC activity coincided with a 280-nm absorption. On SDS-PAGE this peak contained a single polypeptide band, indicating that the rice membrane-bound PLC was purified to an apparently homogeneous polypeptide. A summary of the stepwise purification of rice membrane-bound PLC is given in Table I. The specific activity of the purified enzyme increased 220-fold in comparison with that of the crude enzyme solubilized from microsomal membranes. V,,, and K , values toward PI were determined to be 2.9 gmol min-' mg-' on protein and 1.2 mM, respectively. The molecular mass of the enzyme was estimated to be 42,000 D by SDS-PAGE and to be 19,000 D by G3000 SW gel filtration (Fig. 1). The discrepancy between the two molecular mass estimations may have resulted from a weak affinity of the enzyme to the G3000 SW column. The isoelectric point was shown to be 5.1 by an electrofocusing column (data not shown). The pH dependence and stability of the membranebound enzyme is shown in Figure 2. The optimum pH for both enzyme activity and stability was determined to be approximately 6.5. The membrane-bound PLC was a Ca2+dependent enzyme (Table 11). Sr2+ also activated the membrane-bound enzyme to the same extent as Ca2+(Table 11). Properties of the membrane-bound and soluble PLCs are summarized and compared in Table 111. These results clearly indicate that the molecular structure and catalytic properties of the membrane-bound inositol phospholipid-specific PLC differed from those of the soluble PLC. ldentification of a Regulatory Factor

Our previous study demonstrated that the PLC purified from the soluble fraction of rice cells does not hydrolyze PIP2

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Yotsushima et al.

®


origin-

100

kDa 66-

15-

D

50

36292Hbottom- 1

19 20 21 22

CH

Figure 2. Effects of pH on activity (A, A) and stability (O, •) of rice membrane-bound PLC. The pH dependency of membrane-bound PLC was examined using buffered solutions of 50 mM sodium acetate (pH 4.0-5.5, O, A) and 50 mM Tris-malate-NaOH (pH 5.5 to 9.0, •, A). The maximum activity of PLC (1.95 ^mol min"1 mg~' protein) was normalized to 100%. To examine the pH stability of PLC, the enzyme was incubated for 24 h at 4°C in the above buffered solutions, and then the enzyme activity was measured under the standard assay conditions. The maximum activity of PLC after the incubation was 1.51 jimol min'1 mg"1 of protein.

10 15 20 Elution Time (min)

Figure 1. Purification of rice membrane-bound PLC by C3000 SW HPLC. PLC separated from the chelate cellulofine column chromatography was subjected to a C3000 SW gel filtration column as described in "Materials and Methods." A, Separation profile of PlPLC activity. The column fractions were assayed for enzyme activity toward PI. B, SDS-PACE pattern of the column fractions. Fractions eluting from 19 to 22 min were subjected to electrophoresis and the gel was visualized by silver staining.

(table IV, in Yotsushima et al., 1992). Because the rice plasma membrane-associated PLC had significant activity toward PIP2, we attempted to purify the membrane-bound PLC. Until the chelate cellulofine step, the membrane-bound PLC was able to hydrolyze PIP2, whereas the final purified PLC lost the PlP2-hydrolyzing activity (Table IV). These results strongly suggest that there is a regulatory factor involved in the expression of PIP2-hydrolyzing activity of the enzyme.

To verify the suggestion, we performed reconstitution experiments. When the purified membrane-bound PLC was added to each fraction separated on the G3000 SW gel filtration column, some fractions (fraction nos. 10-15) exhibited PIP2 hydrolysis activity (Fig. 3), indicating that a regulatory factor had been dissociated from the catalytic subunit of PLC by the G3000 SW gel filtration. The stimulating activity fractions (fraction nos. 10-15) were used as the regulatory factor for PLC afterward. The regulatory factor also induced the PIP2-hydrolyzing activity of the soluble PLC to the same extent as the membrane-bound enzyme (Table V). The stimulating activity of the regulatory factor completely disappeared by heat treatment for 5 min in boiling water (Table V), suggesting that the regulatory factor has a protein component. In addition, the isolated plasma membranes were found to stimulate the PIP2-hydrolyzing activity of both the membrane-bound and soluble PLC (Table V). To analyze products of PIP2 hydrolyzed by the membranebound PLC reconstituted with the regulatory factor, the

Table I. Purification of rice membrane-bound inositol phospholipid-specific PLC

Crude extract DEAE-cellulose Sephadex C-100 Chelate cellulofine C3000 SW HPLC

Volume

Concentration

Total Activity

Specific Activity

Purification

mt

mg of protein mi."1

nmol mm"'

nmol min'' mg"'

-fold

3.03 1.81 0.58 0.42 0.075

74.4 55.7 153.7 1968.4

740

0.46

522 100 265 4

0.047 0.10 0.010

0.0095

8.97

1

8.3 6.2 17.1 219.4


DlSCUSSlON

Table II. Effects of various divalent cations on the activity of rice membrane-boundPLC Relative Activitv” %

1 mM ECTA +1 mM CaClz +1 mM MgC12 +1 mM SrClz +1 mM c O c 1 2 +10 mM CaCI2

O 1O0 O 70 34

89

4-10 mM MgC12 +10 mM SrClz +10 mM COCIz

O

69 O

Enzyme assays were done in reaction mixtures consisting of 1 6.5), 0.08% (w/v) deoxycholate, 1 mM ECTA, and 1 or 10 mM divalent cation as indicated. The enzyme activity in the presence of 1 mM CaClz (1.6 fim01 min-’ mg-’ of protein) was normalized to 10Oo/o. a

mM PI, 50 mM Tris-malate-NaOH (pH

hydrolysate was subjected to a Dowex AG 1-X8 formate form column as described in “Materials and Methods.” The relative percentages of IP, Ir2,and Ir3separated on the column were 41.5, 12.3, and 46.2%, respectively (Fig. 4). The results indicate that at least the reconstituted enzyme can produce I r 3 from PIP2. IP and IP2 produced by the PIP2 hydrolysis may have resulted from the dephosphorylation of PIP2 and/or Ir3 by a phosphatase contaminating the regulatory factor preparation. A PIP2 phosphatase activity was detected in the regulatory factor preparation (data not shown). Characterization of Reconstituted PLC

The effects of Ca2+ on the PIPz- and PI-hydrolyzing activities of the reconstituted enzyme are shown in Figure 5. Both activities were completely inhibited with 1 m~ EGTA (pH 6.5). The maximum activation of the PIP2-hydrolyzing activity by Ca2+was observed at approximately 3 PM, whereas that of the PI-hydrolyzing activity was observed at more than 30 ~ L M . The reconstituted PLC activity was not significantly affected by 50 PM GTP or GTP-y-S (data not shown).

Although there are a few reports concerning the purification and characterization of plant inositol phospholipid-specific PLCs, they have not been purified to homogeneity. In this study, we have succeeded in purifying a membranebound PLC from suspension-cultured rice cells using a series of chromatographic techniques. The membrane-bound and soluble (Yotsushima et al., 1992) PLCs (Table 111)have distinct properties. The apparent molecular masses of the membranebound and soluble enzymes as estimated by SDS-PAGE are 42,000 and 55,000 D, respectively. The membrane-bound PLC has a broad optimum pH of approximately 6.5, whereas the soluble PLC has a sharp optimum pH at 5.2. Sr2+strongly activated the membrane-bound PLC but not the soluble PLC. These different properties clearly indicate that the membrane-bound and soluble PLCs are different enzyme isoforms. Melin et al. (1992) reported the partia1 purification and characterization of polyphosphoinositide PLC from wheat plasma membranes. The enzyme was solubilized in octylglucoside and purified 25-fold by hydroxyapatite and ion exchange chromatography. The partially purified enzyme catalyzes the hydrolysis of PIP and PIPp but not PI. The purified rice membrane-bound PLC has a high degree of specificity toward PI, and even the crude enzyme preferred PI rather than PIP and PIPz (Table IV). Judging from these data, we believe it is likely that the rice membrane-bound PLC is not the same as the wheat plasma membrane-bound enzyme. In this study, the PIP2-hydrolyzing activity of the membrane-bound PLC was lost during the final G3000 SW gel filtration step (Table IV). This suggested that a regulatory factor had dissociated from the catalytic subunit of the membrane-bound PLC, and a11 fractions separated on the G3000 SW column were screened for a potential regulatory factor. G3000 SW fractions and the purified membrane-bound PLC were combined in a reconstitution assay to identify a regulatory factor for the PIPz-hydrolyzing activity of PLC. As shown in Figure 3, there is a stimulating factor for PIPZ-PLC in the rice microsomal membranes. Furthermore, the regulatory factor is not stable in heat treatment for 5 min at 100°C, indicating that this regulatory factor is a protein. It has been reported (Roy et al., 1991) that the high molecular mass form

Table 111. Properties of rice membrane-boundand soluble PLCs Catalytic properties were examined using PI as substrate.

Molecular massb lsoelectric point Optimum pH pH stability Optimum temperature Thermal stability Divalent cation requirement Km

(Pl)

Vmax

(Pl)

169

Membrane-Bound PLC

Soluble PLC”

42,000 5.1

55,000

6.5 6.0-8.0 40°C

O-30°C Caz+ 2 SrZ+ 1.2 mM 2.9 wmol min-’ mg-’

a Data obtained from the previous report (Yotsushima et al., 1992). masses of these enzymes were determined by SDS-PACE.

6.5 5.2 6.5-8.0 50°C O-50°C Ca2+

0.3 m M 5 umol min-’ mg-’

The apparent molecular

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Yotsushima et al.


Table IV. Substrate specificities of rice PLCs Relative Activity” Membrane-bound PLCb

Substrate

Plasma membranes

Soluble PLCc

Chelate

C3000 SW

cellulofine %

PI PI P PIP2

-d

Phosphatidylcholine

-

Phosphatidylethanolamine

-

PhosDhatidvlserine

-

1O0

30

1O0 10 30 O O O

1O0

1O 0

15

13

O O

O O

O O

O

o

a One hundred percent activity was set for each enzyme preparation. Three different enzyme samples were used: first column, the isolated plasma membranes; second column, the enzyme obtained from t h e chelate cellulofine column; third column, the enzyme purified by the G3000 SW column. Assay conditions were 0.1 mM substrates, 50 mM Tris-malate-NaOH (pH 6.5), 0.08% (w/v) sodium deoxycholate, and 3 FM Ca2+(0.5 mM CaCI2plus 1 mM ECTA) at 37°C. The absolute 100% activity values of the above enzymes were 22.9, 153.7, and 1968.4 nmol min-’ mg-’ of protein, -, Not Data obtained from the previous report (Yotsushima et al., 1992). respectively. determined.

(60-70 kD) of the membrane-bound PLC from human spleen has very little activity but that activity can be enhanced by enzyme dissociation. This suggests that the PLC enzyme is bound to a regulatory component that is released from the active enzyme (18 kD) when the charge interactions are disrupted. Neither regulatory factor in the rice and human spleen cells is a GTP-binding protein, because GTP and GTP-7-S did not affect the stimulating activity of the regulatory factors. It seems that a non-GTP-binding factor is involved in the regulation of PLC in both plant and animal cells, although the effects of plant and human regulatory factors on the PLC activity are reversed. Severa1 distinct PLC isoforms (eg. PLCB, PLC-7, and PLC-6) have been identified, and three different activation mechanisms for various PLC isoforms have been proposed in mammalian cells (Rhee and Choi, 1992).

Einspahr et al. (1989) reported that the plasma membrane PIPz-PLC of a unicellular green alga (Dunaliella salina) may be regulated by a G protein, although the green alga PIPzPLC enzyme has not been purified. It is likely that there are a few PLC isoforms and at least two different activation mechanisms for PLCs in plant cells. We also found that the rice regulatory factor induces the PIPz-hydrolyzing activity of the soluble PLC to an extent similar to that of the membrane-bound PLC and that the isolated plasma membranes activate the enzyme activities of both membrane-bound and soluble PLC (Table V). These results strongly suggest that the regulatory factor is localized in the plasma membranes, and it regulates both the membrane-bound and soluble PLCs in the rice cells. The reconstituted PLC with the regulatory factor produces IP3 from PIPz, although some PIPz phosphatases contaminate the regulatory factor preparation (Fig. 4). In addition, the PIPzhydrolyzing activity of membrane-bound PLC reconstituted

Table V. Activation of PLC by regulatory factor and plasma

membranes ExDerimental details were described in ”Materialsand Methods.” PI P2-Hydrolyzing Activity Activators

Membrane-bound PLC

Soluble PLC

nmol min-’ mg-’ oiprotein

Elution Time (minl Figure 3. Separation of regulatory factor for PLC by C3000 SW

HPLC. The PIPz-hydrolyzing activities in the column fractions separated by the G3000 SW gel filtration (Fig. 1 ) were assayed in the presence (O)or absence (O)of the purified membrane-bound PLC (12 nmol min-’ mL-’). The reaction mixture consisted of 0.1 mM PIP2, 50 mM Tris-malate-NaOH (pH 6.5), 0.88% (w/v) sodium deoxycholate, 3 PM Ca2+(0.5 m M CaC12 plus 1 mM ECTA). The arrow represents the position of purified PLC.

None Regulatory factor” Heat-treated regulatory factorb Plasma membranes‘

O 88.7 O

O -d

44.2

20.0

19.6

The regulatory factor had no PIPZ-hydrolyzing activity itself. bThe regulatory factor was heated at 100°C for 5 min. Because the isolated plasma membranes had a n internal PIP2-hydrolyzing activity, the value of internal activity was sub-, Not measured. tracted from the original values. a


171

with the regulatory factor is maximally activated by a nearphysiological intracellular Ca2+concentration (3 PM) (Fig. 5 ) . From a11 these experimental results, we propose a hypothetical model for the regulation of inositol phospholipid turnover in the rice cells. The reconstituted PLC enzymes produce IPs and DAG from PIP2 in the plasma membranes. The resulting IP3 promotes the release of Ca2+from the store organelles to the cytoplasm. Because Ca2+stimulates both the membranebound and soluble PLC enzymes, the PI breakdown is amplified by the released Ca2+.Finally, Ca2+controls the cellular events such as a-amylase secretion (Mitsui et al., 1984; Hayashi et al., 1989). At present, it is unclear whether protein phosphorylation or a cell surface receptor is involved in regulating interaction between PLC and its regulatory factor. A more detailed mechanism for the regulation of inositol phospholipid turnover in the rice cells requires further investigation.

1°t 8

a 6

. n r

o

e 4 2

O

Fraction Number

Figure 4. Elution profiles on a Dowex AC 1-X8 column of products of PIPz hydrolysis by membrane-bound PLC reconstituted with regulatory factor. Experimental details are described in "Materials and Methods." The anion exchange column was eluted sequentially with water (a),5 m M disodium tetraborate-60 mM sodium formate (b), 0.1 M formic acid/0.2 M ammonium formate (c), 0.1 M formic acid/0.2 M ammonium formate (d), and 0.1 M formic acid/0.8 M ammonium formate (e)

ACKNOWLEDCMENT

We thank Dr. G.C. Scott-Woofor critica1reading of the manuscript and helpful discussion. Received November 9, 1992; accepted January 17, 1993. Copyright Clearance Center: 0032-0889/93/102/0l65/08. LITERATURE ClTED

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361-366 -1'

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1mM EGTA

7

6

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4

Kamada Y, Muto S (1991) Ca2+regulation of phosphatidylinositol 3

oca

Figure 5. Effects of different concentrations of Ca2+on PI (A) and PIPz (O) hydrolyzing activities of membrane-bound PLC reconsti-

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