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Phosphatidylinositol-specific phospholipase C of murine lymphocytes
ARCHIVES
Vol.
OF BIOCHEMISTRY
AND
249, No. 2, September,
BIOPHYSICS
pp. 569-578,1986
Phosphatidylinositol-Specific YASUSHI Division
KAMISAKA; of Chemical
Phospholipase SATOSHI
Toxicology University
Received
January
C of Murine Lymphocytes’
TOYOSHIMA,
and Immunochemistry, of Tokyo, Bunkyrrku,
AND
TOSHIAKI
Faculty of Pharmaceutical Tokyo 113, Japan,
28, 1986, and in revised
form
May
OSAWA3 Sciences,
27, 1986
Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol4-phosphate, and phosphatidylinositol4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. (1) The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. (2) The activity of the cytosolic enzyme was first detected at 1 PM Ca”+, and maximum activity was observed at 100 PM Ca2+, while the membranebound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. (3) The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate. (4) A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an a-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. (5) Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity. 0 1986 Academic
Press. Inc.
It has been reported that marked inositol phospholipid turnover occurs in a variety of cells and tissues in response to external stimuli and that this phenomenon may
play a vital role in cell activation (l-3). The importance of this effect has been reported, for example, in Ca2+ gating (l), intracellular Ca2+ mobilization (2, 3), Ca2+ regulation, phospholipid-dependent protein kinase activity (4), and arachidonic acid metabolism (5). Though it remains unclear how external stimuli induce inositol phospholipid turnover, there is general agreement that the external stimulus-induced breakdown of inositol phospholipid is mediated through the action of phosphatidylinositol-specific phospholipase C (PI-
‘This investigation was supported by research grants from the Ministry of Education, Science and Culture of Japan, and the Sankyo Foundation for Life Sciences. a Present address: Bioorganic Chemistry Division, National Chemical Laboratory for Industry, Yatabe, Ibaraki 305, Japan. 3 To whom all correspondence should be addressed.
569
0003-9861186 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
570
KAMISAKA,
TOYOSHIMA,
phospholipase C)* (l-3). Since this enzyme acts at a very early stage of receptor-mediated cell activation and the site of PI breakdown seems to be in the plasma membrane, it appears that PI-phospholipase C exists in the plasma membrane of cells. However, PI-phospholipase C activity was primarily detected in the cytosolic fraction of cells and only a few reports suggested the presence of a membranebound PI-phospholipase C (6-8). Furthermore, it was reported that PI-phospholipase C activity detected in platelet particulates was due to artificial contamination of the cytosolic enzyme (9). Allan and Michell (10) also showed that in lymphocytes most of this enzyme was cytosolic with only small amounts in the particulate fraction. However, in the present study we were able to detect significant amounts of PI-phospholipase C activity in the plasma membrane of murine splenic lymphocytes and to show some differences between the properties of the membrane-bound and cytosolic phospholipases C. MATERIALS
AND
METHODS
Cells. Murine spleens from 8- to IO-week-old female ICR mice (Charles River Japan, Kanagawa, Japan) were removed and teased apart on ice in Eagle’s minimal essential medium, pH 7.2 (MEM, Nissui Co., Tokyo, Japan) containing 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes, Wako Pure Chemical Co., Tokyo, Japan). After removal of clumps by passage through a stainless steel mesh, lymphocytes were separated by the Ficoll-Urografin method (11). Lymphocytes were further fractionated on a nylon wool column (12), and the nylon wool nonadherent cells were obtained as a T-cell-enriched fraction. Macrophages were removed by passage through a Se-
* Abbreviations used: PI, phosphatidylinositol; PIphospholipase C, PI-specific phospholipase C; Hepes, N-2-hydroxyethylpiperazine-iV’-2-ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PI-I-P, phosphatidylinositol 4-phosphate; PI-4,5-Pz, phosphatidylinositol4,5-bisphosphate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; MEM, Eagle’s minimal essential medium (pH 7.2); EGTA, ethylene glycol bis(2aminoethyl ether) iV,N’-tetraacetic acid, a-MM, methyl-a-D-mannopyranoside.
AND
OSAWA
phadex G-10 column (macrophage-depleted fraction) (13). For enrichment of B cells, lymphocytes were treated with appropriately absorbed rabbit antimouse brain-associated 0 antigen serum (anti-BAT) (14) and guinea pig complement, and then applied to a Ficoll-Urografin layer to remove dead cells. The resulting population of B cells (B-cell-enriched fraction) contained more than 95% Ig-bearing cells, SubceUularfractionatim Microsomes were prepared according to the method of Makishima et al, (15) with minor modifications. The cells were suspended in 10 mM Tris-HCl buffer (pH 7.4), containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Chemical Co., St. Louis, MO.), 0.02% NaNa, and 1 mM ethylenediaminetetraacetate (EDTA, Sigma), and allowed to stand at 4°C for 5 min. Then the cells were homogenized with a Potter-Elvehjem homogenizer. After 1 ml of 10 mM Tris/HCl buffer (pH 7.3) containing 1.5 M NaCl and 1 mM EDTA was added to make the NaCl concentration 0.15 M, the mixture was centrifuged at 300g for 15 min to remove cell debris and nuclei. The supernatant was collected and centrifuged at 60009 for 10 min to remove mitochondria, lysosomes, etc. The supernatant was then centrifuged at 100,OOOg for 1 h. The pellet was dissolved in 10 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and then centrifuged at 105,OOOg for 1 h. This step was performed again and the final pellets were suspended in 10 mM TrisHCl buffer (pH 7.4) containing 0.15 M NaCl (microsome fraction). The specific activity of 5’-nucleotidase, a marker enzyme of plasma membranes, was about 6.1 times higher in this microsome fraction than in the homogenate. To examine various properties of PI-phospholipase C, the simplified technique of Maeda et al. (16) was employed to separate the plasma membrane and cytosol. Briefly, the cells were suspended in 10 mM TrisHCl buffer (pH 7.4) containing 30 mM NaCl, 1 mM MgCl,, 1 mM PMSF, and 0.02% NaN$, and then homogenized with a Potter-Elvehjem Teflon homogenizer. The homogenate was layered on a 41% sucrose solution in the homogenization buffer and centrifuged at 95,000g for 1 h. After centrifugation, the interfacial band of membranes was collected and washed three times with the homogenization buffer, and the supernatant (top layer) was used as a cytosolic fraction in the subsequent experiments. The specific activity of 5’-nucleotidase, a marker enzyme of plasma membrane, was about 22 times higher in this plasma membrane fraction than in the homogenate. The plasma membrane fraction thus obtained seemed to be purer than the microsome fraction. To investigate the subcellular distribution of PIphospholipase C, a continuous sucrose density gradient (30-50%)was used. The lymphocyte homogenate was centrifuged at 400g for 15 min. The supernatant was centrifuged at 100,000~ for 1 h and then the pellet
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was washed twice with 10 mM Tris-WC1 buffer (pH 7.4) containing 0.15 M NaCl. The washed pellet was suspended in the washing buffer and layered on a sucrose gradient solution (30-5O%,w/v) with a 60% (WI v) sucrose solution at the bottom. After centrifugation at 40,OOOg for 3 h, fractions were collected from the bottom of the tube. Try&z treatment. Intact lymphocytes were treated with 0.1% trypsin (Worthington Biochemicals, Freehold, N.J.) in MEM for 30 min at 25°C. The reaction was stopped by adding soybean trypsin inhibitor (Sigma) solution (1 mg/ml), and then the cells were washed three times with MEM. Cell viability was not affected by this treatment. The plasma membrane and cytosolic fractions were also hydrolyzed with 0.1% trypsin for 30 min at 25°C. To further confirm the presence of PI-phospholipase C in the lymphocyte plasma membrane, the plasma membrane fraction from trypsin-treated lymphocytes or untreated lymphocytes was layered on a continuous sucrose density gradient (20-50%, w/v), and after centrifugation at 40,OOOg for 3 h, the PI-phospholipase C activity in each sucrose density fraction was assayed. Assay for PI-phospholipase C activity. The assay mixture, on ice, contained 10 pM soybean PI, 0.01 &i L-a-phosphatidyl[2-3H]inositol (sp radioact, 15.6 Ci/ mmol; The Radiochemical Centre, Amersham, U.K.), as a liposome suspension prepared by brief sonication, and, unless otherwise stated, 10 fig of enzyme in 250 ~1 of 0.1 M acetate buffer (pH 6.0) containing 1 mM Ca2+ and 0.05% (w/w) sodium cholate. The reaction was performed at 37°C for 90 min and terminated by adding 1 ml of chloroform/methanol/concentrated HCl (100/100/0.6) and 0.3 ml of 1 N HCI containing 5 mM ethylene glycol bis(2-aminoethyl ether) N,N’-tetraacetic acid (EGTA). Phospholipase activity was determined by measuring the radioactivity in the aqueous phase. Under the above conditions, the hydrolyzed products linearly increased with incubation time or the amount of protein. To examine the substrate specificity of phospholipase C, instead of radioactive PI and cold PI, L-a-dipalmitoylphosphatidyl[N-methyl-‘4C]choline (sp radioact 153 mCi/mmol; The Radiochemical Centre), L-ol-dioleoylphosphatidyl[2-‘4C]ethanolamine(spradioact44mCi/mmol;The Radiochemical Centre), or L-a-dioleoylphosphatidyl[U-‘4C]serine (sp radioact 60 mCi/mmol; The Radiochemical Centre) and the corresponding cold phospholipid were added to the reaction mixture. The hydrolyzed products in the aqueous phase were analyzed by descending paper chromatography on Whatman No. 1 paper in ethanoVl3.5 M NH,OH (3/2, v/v) as described by Dawson et al (17). For assay of phosphatidylinositol 4-phosphate (PI-4-P) and phosphatidylinositol 4,5-bisphosphate (PI-4,5-P,) hydrolyses, the reaction mixture contained 500-2000 cpm [3zP]PI-
PHOSPHOLIPASE
C
571
4-P or [3*P]PI-4,5-P2 instead of rH]PI. [32P]PI-4-P and [3ZP]PI-4,5-PZ were isolated from extracts of [32P]orthophosphate-labeled human erythrocyte ghosts (18). Assays for other enzyme activities. 5’-Nucleotidase, a marker enzyme of plasma membranes, was assayed as described by Song and Bodansky (19); lactate dehydrogenase, a marker enzyme of the cytosol, as described by Kornberg (20); and P-glucuronidase, a marker enzyme of lysosomes, as described by Ferber et al. (21). Analytical methods. Proteins were determined by the method of Lowry et al. (22) with bovine serum albumin as standard. Phospholipid phosphorus was measured by the method of Ames and Rubin (23). The free Ca2+ concentration of micromolar order in the assay medium was regulated by using Ca’+-EGTA buffers and calculated according to Portzehl et al. (24). Column chromatography on Sephadex G-100 and concanavalin A-Sepharose. Lymphocyte membranes (about 10 mg) were solubilized at 4°C with 2 ml of 10 mM Tris-HCI buffer (pH 7.4) containing 0.02 M NaCI, 0.02% NaN$, and 0.4% deoxycholate (Sigma). The solubilized membranes were immediately applied to a Sephadex G-100 column (1.6 X 92 cm) equilibrated with the solubilization buffer. The lymphocyte cytosol was also applied to a Sephadex G-100 column (1.6 X 140 cm) equilibrated with 10 mM Tris-HCI buffer (pH 7.4) containing 0.02 M NaCl and 0.02% NaN,. The cytosol and the solubilized membranes were dialyzed against the column equilibration buffer before application to the Sephadex G-100 column. Concanavalin A (Con A, Honen Oil Co., Tokyo, Japan) was coupled to CH-Sepharose (Pharmacia, Uppsala, Sweden) using 1-ethyl-3-dimethylaminopropylcarbodiimide (25). About 15 mg of protein was found to be immobilized per 1 ml of resin. The active fractions obtained on Sephadex G-100 column chromatography of the membranes and the cytosol were applied to a Con A-Sepharose column (1 ml resin) equilibrated with 10 mM Tris-HC1 buffer (pH 7.4) containing 0.4% deoxycholate, 0.15 M NaCI, 0.02% NaNa, 0.1 mM MnCl,, and 0.1 mM CaCI,. The elution was started with the equilibration buffer, followed by the same buffer containing 0.3 M methyl-a-D-mannopyranoside (a-MM).
RESULTS
Subcellular distribution of phospholipase C. As shown in Table I, phospholipase C activity in lymphocytes was primarily distributed in the cytosolic fraction (supernatant). However, a significant amount of phospholipase C activity was detected in the microsome fraction. In spite of the
572
KAMISAKA,
TOYOSHIMA, TABLE
SUBCELLULAR
DISTRIBUTION
Homogenate Nuclei Mitochondria Microsome Supernatant
Protein (mg)
Specific activityb
11 5.2 0.68 0.78 4.1
a Similar results were obtained b nmol min-’ mg protein-‘. ’ nmol mini (% of homogenate).
1.4 0.27 0.72 0.80 2.5 in four
C
Total activityC 15 1.4 0.49 0.62 10
(100) (9.3) (3.3) (4.1) (67)
Fract
FIG.
I C ACTIVITY
Lactate
dehydrogenase
Specific activityb
Total activity’
18 1.7 2.1 0.54 38
200 8.8 1.4 0.42 160
(100) (4.4) (0.71) (0.21) (79)
a
5’-Nucleotidase Specific activityb 0.57 0.25 1.2 3.5 0.38
Total activity” 6.3 1.3 0.92 2.7 1.6
(100) (21) (13) (43) (25)
experiments.
great increase in the specific activity of 5’nucleotidase, a marker enzyme of plasma membrane, in the microsome fraction as compared with the homogenate, the specific activity of phospholipase C in the microsome fraction was still more than half of that in the homogenate. About 4% of the total phospholipase C activity was recovered in the microsome fraction, but the recovery of lactate dehydrogenase, a marker enzyme of the cytosol, was only 0.21%. These results suggest that phospholipase C activity in the microsome fraction is not due to contamination by the cytosolic phospholipase C. The subcellular distribution of phospholipase C was further examined by means of continuous sucrose density gradient
density
OSAWA
OF PI-PHOSPHOLIPASE
PI-phospholipase
Fraction
AND
!on
centrifugation of the 100,OOOg pellet from a lymphocyte homogenate as described under Materials and Methods. As shown in Fig. 1, mainly one peak of phospholipase C activity was observed. This peak was distinct from that of /3-glucuronidase, a marker enzyme for lysosomes, which were possibly impurities in the applied crude membrane fraction. Lactate dehydrogenase activity was not detected in any of the fractions in Fig. 1. The main peak of phospholipase C activity did not coincide with the main peak of 5’-nucleotidase, a marker enzyme of plasma membranes, which was observed in a lower density region, but 5’nucleotidase activity was also detected in the high density region together with phospholipase C activity, which suggested
number
1. Fractionation of the 100,OOOg pellet from gradient centrifugation. The experimental
a lymphocyte homogenate by continuous details are given in the text.
sucrose
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the presence of high density plasma membranes. When cell surface proteins were labeled with lz51 by the lactoperoxidase method before preparation of the 100,OOOg pellet and their distribution on sucrose density gradient centrifugation was investigated, the radioactivity was found to be widely distributed in the region in which the major phospholipase C peak and the major 5’-nucleotidase peak were included (data not shown). It seemed that this phospholipase C activity was bound to plasma membranes with a density higher than that of the plasma membrane showing high 5’nucleotidase activity. Furthermore, the presence of PI-phospholipase C in the plasma membrane was confirmed by trypsin treatment of intact lymphocytes. Trypsin treatment decreased the phospholipase C activity in the membrane fraction, but no significant effect was observed on this activity in the cytosolic fraction (Table II). This suggests that at least some of PI-phospholipase C molecules are exposed on the cell surface. The membrane-bound and cytosolic phospholipases C were confirmed to be indeed phospholipases C by the analysis of the water-soluble product formed from radioactive PI by descending paper chromatography according to the method of Dawson et al. (17). In both cases, on paper
TABLE
II
EFFECT OF TRYPSIN TREATMENT ON PI-PHOSPHOLIPASE C ACTIVITIES IN THE MEMBRANE AND CYTOSOLIC FRACTIONS PI-phospholipase
Fraction Membrane Cytosolic
Untreated
cells
987 f 61 5303 f 221
C activity” Trypsin-treated cellsb 451 f 95 4655 + 566
a Radioactivity (dpm) released from phosphatidyl[2‘Hlinositol; means f SD of triplicate experiments. bIntact cells were treated with trypsin and then the plasma membrane and the cytosolic fractions were separated as described under Materials and Methods.
PHOSPHOLIPASE
573
C TABLE
III
PI-PHOSPHOLIPASE C ACXIVITIY OF T- AND B-CELL-ENRICHED
Fraction T-Cell-enriched B-Cell-enriched Macrophage-depleted
IN MICROSOMES FRACTIONS~
Phospholipase C activityb (% of control) 87 k 1 75 f 7 88 + 3
“Amount of protein of each microsome sample added was 5 yg. * Microsomes from unseparated lymphocytes were used as a control. The specific activity of the 100% control was 0.57 nmol min-’ mg protein’. Values are means + SD (n = 3).
chromatography, the water-soluble product was identical to that formed from radioactive PI by treatment with Bacillus cereus phospholipase C (Sigma) (data not shown). Cellular origin of membrane-bound phospholipase C. The murine spleen lymphocyte
fraction separated by the Ficoll-Urografin method contained T cells, B cells, and macrophages. To determine which kind of lymphocytes possess membrane-bound phospholipase C, phospholipase C activity in microsomes from a T-cell-enriched fraction, a B-cell-enriched fraction, and a macrophage-depleted fraction was measured. As shown in Table III, the microsome phospholipase C activity in these cell fractions was only slightly lower than that in unseparated spleen lymphocytes (control). Furthermore, no significant difference in phospholipase C activity was observed among any of these cell fractions. These results suggest that membrane-bound phospholipase C is present in both T cells and B cells. Comparison of some properties of the cytosolic and membrane-bound phospholipases
C. Table IV shows the specificity of the membrane-bound and cytosolic phospholipases C toward various phospholipids. Both enzymes showed higher reactivity toward PI than phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Hydro-
574
KAMISAKA, TABLE
TOYOSHIMA,
AND
OSAWA
IV
SUBSTRATE SPECIFICITY OF MEMBRANE-BOUND AND CYTOSOLIC PI-PHOSPHOLIPASES C” PI-phospholipase C (nmol mini mg protein-‘)
Substrate PI PC PE PS
Membranebound (6.0 f 0.4) x lo-’ (1.4 f 0.1) x 10-z (2.7 IL 0.1) x W3 0
Cytosol 4.1 + 0.2 (3.1 f 0.2) x 10-z (2.9 + 0.1) x W 0
“Phospholipase C activity was measured as described under Materials and Methods. Each value is the mean of triplicate assays + SD. Each tube contained 10 Fg of enzyme, 0.01 &i of [aH]PI, [i4C]PC, [‘*C]PE, or [14C]PS and 10 FM of the corresponding cold phospholipid. The mixture was incubated at 3’7°C for 90 min.
lysis of phosphatidylserine (PS) was not detected under the assay conditions used here. Since the isotopical specific activities were corrected, the results indicate that these enzymes react preferentially with PI. PI-4-P and PI-4,5-PZ were also hydrolyzed by these enzymes, but the specific activity of the enzyme toward [32P]PI-4-P and [32P]PI-4,5-Pz could not be determined because of the very low specific radioactivities of the [32P]PI-4-P and [32P]PI-4,5-P2 employed. The effect of pH on the cytosolic and membrane-bound enzymes is shown in Fig. 2. In the presence of deoxycholate, the cytosolic enzyme was maximally active at pH 6.0 and the membrane-bound enzyme showed two pH optima between pH 5.0 and 7.0. Addition of deoxycholate to the assay buffer induced a marked increase in the activities of these enzymes, especially that of the cytosolic enzyme. These discrepancies in the pH optimum and the deoxycholate effect suggest that plural enzymes with similar phospholipase C activities may be present in spleen lymphocytes, and that the enzyme activity in the membrane fraction is not due to contamination by a soluble enzyme. Effects of various phospholipids on the membrane-bound and cytosolic enzymes
FIG. 2. pH dependency of PI-phospholipase C activity in the cytosolic (A) and membrane (B) fractions. The buffers used were sodium acetate-acetic acid (pH 4-6.4) and Tris-acetate (pH 7-9). The assays were performed in the presence (0) or absence (e) of 0.05% deoxycholate in triplicate. Each value represents the mean f SD. The same experiment was repeated twice and the results were reproducible.
were also examined and the results are shown in Table V. PC, PE, and PS markedly inhibited the activity of both enzymes. However, phosphatidic acid (PA) showed TABLE
V
EFFECTS OF DIFFERENT PHOSPHOLIPIDS Two PI-PHOSPHOLIPASES C” Activity Phospholipid PC PE PS PA
Membrane 315 9 23 f 10 23+ 9 121 f 12
ON THE
(% of control)b Supernatant llf 2 26 f 16 23 f 18 69 rt_ 20
“Phospholipase C activity was measured as described under Materials and Methods. Each value is the mean of triplicate experiments f SD. b PC, PE, PS, or PA (10 rg) was added to a control assay tube containing 10 pg enzyme, 0.01 &i [3H]PI, and 5 pg PI. The specific activities of the 100% control for membrane-bound and cytosolilc enzymes were 0.71 and 2.2 nmol min-’ mg protein-‘, respectively.
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different effects on these two enzymes. The membrane-bound phospholipase C activity was enhanced, while the cytosolic enzyme was inhibited. The activity of the membrane-bound enzyme increased gradually with increasing calcium concentration (up to IO-’ M). On the other hand, the activity of the cytosolic phospholipase C was maximum at 100 yM Ca2+ (Fig. 3). Both enzymes showed similar heat stability. The activities of membrane-bound and cytosolic phospholipases C were completely abolished on heating at 50°C for 10 min. Column chromatography of the membrane-bound and cytosolic phospholipases C on Sephadex G-100 and concanavalin A-Sepharose. Before column chromatog-
raphy, the solubilization conditions for membrane-bound phospholipase C were examined. As shown in Table VI, 1% deoxycholate was most effective for solubilizing PI-phospholipase C from lymphocyte membranes. A lower concentration of cholate (0.4%) was also effective, solubilizing about 70% of the membrane-bound phospholipase activity (data not shown). On the other hand, a significant amount of PI-
FIG. 3. Effect of Ca2+ on the cytosolic (A) and membrane (B) phospholipase C activities. The free calcium concentration in the assay buffer was adjusted by adding Caa+-EGTA buffer (24).
PHOSPHOLIPASE
575
C TABLE
VI
SOLUBILIZATIONOFMEMBRANEPHOSPHOLIPASEC BYVARIOUSTREATMENTS % Solubilized
Treatment
Protein
1% Deoxycholate 1 M NaCl 1 mM EDTA 2 M KSCN
53 23 Trace 55
PI-phospholipase activity’
C
79 Trace 5 ND*
“The membrane fraction was treated with 1% deoxycholate, 1 M NaCl, 1 mM EDTA, or 2 M KSCN at 4°C for 2 h, and then centrifuged at lOO,OOO(l for 1 h. The supernatant was assayed for PI-phospholipase C activity. *Not detected.
phospholipase C activity was not solubilized with 1 M NaCl or 1 mM EDTA. More than 50% of the membrane proteins were solubilized with 2 M KSCN, but the enzyme activity was not detected in the resultant solubilized solution. However, since its addition completely abolished the phospholipase C activity in a cholate extract, KSCN appears to be unsuitable for solubilization of the enzyme. The cholate extract of lymphocyte membranes was applied to a column of Sephadex G-100. The elution profile is shown in Fig. 4B. Phospholipase C activity was eluted as a single peak with a shoulder. An approximate molecular weight of 70,000 was calculated for the membrane phospholipase C from a curve of elution volumes against logarithms of molecular weights of various standard proteins. However, gel filtration analysis in the presence of detergent was not suitable for the estimation of molecular weight. Lymphocyte cytosol was also applied to a column of Sephadex G-100. The elution profile of the cytosolic phospholipase C was similar to that of the membrane-bound phospholipase C, and the molecular weight of the cytosolic enzyme was also found to be about 70,000 (Fig. 4A). For further comparison of the membrane-bound and cytosolic phospholipases C, the pooled peak fractions of phospholi-
576
KAMISAKA,
Fraction
number
TOYOSHIMA,
(3mlltube)
FIG. 4. Gel filtration of cytosolic (A) and membranebound (B) phospholipases C on Sephadex G-100. The experimental details are given in the text.
pase C activity from a Sephadex G-100 column were applied to a column of Con ASepharose. As shown in Fig. 5, a portion of the membrane-bound phospholipase C activity bound to Con A-Sepharose and was eluted with the elution buffer containing (w-MM, whereas none of the cytosolic phospholipase C activity bound to Con A-Sepharose. The recovery of the membranebound enzyme activity in the specifically eluted fraction was approximately 20 to 30%. These results suggest that a part of the membrane-bound phospholipase C is a Con A-binding glycoprotein or associates with Con A-binding sites on the cell surface.
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pholipase C was also reported in the brain (6). However, it has recently been claimed that the phospholipase C activity detected in the membrane fraction can be adequately accounted for by contamination by a cytosolic enzyme (26,27). In the present study, we were able to detect the presence of a membrane-bound phospholipase C (Fig. 1, Table I). PI-Phospholipase C activity in the membrane fraction could be extracted only with cholate (Table VI). Trypsin treatment of intact lymphocytes caused an about 50% decrease in phospholipase C activity (Table II). Furthermore, a portion of the phospholipase C activity extracted from the membrane fraction bound to Con A-Sepharose (Fig. 5). These results suggest that a portion of the membrane-bound phospholipase C is expressed on the cell surface and that it is a glycoprotein or a protein firmly associated with membrane glycoprotein. Some properties of the cytosolic and the membrane-bound phospholipases C were studied. They have the same substrate specificity for PI (Table IV) and require Ca2+ for their activity (Fig. 3). They differ in pH optima (Fig. 2), in the Ca2+ concentration required for their optimum activity (Fig. 3), and in the effect of PA on their enzymatic activity. However, changes in and PA concenpH, Ca2+ concentration, tration may alter the activity by influenc-
DISCUSSION Fraction
PI-phospholipase C, which catalyzes the initial step of PI turnover, has been found primarily in the cytosolic fraction of mammalian cells. A membrane-bound PI-phos-
number
(1 Smlltubd
FIG. 5. Affinity column chromatography of cytosolic (A) and membrane-bound (B) phospholipases C on Con A-Sepharose. The experimental details are given in the text.
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ing the susceptibility of PI to the enzyme rather than by having direct effects on the enzymes per se because of the high sensitivity of the enzymes to changes in the physical nature of the substrate. Therefore, it is difficult to come to a definite conclusion as to the physiological significance of the differences observed between two enzymes. Recently, Dawson et al. (28) and other groups (29-31) suggested possible mechanisms for both the suppression and the activation of the soluble phospholipase C by various membrane lipids. Similar modification of PI-phospholipase C activity by membrane lipids was also noted in the present study. We found inhibition of both the membrane-bound and cytosolic phospholipases C by PC, similar to that reported by other investigators (28-31). The effects we observed with other lipids were different from the results of others. In lymphocytes, PE and PS were inhibitory for both the membrane-bound and cytosolic phospholipases C of lymphocytes (Table V), but they only had a moderate effect on the PI-phospholipase C activity of sheep seminal vesicular gland (30). Furthermore, the effects of PA on the membrane-bound and cytosolic enzymes were dichotomous (Table V). These differences might be partly due to changes in substrate concentration and Ca2+ concentration. However, in the case of membrane-bound phospholipase C, they are more difficult to explain because membrane preparations contain various kinds of lipids. Recently, it has become apparent that polyphosphoinositides undergo agonistinduced hydrolysis by PI-phospholipase C to yield diglyceride and inositol phosphates (2,3), and this hydrolysis appears to reflect a signal-generating system. In thymocytes, a mitogen also induces an increase in inositol phosphates within a minute of stimulation (32), which suggests the presence of polyphosphoinositide degradation enzymes. We found that a hydrolytic activity for polyphosphoinositides was present in both the membrane and cytosolic fractions from murine lymphocytes. Recent reports suggest that the mitogenie signals generated after mitogen-re-
PHOSPHOLIPASE
C
577
ceptor interaction may be transduced via hydrolysis of inositol phospholipids not only in T-cell activation but also in B-cell activation (32-35). Therefore, it seems reasonable that PI-phospholipase C activity was detected in both T-cell-enriched and B-cell-enriched fractions (Table III). REFERENCES 1. MICHELL, R. H. (1975) Biochim. Biophys. Acta 415, 81-147. 2. BERRIDGE, M. J. (1984) &~&em. J. 220,345-360. 3. BERRIDGE, M. J., AND IRVINE, R. F. (1984) Nature (Lmdon) 312, 315-321. 4. NISHIZUKA, Y. (1984) Science 225,1365-1370. 5. HAMBERG, M., SVENSSON, J., AND SAMUELSSON, B. (197.5) Proc. Natl. Acad. Sci. USA 72,2994-2998. 6. LAPETINA, E. G., AND MICHELL, R. H. (1973) Biochem. J. 131,433-442. 7. FRIEDEL, R. O., BROWN, J. D., AND DURELL, J. (1969) J. Neurochem. 16,371-3’78. 8. ROSENBERG, R. J., MASKOWITZ, R. W., AND MALEMUD, C. J. (1983) Biochem. Biophys. Res. Comrnul?, 115,331-338. 9. SIESS, W., AND LAPETINA, E. G. (1983) Biochim. Biophys. Acta 752,329-338. 10. ALLAN, D., AND MICHELL, R. H. (1974) B&hem. J. 142,591-597. 11. KAWAGUCHI, T., MATSUMOTO, I., AND OSAWA, T. (1974) J. Biol. Chem. 249,2786-2792. 12. JULIUS, M. H., SINPSON, E., AND HERZENBERG, L. A. (1973) Eur. J. Immunol. 3,645-649. 13. LY, I. A., AND MICHELL, R. H. (1974) .I Immunol. Methods 5,239-247. 14. GOLUB, E. A. (1972) J. Immunol. 109,168-170. 15. MAKISHIMA, F., TOYOSHIMA, S., AND OSAWA, T. (1985) Arch. Biochem. Biophys. 238, 315-324. 16. MAEDA, T., BALAKRISHNAN, K., AND MEHDI, S. 0. (1983) B&him. Biophys. Acta 731, 115-120. 17 _.. DAWSON, R. M. C., AND CLARKE, N. (1972) Biodem. J. 127,113-118. 18. DOWNES, C. P., AND MICHELL, R. H. (1981) B&hem. J 198,133-140. 19. SONG, C. S., AND BODANSKY, 0. (1967) J. Biol Chem. 242,694-699. 20. KORNBERG, A. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 441-443, Academic Press, New York. 21. FERBER, E., RESCH, K., WALLACH, D. F. H., AND IMM, W. (1972) Biochim. Biophys. Acta 266,494504. 22. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1961) J. Biol. Chem. 193, 265-275.
578
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TOYOSHIMA,
23. AMES, B. N., AND RUBIN, 0. T. (1960) J. Biol. Chem 235,769-775. 24. PORTZEHL, H., CALDWELL, P. C., AND ROEGG, J. C. (1964) Biochim Biophys. Acta 79,581-591. 25. MATSUMOTO, I., MIZUNO, Y., AND SENO, N. (1979) J. Biochem. 85,1091-1098. 26. IRVINE, R. F., AND DAWSON, R. M. C. (1978) J. Neurochem. 31,1427-1434. 27. SIESS, W., AND LAPETINA, E. G. (1983) Biochim Biqphgs. Acta 752,329-338. 28. DAWSON, R. M. C., HEMINGTON, N., AND IRVINE, R. F. (1980) Eur. J. Biochem 112,33-38. 29. TAKENAWA, T., AND NAGAI, Y. (1981) J. Bid Chem 256,6769-6975.
AND
OSAWA
30. HOFMANN, S. L., AND MAJERUS, Biol. Chem. 257,14359-14364. 31. CHAU, L.-Y., AND TAI, H.-H. phys. Acta 713,344-351.
(1982)
P. W. (1982) Bio&im
J. Bi+
32. TAYLER, M. V., METCALFE, J. C., HESKETH, T. R., SMITH, G. A., AND MOOR, J. P. (1934) Nature (Lmdon) 312,462-465. 33. COGGESHALL, Immund
K. M., AND CAMBIER, 133,3382-3386.
34. MAINO, V. C., HAYMAN, M. J. (1975) B&hem 35. MASUZAWA, S. (1973)
J. C. (1934)
M. J., AND CRUMPTON, .I 146,247-252.
Y., OSAWA, T., INOUE, K., AND NOJIU, B&him. Biqphys. Acta 326,339-344.
J.
Vol.
OF BIOCHEMISTRY
AND
249, No. 2, September,
BIOPHYSICS
pp. 569-578,1986
Phosphatidylinositol-Specific YASUSHI Division
KAMISAKA; of Chemical
Phospholipase SATOSHI
Toxicology University
Received
January
C of Murine Lymphocytes’
TOYOSHIMA,
and Immunochemistry, of Tokyo, Bunkyrrku,
AND
TOSHIAKI
Faculty of Pharmaceutical Tokyo 113, Japan,
28, 1986, and in revised
form
May
OSAWA3 Sciences,
27, 1986
Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol4-phosphate, and phosphatidylinositol4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. (1) The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. (2) The activity of the cytosolic enzyme was first detected at 1 PM Ca”+, and maximum activity was observed at 100 PM Ca2+, while the membranebound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. (3) The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate. (4) A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an a-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. (5) Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity. 0 1986 Academic
Press. Inc.
It has been reported that marked inositol phospholipid turnover occurs in a variety of cells and tissues in response to external stimuli and that this phenomenon may
play a vital role in cell activation (l-3). The importance of this effect has been reported, for example, in Ca2+ gating (l), intracellular Ca2+ mobilization (2, 3), Ca2+ regulation, phospholipid-dependent protein kinase activity (4), and arachidonic acid metabolism (5). Though it remains unclear how external stimuli induce inositol phospholipid turnover, there is general agreement that the external stimulus-induced breakdown of inositol phospholipid is mediated through the action of phosphatidylinositol-specific phospholipase C (PI-
‘This investigation was supported by research grants from the Ministry of Education, Science and Culture of Japan, and the Sankyo Foundation for Life Sciences. a Present address: Bioorganic Chemistry Division, National Chemical Laboratory for Industry, Yatabe, Ibaraki 305, Japan. 3 To whom all correspondence should be addressed.
569
0003-9861186 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
570
KAMISAKA,
TOYOSHIMA,
phospholipase C)* (l-3). Since this enzyme acts at a very early stage of receptor-mediated cell activation and the site of PI breakdown seems to be in the plasma membrane, it appears that PI-phospholipase C exists in the plasma membrane of cells. However, PI-phospholipase C activity was primarily detected in the cytosolic fraction of cells and only a few reports suggested the presence of a membranebound PI-phospholipase C (6-8). Furthermore, it was reported that PI-phospholipase C activity detected in platelet particulates was due to artificial contamination of the cytosolic enzyme (9). Allan and Michell (10) also showed that in lymphocytes most of this enzyme was cytosolic with only small amounts in the particulate fraction. However, in the present study we were able to detect significant amounts of PI-phospholipase C activity in the plasma membrane of murine splenic lymphocytes and to show some differences between the properties of the membrane-bound and cytosolic phospholipases C. MATERIALS
AND
METHODS
Cells. Murine spleens from 8- to IO-week-old female ICR mice (Charles River Japan, Kanagawa, Japan) were removed and teased apart on ice in Eagle’s minimal essential medium, pH 7.2 (MEM, Nissui Co., Tokyo, Japan) containing 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes, Wako Pure Chemical Co., Tokyo, Japan). After removal of clumps by passage through a stainless steel mesh, lymphocytes were separated by the Ficoll-Urografin method (11). Lymphocytes were further fractionated on a nylon wool column (12), and the nylon wool nonadherent cells were obtained as a T-cell-enriched fraction. Macrophages were removed by passage through a Se-
* Abbreviations used: PI, phosphatidylinositol; PIphospholipase C, PI-specific phospholipase C; Hepes, N-2-hydroxyethylpiperazine-iV’-2-ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PI-I-P, phosphatidylinositol 4-phosphate; PI-4,5-Pz, phosphatidylinositol4,5-bisphosphate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid; MEM, Eagle’s minimal essential medium (pH 7.2); EGTA, ethylene glycol bis(2aminoethyl ether) iV,N’-tetraacetic acid, a-MM, methyl-a-D-mannopyranoside.
AND
OSAWA
phadex G-10 column (macrophage-depleted fraction) (13). For enrichment of B cells, lymphocytes were treated with appropriately absorbed rabbit antimouse brain-associated 0 antigen serum (anti-BAT) (14) and guinea pig complement, and then applied to a Ficoll-Urografin layer to remove dead cells. The resulting population of B cells (B-cell-enriched fraction) contained more than 95% Ig-bearing cells, SubceUularfractionatim Microsomes were prepared according to the method of Makishima et al, (15) with minor modifications. The cells were suspended in 10 mM Tris-HCl buffer (pH 7.4), containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Chemical Co., St. Louis, MO.), 0.02% NaNa, and 1 mM ethylenediaminetetraacetate (EDTA, Sigma), and allowed to stand at 4°C for 5 min. Then the cells were homogenized with a Potter-Elvehjem homogenizer. After 1 ml of 10 mM Tris/HCl buffer (pH 7.3) containing 1.5 M NaCl and 1 mM EDTA was added to make the NaCl concentration 0.15 M, the mixture was centrifuged at 300g for 15 min to remove cell debris and nuclei. The supernatant was collected and centrifuged at 60009 for 10 min to remove mitochondria, lysosomes, etc. The supernatant was then centrifuged at 100,OOOg for 1 h. The pellet was dissolved in 10 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and then centrifuged at 105,OOOg for 1 h. This step was performed again and the final pellets were suspended in 10 mM TrisHCl buffer (pH 7.4) containing 0.15 M NaCl (microsome fraction). The specific activity of 5’-nucleotidase, a marker enzyme of plasma membranes, was about 6.1 times higher in this microsome fraction than in the homogenate. To examine various properties of PI-phospholipase C, the simplified technique of Maeda et al. (16) was employed to separate the plasma membrane and cytosol. Briefly, the cells were suspended in 10 mM TrisHCl buffer (pH 7.4) containing 30 mM NaCl, 1 mM MgCl,, 1 mM PMSF, and 0.02% NaN$, and then homogenized with a Potter-Elvehjem Teflon homogenizer. The homogenate was layered on a 41% sucrose solution in the homogenization buffer and centrifuged at 95,000g for 1 h. After centrifugation, the interfacial band of membranes was collected and washed three times with the homogenization buffer, and the supernatant (top layer) was used as a cytosolic fraction in the subsequent experiments. The specific activity of 5’-nucleotidase, a marker enzyme of plasma membrane, was about 22 times higher in this plasma membrane fraction than in the homogenate. The plasma membrane fraction thus obtained seemed to be purer than the microsome fraction. To investigate the subcellular distribution of PIphospholipase C, a continuous sucrose density gradient (30-50%)was used. The lymphocyte homogenate was centrifuged at 400g for 15 min. The supernatant was centrifuged at 100,000~ for 1 h and then the pellet
MURINE
LYMPHOCYTE
was washed twice with 10 mM Tris-WC1 buffer (pH 7.4) containing 0.15 M NaCl. The washed pellet was suspended in the washing buffer and layered on a sucrose gradient solution (30-5O%,w/v) with a 60% (WI v) sucrose solution at the bottom. After centrifugation at 40,OOOg for 3 h, fractions were collected from the bottom of the tube. Try&z treatment. Intact lymphocytes were treated with 0.1% trypsin (Worthington Biochemicals, Freehold, N.J.) in MEM for 30 min at 25°C. The reaction was stopped by adding soybean trypsin inhibitor (Sigma) solution (1 mg/ml), and then the cells were washed three times with MEM. Cell viability was not affected by this treatment. The plasma membrane and cytosolic fractions were also hydrolyzed with 0.1% trypsin for 30 min at 25°C. To further confirm the presence of PI-phospholipase C in the lymphocyte plasma membrane, the plasma membrane fraction from trypsin-treated lymphocytes or untreated lymphocytes was layered on a continuous sucrose density gradient (20-50%, w/v), and after centrifugation at 40,OOOg for 3 h, the PI-phospholipase C activity in each sucrose density fraction was assayed. Assay for PI-phospholipase C activity. The assay mixture, on ice, contained 10 pM soybean PI, 0.01 &i L-a-phosphatidyl[2-3H]inositol (sp radioact, 15.6 Ci/ mmol; The Radiochemical Centre, Amersham, U.K.), as a liposome suspension prepared by brief sonication, and, unless otherwise stated, 10 fig of enzyme in 250 ~1 of 0.1 M acetate buffer (pH 6.0) containing 1 mM Ca2+ and 0.05% (w/w) sodium cholate. The reaction was performed at 37°C for 90 min and terminated by adding 1 ml of chloroform/methanol/concentrated HCl (100/100/0.6) and 0.3 ml of 1 N HCI containing 5 mM ethylene glycol bis(2-aminoethyl ether) N,N’-tetraacetic acid (EGTA). Phospholipase activity was determined by measuring the radioactivity in the aqueous phase. Under the above conditions, the hydrolyzed products linearly increased with incubation time or the amount of protein. To examine the substrate specificity of phospholipase C, instead of radioactive PI and cold PI, L-a-dipalmitoylphosphatidyl[N-methyl-‘4C]choline (sp radioact 153 mCi/mmol; The Radiochemical Centre), L-ol-dioleoylphosphatidyl[2-‘4C]ethanolamine(spradioact44mCi/mmol;The Radiochemical Centre), or L-a-dioleoylphosphatidyl[U-‘4C]serine (sp radioact 60 mCi/mmol; The Radiochemical Centre) and the corresponding cold phospholipid were added to the reaction mixture. The hydrolyzed products in the aqueous phase were analyzed by descending paper chromatography on Whatman No. 1 paper in ethanoVl3.5 M NH,OH (3/2, v/v) as described by Dawson et al (17). For assay of phosphatidylinositol 4-phosphate (PI-4-P) and phosphatidylinositol 4,5-bisphosphate (PI-4,5-P,) hydrolyses, the reaction mixture contained 500-2000 cpm [3zP]PI-
PHOSPHOLIPASE
C
571
4-P or [3*P]PI-4,5-P2 instead of rH]PI. [32P]PI-4-P and [3ZP]PI-4,5-PZ were isolated from extracts of [32P]orthophosphate-labeled human erythrocyte ghosts (18). Assays for other enzyme activities. 5’-Nucleotidase, a marker enzyme of plasma membranes, was assayed as described by Song and Bodansky (19); lactate dehydrogenase, a marker enzyme of the cytosol, as described by Kornberg (20); and P-glucuronidase, a marker enzyme of lysosomes, as described by Ferber et al. (21). Analytical methods. Proteins were determined by the method of Lowry et al. (22) with bovine serum albumin as standard. Phospholipid phosphorus was measured by the method of Ames and Rubin (23). The free Ca2+ concentration of micromolar order in the assay medium was regulated by using Ca’+-EGTA buffers and calculated according to Portzehl et al. (24). Column chromatography on Sephadex G-100 and concanavalin A-Sepharose. Lymphocyte membranes (about 10 mg) were solubilized at 4°C with 2 ml of 10 mM Tris-HCI buffer (pH 7.4) containing 0.02 M NaCI, 0.02% NaN$, and 0.4% deoxycholate (Sigma). The solubilized membranes were immediately applied to a Sephadex G-100 column (1.6 X 92 cm) equilibrated with the solubilization buffer. The lymphocyte cytosol was also applied to a Sephadex G-100 column (1.6 X 140 cm) equilibrated with 10 mM Tris-HCI buffer (pH 7.4) containing 0.02 M NaCl and 0.02% NaN,. The cytosol and the solubilized membranes were dialyzed against the column equilibration buffer before application to the Sephadex G-100 column. Concanavalin A (Con A, Honen Oil Co., Tokyo, Japan) was coupled to CH-Sepharose (Pharmacia, Uppsala, Sweden) using 1-ethyl-3-dimethylaminopropylcarbodiimide (25). About 15 mg of protein was found to be immobilized per 1 ml of resin. The active fractions obtained on Sephadex G-100 column chromatography of the membranes and the cytosol were applied to a Con A-Sepharose column (1 ml resin) equilibrated with 10 mM Tris-HC1 buffer (pH 7.4) containing 0.4% deoxycholate, 0.15 M NaCI, 0.02% NaNa, 0.1 mM MnCl,, and 0.1 mM CaCI,. The elution was started with the equilibration buffer, followed by the same buffer containing 0.3 M methyl-a-D-mannopyranoside (a-MM).
RESULTS
Subcellular distribution of phospholipase C. As shown in Table I, phospholipase C activity in lymphocytes was primarily distributed in the cytosolic fraction (supernatant). However, a significant amount of phospholipase C activity was detected in the microsome fraction. In spite of the
572
KAMISAKA,
TOYOSHIMA, TABLE
SUBCELLULAR
DISTRIBUTION
Homogenate Nuclei Mitochondria Microsome Supernatant
Protein (mg)
Specific activityb
11 5.2 0.68 0.78 4.1
a Similar results were obtained b nmol min-’ mg protein-‘. ’ nmol mini (% of homogenate).
1.4 0.27 0.72 0.80 2.5 in four
C
Total activityC 15 1.4 0.49 0.62 10
(100) (9.3) (3.3) (4.1) (67)
Fract
FIG.
I C ACTIVITY
Lactate
dehydrogenase
Specific activityb
Total activity’
18 1.7 2.1 0.54 38
200 8.8 1.4 0.42 160
(100) (4.4) (0.71) (0.21) (79)
a
5’-Nucleotidase Specific activityb 0.57 0.25 1.2 3.5 0.38
Total activity” 6.3 1.3 0.92 2.7 1.6
(100) (21) (13) (43) (25)
experiments.
great increase in the specific activity of 5’nucleotidase, a marker enzyme of plasma membrane, in the microsome fraction as compared with the homogenate, the specific activity of phospholipase C in the microsome fraction was still more than half of that in the homogenate. About 4% of the total phospholipase C activity was recovered in the microsome fraction, but the recovery of lactate dehydrogenase, a marker enzyme of the cytosol, was only 0.21%. These results suggest that phospholipase C activity in the microsome fraction is not due to contamination by the cytosolic phospholipase C. The subcellular distribution of phospholipase C was further examined by means of continuous sucrose density gradient
density
OSAWA
OF PI-PHOSPHOLIPASE
PI-phospholipase
Fraction
AND
!on
centrifugation of the 100,OOOg pellet from a lymphocyte homogenate as described under Materials and Methods. As shown in Fig. 1, mainly one peak of phospholipase C activity was observed. This peak was distinct from that of /3-glucuronidase, a marker enzyme for lysosomes, which were possibly impurities in the applied crude membrane fraction. Lactate dehydrogenase activity was not detected in any of the fractions in Fig. 1. The main peak of phospholipase C activity did not coincide with the main peak of 5’-nucleotidase, a marker enzyme of plasma membranes, which was observed in a lower density region, but 5’nucleotidase activity was also detected in the high density region together with phospholipase C activity, which suggested
number
1. Fractionation of the 100,OOOg pellet from gradient centrifugation. The experimental
a lymphocyte homogenate by continuous details are given in the text.
sucrose
MURINE
LYMPHOCYTE
the presence of high density plasma membranes. When cell surface proteins were labeled with lz51 by the lactoperoxidase method before preparation of the 100,OOOg pellet and their distribution on sucrose density gradient centrifugation was investigated, the radioactivity was found to be widely distributed in the region in which the major phospholipase C peak and the major 5’-nucleotidase peak were included (data not shown). It seemed that this phospholipase C activity was bound to plasma membranes with a density higher than that of the plasma membrane showing high 5’nucleotidase activity. Furthermore, the presence of PI-phospholipase C in the plasma membrane was confirmed by trypsin treatment of intact lymphocytes. Trypsin treatment decreased the phospholipase C activity in the membrane fraction, but no significant effect was observed on this activity in the cytosolic fraction (Table II). This suggests that at least some of PI-phospholipase C molecules are exposed on the cell surface. The membrane-bound and cytosolic phospholipases C were confirmed to be indeed phospholipases C by the analysis of the water-soluble product formed from radioactive PI by descending paper chromatography according to the method of Dawson et al. (17). In both cases, on paper
TABLE
II
EFFECT OF TRYPSIN TREATMENT ON PI-PHOSPHOLIPASE C ACTIVITIES IN THE MEMBRANE AND CYTOSOLIC FRACTIONS PI-phospholipase
Fraction Membrane Cytosolic
Untreated
cells
987 f 61 5303 f 221
C activity” Trypsin-treated cellsb 451 f 95 4655 + 566
a Radioactivity (dpm) released from phosphatidyl[2‘Hlinositol; means f SD of triplicate experiments. bIntact cells were treated with trypsin and then the plasma membrane and the cytosolic fractions were separated as described under Materials and Methods.
PHOSPHOLIPASE
573
C TABLE
III
PI-PHOSPHOLIPASE C ACXIVITIY OF T- AND B-CELL-ENRICHED
Fraction T-Cell-enriched B-Cell-enriched Macrophage-depleted
IN MICROSOMES FRACTIONS~
Phospholipase C activityb (% of control) 87 k 1 75 f 7 88 + 3
“Amount of protein of each microsome sample added was 5 yg. * Microsomes from unseparated lymphocytes were used as a control. The specific activity of the 100% control was 0.57 nmol min-’ mg protein’. Values are means + SD (n = 3).
chromatography, the water-soluble product was identical to that formed from radioactive PI by treatment with Bacillus cereus phospholipase C (Sigma) (data not shown). Cellular origin of membrane-bound phospholipase C. The murine spleen lymphocyte
fraction separated by the Ficoll-Urografin method contained T cells, B cells, and macrophages. To determine which kind of lymphocytes possess membrane-bound phospholipase C, phospholipase C activity in microsomes from a T-cell-enriched fraction, a B-cell-enriched fraction, and a macrophage-depleted fraction was measured. As shown in Table III, the microsome phospholipase C activity in these cell fractions was only slightly lower than that in unseparated spleen lymphocytes (control). Furthermore, no significant difference in phospholipase C activity was observed among any of these cell fractions. These results suggest that membrane-bound phospholipase C is present in both T cells and B cells. Comparison of some properties of the cytosolic and membrane-bound phospholipases
C. Table IV shows the specificity of the membrane-bound and cytosolic phospholipases C toward various phospholipids. Both enzymes showed higher reactivity toward PI than phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Hydro-
574
KAMISAKA, TABLE
TOYOSHIMA,
AND
OSAWA
IV
SUBSTRATE SPECIFICITY OF MEMBRANE-BOUND AND CYTOSOLIC PI-PHOSPHOLIPASES C” PI-phospholipase C (nmol mini mg protein-‘)
Substrate PI PC PE PS
Membranebound (6.0 f 0.4) x lo-’ (1.4 f 0.1) x 10-z (2.7 IL 0.1) x W3 0
Cytosol 4.1 + 0.2 (3.1 f 0.2) x 10-z (2.9 + 0.1) x W 0
“Phospholipase C activity was measured as described under Materials and Methods. Each value is the mean of triplicate assays + SD. Each tube contained 10 Fg of enzyme, 0.01 &i of [aH]PI, [i4C]PC, [‘*C]PE, or [14C]PS and 10 FM of the corresponding cold phospholipid. The mixture was incubated at 3’7°C for 90 min.
lysis of phosphatidylserine (PS) was not detected under the assay conditions used here. Since the isotopical specific activities were corrected, the results indicate that these enzymes react preferentially with PI. PI-4-P and PI-4,5-PZ were also hydrolyzed by these enzymes, but the specific activity of the enzyme toward [32P]PI-4-P and [32P]PI-4,5-Pz could not be determined because of the very low specific radioactivities of the [32P]PI-4-P and [32P]PI-4,5-P2 employed. The effect of pH on the cytosolic and membrane-bound enzymes is shown in Fig. 2. In the presence of deoxycholate, the cytosolic enzyme was maximally active at pH 6.0 and the membrane-bound enzyme showed two pH optima between pH 5.0 and 7.0. Addition of deoxycholate to the assay buffer induced a marked increase in the activities of these enzymes, especially that of the cytosolic enzyme. These discrepancies in the pH optimum and the deoxycholate effect suggest that plural enzymes with similar phospholipase C activities may be present in spleen lymphocytes, and that the enzyme activity in the membrane fraction is not due to contamination by a soluble enzyme. Effects of various phospholipids on the membrane-bound and cytosolic enzymes
FIG. 2. pH dependency of PI-phospholipase C activity in the cytosolic (A) and membrane (B) fractions. The buffers used were sodium acetate-acetic acid (pH 4-6.4) and Tris-acetate (pH 7-9). The assays were performed in the presence (0) or absence (e) of 0.05% deoxycholate in triplicate. Each value represents the mean f SD. The same experiment was repeated twice and the results were reproducible.
were also examined and the results are shown in Table V. PC, PE, and PS markedly inhibited the activity of both enzymes. However, phosphatidic acid (PA) showed TABLE
V
EFFECTS OF DIFFERENT PHOSPHOLIPIDS Two PI-PHOSPHOLIPASES C” Activity Phospholipid PC PE PS PA
Membrane 315 9 23 f 10 23+ 9 121 f 12
ON THE
(% of control)b Supernatant llf 2 26 f 16 23 f 18 69 rt_ 20
“Phospholipase C activity was measured as described under Materials and Methods. Each value is the mean of triplicate experiments f SD. b PC, PE, PS, or PA (10 rg) was added to a control assay tube containing 10 pg enzyme, 0.01 &i [3H]PI, and 5 pg PI. The specific activities of the 100% control for membrane-bound and cytosolilc enzymes were 0.71 and 2.2 nmol min-’ mg protein-‘, respectively.
MURINE
LYMPHOCYTE
different effects on these two enzymes. The membrane-bound phospholipase C activity was enhanced, while the cytosolic enzyme was inhibited. The activity of the membrane-bound enzyme increased gradually with increasing calcium concentration (up to IO-’ M). On the other hand, the activity of the cytosolic phospholipase C was maximum at 100 yM Ca2+ (Fig. 3). Both enzymes showed similar heat stability. The activities of membrane-bound and cytosolic phospholipases C were completely abolished on heating at 50°C for 10 min. Column chromatography of the membrane-bound and cytosolic phospholipases C on Sephadex G-100 and concanavalin A-Sepharose. Before column chromatog-
raphy, the solubilization conditions for membrane-bound phospholipase C were examined. As shown in Table VI, 1% deoxycholate was most effective for solubilizing PI-phospholipase C from lymphocyte membranes. A lower concentration of cholate (0.4%) was also effective, solubilizing about 70% of the membrane-bound phospholipase activity (data not shown). On the other hand, a significant amount of PI-
FIG. 3. Effect of Ca2+ on the cytosolic (A) and membrane (B) phospholipase C activities. The free calcium concentration in the assay buffer was adjusted by adding Caa+-EGTA buffer (24).
PHOSPHOLIPASE
575
C TABLE
VI
SOLUBILIZATIONOFMEMBRANEPHOSPHOLIPASEC BYVARIOUSTREATMENTS % Solubilized
Treatment
Protein
1% Deoxycholate 1 M NaCl 1 mM EDTA 2 M KSCN
53 23 Trace 55
PI-phospholipase activity’
C
79 Trace 5 ND*
“The membrane fraction was treated with 1% deoxycholate, 1 M NaCl, 1 mM EDTA, or 2 M KSCN at 4°C for 2 h, and then centrifuged at lOO,OOO(l for 1 h. The supernatant was assayed for PI-phospholipase C activity. *Not detected.
phospholipase C activity was not solubilized with 1 M NaCl or 1 mM EDTA. More than 50% of the membrane proteins were solubilized with 2 M KSCN, but the enzyme activity was not detected in the resultant solubilized solution. However, since its addition completely abolished the phospholipase C activity in a cholate extract, KSCN appears to be unsuitable for solubilization of the enzyme. The cholate extract of lymphocyte membranes was applied to a column of Sephadex G-100. The elution profile is shown in Fig. 4B. Phospholipase C activity was eluted as a single peak with a shoulder. An approximate molecular weight of 70,000 was calculated for the membrane phospholipase C from a curve of elution volumes against logarithms of molecular weights of various standard proteins. However, gel filtration analysis in the presence of detergent was not suitable for the estimation of molecular weight. Lymphocyte cytosol was also applied to a column of Sephadex G-100. The elution profile of the cytosolic phospholipase C was similar to that of the membrane-bound phospholipase C, and the molecular weight of the cytosolic enzyme was also found to be about 70,000 (Fig. 4A). For further comparison of the membrane-bound and cytosolic phospholipases C, the pooled peak fractions of phospholi-
576
KAMISAKA,
Fraction
number
TOYOSHIMA,
(3mlltube)
FIG. 4. Gel filtration of cytosolic (A) and membranebound (B) phospholipases C on Sephadex G-100. The experimental details are given in the text.
pase C activity from a Sephadex G-100 column were applied to a column of Con ASepharose. As shown in Fig. 5, a portion of the membrane-bound phospholipase C activity bound to Con A-Sepharose and was eluted with the elution buffer containing (w-MM, whereas none of the cytosolic phospholipase C activity bound to Con A-Sepharose. The recovery of the membranebound enzyme activity in the specifically eluted fraction was approximately 20 to 30%. These results suggest that a part of the membrane-bound phospholipase C is a Con A-binding glycoprotein or associates with Con A-binding sites on the cell surface.
AND
OSAWA
pholipase C was also reported in the brain (6). However, it has recently been claimed that the phospholipase C activity detected in the membrane fraction can be adequately accounted for by contamination by a cytosolic enzyme (26,27). In the present study, we were able to detect the presence of a membrane-bound phospholipase C (Fig. 1, Table I). PI-Phospholipase C activity in the membrane fraction could be extracted only with cholate (Table VI). Trypsin treatment of intact lymphocytes caused an about 50% decrease in phospholipase C activity (Table II). Furthermore, a portion of the phospholipase C activity extracted from the membrane fraction bound to Con A-Sepharose (Fig. 5). These results suggest that a portion of the membrane-bound phospholipase C is expressed on the cell surface and that it is a glycoprotein or a protein firmly associated with membrane glycoprotein. Some properties of the cytosolic and the membrane-bound phospholipases C were studied. They have the same substrate specificity for PI (Table IV) and require Ca2+ for their activity (Fig. 3). They differ in pH optima (Fig. 2), in the Ca2+ concentration required for their optimum activity (Fig. 3), and in the effect of PA on their enzymatic activity. However, changes in and PA concenpH, Ca2+ concentration, tration may alter the activity by influenc-
DISCUSSION Fraction
PI-phospholipase C, which catalyzes the initial step of PI turnover, has been found primarily in the cytosolic fraction of mammalian cells. A membrane-bound PI-phos-
number
(1 Smlltubd
FIG. 5. Affinity column chromatography of cytosolic (A) and membrane-bound (B) phospholipases C on Con A-Sepharose. The experimental details are given in the text.
MURINE
LYMPHOCYTE
ing the susceptibility of PI to the enzyme rather than by having direct effects on the enzymes per se because of the high sensitivity of the enzymes to changes in the physical nature of the substrate. Therefore, it is difficult to come to a definite conclusion as to the physiological significance of the differences observed between two enzymes. Recently, Dawson et al. (28) and other groups (29-31) suggested possible mechanisms for both the suppression and the activation of the soluble phospholipase C by various membrane lipids. Similar modification of PI-phospholipase C activity by membrane lipids was also noted in the present study. We found inhibition of both the membrane-bound and cytosolic phospholipases C by PC, similar to that reported by other investigators (28-31). The effects we observed with other lipids were different from the results of others. In lymphocytes, PE and PS were inhibitory for both the membrane-bound and cytosolic phospholipases C of lymphocytes (Table V), but they only had a moderate effect on the PI-phospholipase C activity of sheep seminal vesicular gland (30). Furthermore, the effects of PA on the membrane-bound and cytosolic enzymes were dichotomous (Table V). These differences might be partly due to changes in substrate concentration and Ca2+ concentration. However, in the case of membrane-bound phospholipase C, they are more difficult to explain because membrane preparations contain various kinds of lipids. Recently, it has become apparent that polyphosphoinositides undergo agonistinduced hydrolysis by PI-phospholipase C to yield diglyceride and inositol phosphates (2,3), and this hydrolysis appears to reflect a signal-generating system. In thymocytes, a mitogen also induces an increase in inositol phosphates within a minute of stimulation (32), which suggests the presence of polyphosphoinositide degradation enzymes. We found that a hydrolytic activity for polyphosphoinositides was present in both the membrane and cytosolic fractions from murine lymphocytes. Recent reports suggest that the mitogenie signals generated after mitogen-re-
PHOSPHOLIPASE
C
577
ceptor interaction may be transduced via hydrolysis of inositol phospholipids not only in T-cell activation but also in B-cell activation (32-35). Therefore, it seems reasonable that PI-phospholipase C activity was detected in both T-cell-enriched and B-cell-enriched fractions (Table III). REFERENCES 1. MICHELL, R. H. (1975) Biochim. Biophys. Acta 415, 81-147. 2. BERRIDGE, M. J. (1984) &~&em. J. 220,345-360. 3. BERRIDGE, M. J., AND IRVINE, R. F. (1984) Nature (Lmdon) 312, 315-321. 4. NISHIZUKA, Y. (1984) Science 225,1365-1370. 5. HAMBERG, M., SVENSSON, J., AND SAMUELSSON, B. (197.5) Proc. Natl. Acad. Sci. USA 72,2994-2998. 6. LAPETINA, E. G., AND MICHELL, R. H. (1973) Biochem. J. 131,433-442. 7. FRIEDEL, R. O., BROWN, J. D., AND DURELL, J. (1969) J. Neurochem. 16,371-3’78. 8. ROSENBERG, R. J., MASKOWITZ, R. W., AND MALEMUD, C. J. (1983) Biochem. Biophys. Res. Comrnul?, 115,331-338. 9. SIESS, W., AND LAPETINA, E. G. (1983) Biochim. Biophys. Acta 752,329-338. 10. ALLAN, D., AND MICHELL, R. H. (1974) B&hem. J. 142,591-597. 11. KAWAGUCHI, T., MATSUMOTO, I., AND OSAWA, T. (1974) J. Biol. Chem. 249,2786-2792. 12. JULIUS, M. H., SINPSON, E., AND HERZENBERG, L. A. (1973) Eur. J. Immunol. 3,645-649. 13. LY, I. A., AND MICHELL, R. H. (1974) .I Immunol. Methods 5,239-247. 14. GOLUB, E. A. (1972) J. Immunol. 109,168-170. 15. MAKISHIMA, F., TOYOSHIMA, S., AND OSAWA, T. (1985) Arch. Biochem. Biophys. 238, 315-324. 16. MAEDA, T., BALAKRISHNAN, K., AND MEHDI, S. 0. (1983) B&him. Biophys. Acta 731, 115-120. 17 _.. DAWSON, R. M. C., AND CLARKE, N. (1972) Biodem. J. 127,113-118. 18. DOWNES, C. P., AND MICHELL, R. H. (1981) B&hem. J 198,133-140. 19. SONG, C. S., AND BODANSKY, 0. (1967) J. Biol Chem. 242,694-699. 20. KORNBERG, A. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 441-443, Academic Press, New York. 21. FERBER, E., RESCH, K., WALLACH, D. F. H., AND IMM, W. (1972) Biochim. Biophys. Acta 266,494504. 22. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1961) J. Biol. Chem. 193, 265-275.
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