- Email: [email protected]
[50] Phosphatidylinositol-specific phospholipase C from human platelets
518
PHOSPHOLIPASE C
[50]
At pH 7.0 and 100/zM C a 2+ , apparent K m values for PI and PIP 2 were 110 and 160 /xM, respectively. Since the tissue distribution of PLC-yj and PLC-y2 is different, PLC-y2 may play roles similar to those of PLC-y~ in different cells.
[50] Phosphatidylinositol-Specific Phospholipase C f r o m H u m a n Platelets
By YOSHINORI NOZAWAand YOSHIKO BANNO Introduction Phosphoinositide (PI)-specific phospholipase C (PLC) plays a crucial role in transmembrane signaling in cells exposed to various extracellular agonists, such as hormones, neurotransmitters, autacoids, and growth factors. The activated PI-PLC generates the second messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which mobilize calcium ions from intracellular stores and activates protein kinase C, respectively. These initial biochemical events eventually lead to metabolism, secretion, nerve excitation, muscle contraction, and cell proliferation. Despite numerous data supporting the stimulus-response coupling theory, the precise mechanism at the molecular level for induction of cell functions by occupancy of calcium-mobilizing receptors involving PI-PLC is poorly understood. Recent substantial evidence indicates existence of several types of PIPLC isozymes in mammalian cells. 1The physiological significance of such a multiplicity of PI-PLC remains to be explored. Platelets also contain multiple forms of PI-PLC in both membrane and cytosol fractions. In this chapter, we describe the purification and partial characterization of PIPLCs of human platelets. Preparation of Membrane and Cytosol Fractions Human platelets are isolated from outdated blood.2 Whole human blood in plastic bags are centrifuged at 1400 g for 5 min to remove erythrocytes and leukocytes. The supernatant (platelet-rich plasma) is then centrifuged 1 S. G. Rhee, P.-G. Suh, S.-H. Ryu, and S. Y. Lee, Science 244, 546 (1989). 2 N. L. Baenziger and P. W. Majerus, this series, Vol. 31, p. 149.
METHODS IN ENZYMOLOGY,VOL. 197
Copyright © 1991by Academic Press, Inc. All rights of reproductionin any form reserved.
[50]
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS
519
at 2500 g for 5 min at 4°, and the resulting platelet pellet is suspended in plasma. The platelets thus obtained are washed twice with Tris-citrate-bicarbonate buffer, 3 pH 7.0, containing 5 mM EGTA by centrifugation for 15 min at 2000 g. Washed platelets are resuspended to a final concentration of 5 × 109 cells/ml in lysis buffer [20 mM Tris-HC1 buffer, pH 7.4, 20 mM EGTA, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] and allowed to stand on ice for 1 hr. The platelet suspensions are disrupted by sonication on ice for a total of 5 min with 15-sec bursts of a probe-type sonicator (Branson sonifier, B-12). After removing unbroken platelets by centrifugation at 2400 g for 10 min, the supernatant is subjected to centrifugation at 105,000 g for 60 min. The supernatant (cytosol fraction) is withdrawn, and the pellet (membrane fraction) is resuspended in a small volume of buffer A [20 mM Tris-HCl, pH 7.4, 20 mM EGTA, 1 mMEDTA, 1 mM dithiothreitol (DTT), and 0.5 mM PMSF] and stored at - 8 0 °.
Assay for Phosphoinositide-Specific Phospholipase C Activity PI-PLC activity is assayed by measuring the formation of radioactive inositol phosphates from 250/zM [3H]PI [15,000 disintegrations per minute (dpm)] or 200/zM [3H]PIP2 (20,000 dpm) prepared from [inositol-2-3H(N)] phosphatidylinositol (16.6 Ci/mmol) or [ i n o s i t o l - 2 - 3 H ( N ) ] p h o s p h a t i d y l inositol 4,5-bisphosphate (6.7 Ci/mmol) (New England Nuclear, Boston, MA), as described previously.4 A s s a y M i x t u r e . The final concentrations of components of the mixture (total volume 50/zl) are as follows: 1. 250/~M PI containing [3H]PI (15,000 dpm) CaC1z, 2 mM Tris-maleate buffer, 25 mM, pH 5.5 KCI, 80 mM 2. 250 brM PI containing [3H]PI (15,000 dpm) CaC12, 2 mM Tris-maleate buffer, 25 mM, pH 7.0 Sodium deoxycholate, 1 mg/ml KCI, 80 mM 3. 200/zM PIPz containing [3H]PIP2 (20,000 dpm) or PIPJphosphatidylethanolamine (PE), 40/200 /zM containing [3H]PIP2 (15,000 dpm) CaCI2/EGTA, 10/xM (free Ca 2÷ concentration) Tris-maleate buffer, 25 mM, pH 6.5 3 S. E. Rittenhouse-Simmons, J. Clin. Invest. 8, 580 (1979). 4 y . Banno and Y. Nozawa, Biochem. J. 248, 95 (1987).
520
PHOSPHOLIPASE C
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Sodium deoxycholate, 1 mg/ml KCI, 80 mM Procedure. Free Ca 2+ concentrations are adjusted to the desired levels using Ca 2÷/EGTA buffers containing 2 mM EGTA final concentration and the appropriate amount of CaCI 2.5 To prepare substrates for kinetics study, [3H]PI or [3H]PIP2 dissolved in chloroform, enough for several assays, is kept under a stream of nitrogen, and the residues are dissolved in distilled water in a sonicator bath. Over the course of purification steps on various columns, PLC activity is assayed using [3H]PI as substrate at pH 5.5 (assay mixture 1) or at pH 7.0 (assay mixture 2) and with [3H]PIP2/PE (1:5 molar ratio) as substrate (assay mixture 3). Small unilamellar vesicles of [3H]PIP2/PE are prepared as follows: the lipids are dispersed into the assay buffer by vigorous vortexing and then sonicated for 2 min to yield 2 nmol of [3H]PIP2 (15,000 dpm) with 10 nmol of PE/assay. The assay is initiated by the addition of enzyme solution. After incubation for 10 min at 37°, the reaction is terminated by addition of 0.25 ml of chloroform/methanol/concentrated HC1 (100:100:0.6, by volume) and 0. I ml of a 5 mM EGTA, 1 N HCI solution. The mixtures are vortex-mixed and then centrifuged for 10 min at 2000 g at 25°. A 0.2-ml portion of the upper aqueous phase is carefully transferred to a vial, mixed with 6 ml of scintillation fluid, and the radioactivity determined in a liquid scintillation counter (Beckman LS-9000). The water-soluble reaction products from incubation of the purified PLCs with [3H]PI and [3H]PIP2 are analyzed by chromatography on a column of Dowex AG1-X8 according to the method of Downes and Michell. 6More than 90% of the product derived from [3H]PI and [3H]PIP 2hydrolysis corresponds to the carrier inositol monophosphate and inositol 1,4,5-trisphosphate, respectively.
Purification of Membrane-Bound Phospholipase C The study of distribution of PIP2-hydrolyzing activity in human platelets has shown that 20% of the total activity of the homogenate is associated with the particulate fraction. 4 The frozen platelet membrane fractions are thawed, resuspended in buffer A, and spun down at 105,000 g for 60 min. The washed pellet is resuspended in 2 M KC1 in buffer A and stirred for 2 hr at 4 °. Buffer A is then added to adjust the KC1 concentration to 1 M, and the suspension is centrifuged at 105,000 g for 60 min. The residual pellet is suspended in buffer A and extracted with an equal volume of 5 j. Raaflaub, Methods Biochem. Anal. 3, 301 0950). 6 C. P. Downes and R. H. Michell, Biochem. J. 198, 133 (1981).
[50]
521
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS Ht~m~a~le~
t Membrmae
~sol
I I
2M KC1 extract
Fast Q - ~ p h a r o s e
I PeakI
I
Heperin-Sepharose
L
Ultrogel AoA 44
I Peak II
'
Hepsxin-Selxharose I ~ IIb Peak lla I 1 U l t r o g e l AcA 44
~o0~
l
l
l
1st Mono Q HPLC
Heparin-agarose HPI~
[
Mono S ItPI/3
I
e.l~..~I
L
I Peak I I c
L
L
Phenyl -Settmrose HPLC Hono Q
}[ydroxTKnn t i t e
r
e.l~III
N
I
Peak II
~Ultrogel AcA 44 L ~ Mono Q [tPLC L
L
L
~
L
mIMi:-I
mPIZ~II
Superose 12 HPhC
2rid Mono Q HPI/3
ctt~II
HeIwirin?ephB/~se
Superose 6/12 HPLC
Hydroxyapati t e HPIJ3
L
extract
Fast Q-Sepharose
t
I Pher~l-Sepharose HPI/3 Hydroxzal~tite HPLC I I L
C~olate
oPI~-IV
FIG. 1. Purification of phosphatidylinositol-specific phospholipases C from human platelets.
buffer A containing 2% sodium cholate and 200 mM NaC1, pH 7.4. After stirring for 2 hr at 4 °, the suspension is sedimented by centrifugation at 105,000 g for 60 min to obtain the cholate extract for the subsequent purification process (Fig. I). About one-fourth of the particulate-associated [3H]PIP2-hydrolyzing activity is released by extraction with 2 M KC1. When the residual pellet obtained after the repeated KC1 extraction is treated with 1% sodium cholate and 0.1 M NaC1, nearly 80% of the [3H]PIP2-hydrolyzing activity and about 40% of the total membrane protein are released. Fast Q-Sepharose Column Chromatography. The cholate extract is dialyzed overnight against buffer B containing 20 mM Tris-HC1, pH 7.4, 1 mM DTT, I mM EGTA, 0.5 mM PMSF, 0.1 M NaCI, and 0.5% sodium cholate. The dialyzed solution is applied onto a Fast Q-Sepharose column
522
PHOSPHOLIPASEC
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(6.0 × 25 cm) equilibrated with buffer B. After washing the column with 500 ml of the same buffer to remove unbound protein, elution is performed with a linear concentration gradient of NaCI from 0.1 to 0.5 M in 2000 ml of buffer B. The majority of PLC activity for [3H]PI and [3H]PIP2 is eluted between 0.2 and 0.3 M NaC1. The active fractions are pooled and dialyzed overnight against buffer C (20 mM Tris-HC1, pH 7.4, 1 mM EDTA, 0.1 M NaCI, 0.5 M PMSF, 1 mM DTT, and 0.5% sodium cholate). Heparin-Sepharose Column Chromatography. The dialyzate from the Fast Q-Sepharose step is applied to a heparin-Sepharose column (3 × 10 cm) equilibrated with buffer C, and the column is first washed with the same buffer and then eluted with a 1000-ml NaC1 linear gradient (0.1-0.7 M) in buffer C. The two activity peaks, mPLC-I and mPLC-II, are eluted at 0.35-0.45 and 0.5-0.6 M NaC1, respectively. The fractions containing mPLC-! and mPLC-II are pooled separately and concentrated in an AmP con (Danvers, MA) filtration apparatus (YM10 membrane). Ultrogel AcA 44 Column Chromatography. The concentrated samples (mPLC-I and mPLC-II) obtained as above are separately applied to Ultrogel AcA 44 columns (2.5 × 90 cm) equilibrated with buffer C containing 0.3 M NaC1. The active fractions are pooled and concentrated as above. The concentrated mPLC-I and mPLC-II solutions are diluted one-tenth with salt-free buffer D (20 mMTris-HCl, pH 7.4, 1 mMDTT, 1 mMEGTA, and 10% glycerol). Mono Q HPLC. Aliquots of mPLC-I and mPLC-II obtained from the previous step are applied separately to Mono Q columns (HR 10/10) equilibrated with buffer D. The proteins are eluted at a flow rate of 2 ml/ min by successive application of increasing NaCI gradients from 0 to 0.3 M for 5 min, from 0.3 to 0.5 M for 30 min, and from 0.5 to 0.6 M for 5 min using a fast liquid chromatography. The fractions exhibiting PLC activity are pooled and concentrated in a Centricon microconcentrator (Amicon). Superose 6-12 Combination HPLC. The mPLC-I and mPLC-II (0.1 ml) solutions pooled from the Mono Q column step are separately applied to Superose 6-12 combination columns (HR 10/30) connected in series and equilibrated with buffer C containing 0.3 M NaC1. Elution is carried out with the same buffer at a flow rate of 0.4 ml/min, and 0.2-ml fractions are collected for assay of activity. Superose 12 HPLC. The mPLC-II solution obtained from the Superose 6-12 combination column is applied to a Superose 12 column (HR 10/30) equilibrated with buffer C containing 0.15 M NaC1. The column is eluted with the same buffer at a flow rate of 0.4 ml/min, and 0.1-ml fractions are collected. The active fractions are pooled and concentrated for rechromatography on the same column, producing a nearly symmetric activity peak.
[50]
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS
523
Purification of Cytosolic Phospholipase C PI-hydrolyzing activity (98% at pH 5.5 and 90% at pH 7.0) and PIP 2hydrolyzing activity (80%) are found to locate in the cytosolic fraction of human platelets. The cytosolic fraction (1500 ml) is dialyzed against buffer A (20 mM Tris-HCl, pH 7.4, 5 mM EGTA, I mM EDTA, 0.5 mM PMSF, 1 mM DTT, and 10% glycerol) for subsequent purification (Fig. 1). Fast Q-Sepharose. The supernatant obtained after centrifugation of the dialyzate is loaded onto a Fast Q-Sepharose column (6.0 × 20 cm) equilibrated with buffer A. After washing with the same buffer, elution is performed with a linear concentration gradient from 0.1 to 0.4 M NaC1 in buffer A. When assayed for [3H]PI hydrolysis at pH 5.5 and [3H]PIP2 hydrolysis at pH 6.5, the two activity peaks are eluted at between 0.15 and 0.28 and between 0.3 and 0.4 M NaCI. The first peak fraction (peak I) exhibits major [3H]PI-hydrolyzing activity at pH 5.5, and the second activity peak (peak II) shows preferential hydrolysis for [3H]PIP2. The activity for [3H]PI hydrolysis at pH 7.0 coincides with the peak II fraction. The fractions containing peak I and peak II are pooled separately and concentrated in an Amicon concentrator (YM 10 membrane). The concentrated enzyme solutions are dialyzed against buffer B (20 mM Tris-HC1, pH 7.4, 1 mM EDTA, 0.1 mM PMSF, 1 mM DTT, and 10% glycerol). Heparin-Sepharose Column Chromatography. The dialyzates of peak I and peak II from the Fast Q-Sepharose column steps are separately applied to heparin-Sepharose columns (3 x 15 cm) equilibrated with buffer B. The column is first washed with the same buffer, and then elution is performed with a linear gradient ranging from 0.1 to 0.7 M NaCI in buffer B. The [3H]PI-hydrolyzing activity of peak I is eluted in a single peak between 0.2 and 0.3 M NaC1. The [3H]PIP2-hydrolyzing activity of peak II is resolved into three activity peaks (IIa, IIb, IIc) with linear gradient of NaCI (0.1-0.7 M). The first activity peak (IIa) elutes at between 0.2 and 0.35 M, the second (IIb) between 0.35 and 0.55 M, and the third (IIc) between 0.55 and 0.65 M are pooled separately. The ratios of total [3H]PIP2-hydrolyzing activity of peaks IIa, IIb, and IIc are 24.8, 49.6, and 25.6%, respectively. When the PLC activity of the three peaks is measured using [3H]PI as substrate at pH 7.0, activity is detected in both peak IIa and peak IIb, whereas [3H]PI-hydrolyzing activity is hardly detected in peak IIc. Peak I, designated cPLC-I, with high [3H]PI-hydrolyzing activity at 5.5 from the heparin-Sepharose column chromatography step is further purified by Mono Q HPLC, heparin-agarose HPLC, hydroxyapatite HPLC, and Mono S HPLC. The cPLC-I is eluted with 0.15-0.18 M NaC1 from Mono Q, with 0.3 M NaC1 frorn heparin-agarose, with 0.2 M NaC1
524
PHOSPHOLIPASEC
[50]
from Mono S, and with 0.23 M potassium phosphate buffer from the hydroxyapatite column. Peak IIc obtained by heparin-Sepharose column chromatography, which exhibits a preferential specificity for [3H]PIP2 hydrolysis, is further purified as follows. The activity is measured using [3H]PIP2/PE as substrate. Ultrogel AcA 44 Column Chromatography. The concentrated peak IIc solution is applied to an Ultrogel AcA 44 column (4.0 × 90 cm) equilibrated with buffer B containing 0.3 M NaC1. Active fractions are pooled. Phenyl-Sepharose HPLC. Solid KCI is added to the peak IIc solution from the Ultrogel AcA 44 column to give a concentration of 3 M. The enzyme solution is applied to an HPLC Phenyl-Sepharose column (HR 5/5) equilibrated with buffer B containing 3 M KC1. The proteins are eluted at a flow rate of 0.5 ml/min for 5 min, with a decreasing KC1 gradient from 3.0 to 1.8 M for 5 min, and with decreasing KC1 followed by a linear cholate gradient of 0-0.4% in buffer B for 30 min. The active fractions eluted between 0.15 and 0.25% cholate are pooled and diluted to one-fifth with buffer B. Mono Q HPLC. The enzyme solution from the Phenyl-Sepharose column step is applied to a Mono Q column equilibrated with buffer B. The proteins are eluted at a flow rate of 1 ml/min with increasing NaCI from 0 to 0.2 M for 5 min, from 0.2 to 0.35 M for 30 rain, and from 0.35 to 0.7 M for 5 min. The fractions coinciding with PLC activity elute at about 0.3 M NaCI and are pooled and diluted to one-tenth with buffer C (20 mM Tris-HCl, pH 7.4, 1 mM DTT, 0.1 mM EDTA and 10% glycerol). Hydroxyapatite HCA-IO0 HPLC. An aliquot of peak IIc after the Mono Q column step is applied to a hydroxyapatite HCA-100 column (5 × 10 cm) equilibrated with buffer C. The proteins are eluted with a 30-ml linear gradient ranging from 50 to 400 mM potassium phosphate buffer, pH 7.4, at a flow rate of 0.5 ml/min. The activity fractions are pooled and concentrated in a Centricon microconcentrator (cPLC-IV). Peak IIa and peak IIb obtained from the heparin-Sepharose column chromatography step are further purified on Ultrogel AcA 44, the first Mono Q column, phenyl-Sepharose, hydroxyapatite, and the second Mono Q column. By the first mono Q HPLC, IIa is eluted with 0.2-0.28 M NaCI, and IIb is eluted with 0.3-0.4 M NaCI. Peak IIa is eluted with 0.3-0.37% cholate on Phenyl-Sepharose and with 0.25 M potassium phosphate buffer on hydroxyapatite. The elution profiles of peak IIb on these two columns are similar to those of peak IIa. By the second Mono Q column chromatography, IIa (designated as cPLC-II) and IIb (designated as cPLC-III) are eluted with 0.25 and 0.32 M NaCI, respectively.
[50]
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS
525
Properties There are more than four different forms of PLC in the cytosol 7-9 and two forms in the membrane fraction ~°,H of human platelets. The results of identification of human platelet PLC isoforms by antibodies against various types of soluble PLCs (brain PLC-fl, -T1, -8,12'13 uterus PLC-ct, 14 and PLC-y215'~6) indicate that human platelet cPLC-I is recognized by the antibody against PLC-Tz. On the other hand, platelet cPLC-II, -III, and mPLC-II preparations do not react with any other PLC antibodies. The catalytic properties of the platelet PLC isoforms are studied by using either [3H]PI or [3H]PIP2 as substrates. Hydrolysis of both PI and PIP 2 by platelet PLC isoforms is dependent on Ca 2+. However, at low Ca z+ concentrations (10/xM) [3H]PIP2 is the preferred substrate for platelet PLC isoforms. When assayed with [3H]PI as substrate at high calcium concentration (2 mM), cPLC-I activity is most active at pH 5.5; however, at neutral pH the order of specific activity is cPLC-II > cPLC-I > cPLCIII. On the other hand, cPLC-IV has very low activity for PI hydrolysis at both pH values. The order of specific activity is cPLC-IV > cPLC-III > cPLC-II > cPLC-I for [3H]PIP2 hydrolysis, indicating that cPLC-IV is the most specific for [3H]PIP2. The preference for [3H]PIP2 hydrolysis is also observed for the mPLC-I and mPLC-II preparations. The Km values for [3H]PI hydrolysis of cPLC-IV or mPLC-II are higher than those of cPLC-I or cPLC-II, namely, 0.7 and 0.5 mM for cPLC-IV and mPLC-II, and 0.1 and 0.08 mM for cPLC-I and cPLC-II, respectively. The effects of various metal ions on the PIP2-hydrolyzing activities of platelet PLC isoforms have been examined. Platelet cytosolic and membrane PLCs are inhibited by addition of 2 mM EGTA or 2 mM EDTA. The addition of Ca 2÷ (10/xM free) causes activation whereas Mg2÷ does 7 y. Banno, S. Nakashima, and Y. Nozawa, Biochem. Biophys. Res. Commun. 136, 713 (1986). s M. G. Low, R. C. Carroll, and A. C. Cox, Biochem. J. 243, 763 (1987). 9 V. Manne and H. F. Kung, Biochem. J. 243, 763 (1987). 10y . Banno, Y. Yada, and Y. Nozawa, J. Biol. Chem. 263, 11459 (1988). 11 j. j. Baldassare, P. A. Henderson, and G. J. Fisher, Biochemistry 28, 6010 (1989). 12 S. n . Ryu, P.-G. Suh, K. S. Cho, K. Y. Lee, and S. G. Rhee, J. Biol. Chem. 262, 12511 (1987). 13 S. H. Ryu, P.-G. Suh, K. S. Cho, K. Y. Lee, and S. G. Rhee, Proc. Natl. Acad. Sci. U.S.A. 84, 6649 (1987). 14 C. F. Bennett and S. T. Crooke, J. Biol. Chem. 262, 13789 (1987). 15 S. Ohta, A. Matsui, Y. Nozawa, and Y. Kagawa, FEBS Lett. 242, 31 (1988). 16y. Homma, T. Takenawa, Y. Emori, H. Sorimachi, and K. Suzuki, Biochem. Biophys. Res. Commun. 164, 406 (1989).
526
PHOSPHOLIPASEC
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not. At low C a 2+ concentrations (10 -7 M) corresponding to the level in resting platelets, cPLC-IV and mPLC-II act to hydrolyze PIP 2 at 20-26% of maximum hydrolysis, but cPLC-I does not. Other metal ions (50/zM) such as Mn 2÷ , Fe 2÷ , and Cu 2÷ show some inhibition (15-20%). The inhibitory potency of Hg 2÷ differs among PLC isoforms; cPLC-I and mPLC-II are extremely sensitive to Hg 2÷ (•o.5 <1 /zM), whereas cPLC-IV is less sensitive to the ion (10.5 >5/zM).
[51] P u r i f i c a t i o n o f G u i n e a P i g U t e r u s P h o s p h o i n o s i t i d e Specific P h o s p h o l i p a s e C
By C. FRANK BENNETT, MICHAEL P. ANGIOLI, and STANLEY T. CROOKE
Introduction Hydrolysis of membrane phospholipids following occupancy of cell surface receptors is a common signal transduction pathway for a variety of agonists. Hydrolysis of phosphoinositides by phospholipase C (PLC) is well documented to be a major transduction mechanism for calciummobilizing agonists. Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generates two second messenger molecules, inositol 1,4,5-trisphosphate and diacylglycerol. 1,2Purification of the enzymes which hydrolyze the phosphodiester bond of phosphatidylinositol 4,5-bisphosphate (PI-PLC) from a variety of tissues has recently been accomplished. These studies have demonstrated that multiple forms of PIPLC exist within the same tissue or cell type. 3'4 Isolation of the cDNA clones for four PI-PLC isoenzymes demonstrated that the different forms of PI-PLC are distinct gene products with similar enzymatic activity (reviewed in Refs. 3 and 4). In a study of phospholipases in tissues which respond to peptidoleukotrienes, as part of our long-term studies on these agents, we determined that guinea pig uterus was a relatively rich source of phosphoinositidespecific phospholipase C and exhibited moderate levels of a phosphatidylethanolamine-preferring phospholipase A2 activity and low levels of phosphatidylcholine-specific phospholipase C activity. We subsequently puriI M. J. Berridge, Annu. Rev. Biochem. 56, 159 (1987). 2 U. K i k k a w a and Y. Nishizuka, Annu. Rev. Cell Biol. 2, 149 (1986). 3 S. G. Rhee, P.-G. Suh, S.-H. Ryu, and S. Y. Lee, Science 244, 546 (1989). 4 S. T. Crooke a n d C. F. Bennett, Cell Calcium 10, 309 (1989).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
PHOSPHOLIPASE C
[50]
At pH 7.0 and 100/zM C a 2+ , apparent K m values for PI and PIP 2 were 110 and 160 /xM, respectively. Since the tissue distribution of PLC-yj and PLC-y2 is different, PLC-y2 may play roles similar to those of PLC-y~ in different cells.
[50] Phosphatidylinositol-Specific Phospholipase C f r o m H u m a n Platelets
By YOSHINORI NOZAWAand YOSHIKO BANNO Introduction Phosphoinositide (PI)-specific phospholipase C (PLC) plays a crucial role in transmembrane signaling in cells exposed to various extracellular agonists, such as hormones, neurotransmitters, autacoids, and growth factors. The activated PI-PLC generates the second messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which mobilize calcium ions from intracellular stores and activates protein kinase C, respectively. These initial biochemical events eventually lead to metabolism, secretion, nerve excitation, muscle contraction, and cell proliferation. Despite numerous data supporting the stimulus-response coupling theory, the precise mechanism at the molecular level for induction of cell functions by occupancy of calcium-mobilizing receptors involving PI-PLC is poorly understood. Recent substantial evidence indicates existence of several types of PIPLC isozymes in mammalian cells. 1The physiological significance of such a multiplicity of PI-PLC remains to be explored. Platelets also contain multiple forms of PI-PLC in both membrane and cytosol fractions. In this chapter, we describe the purification and partial characterization of PIPLCs of human platelets. Preparation of Membrane and Cytosol Fractions Human platelets are isolated from outdated blood.2 Whole human blood in plastic bags are centrifuged at 1400 g for 5 min to remove erythrocytes and leukocytes. The supernatant (platelet-rich plasma) is then centrifuged 1 S. G. Rhee, P.-G. Suh, S.-H. Ryu, and S. Y. Lee, Science 244, 546 (1989). 2 N. L. Baenziger and P. W. Majerus, this series, Vol. 31, p. 149.
METHODS IN ENZYMOLOGY,VOL. 197
Copyright © 1991by Academic Press, Inc. All rights of reproductionin any form reserved.
[50]
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS
519
at 2500 g for 5 min at 4°, and the resulting platelet pellet is suspended in plasma. The platelets thus obtained are washed twice with Tris-citrate-bicarbonate buffer, 3 pH 7.0, containing 5 mM EGTA by centrifugation for 15 min at 2000 g. Washed platelets are resuspended to a final concentration of 5 × 109 cells/ml in lysis buffer [20 mM Tris-HC1 buffer, pH 7.4, 20 mM EGTA, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] and allowed to stand on ice for 1 hr. The platelet suspensions are disrupted by sonication on ice for a total of 5 min with 15-sec bursts of a probe-type sonicator (Branson sonifier, B-12). After removing unbroken platelets by centrifugation at 2400 g for 10 min, the supernatant is subjected to centrifugation at 105,000 g for 60 min. The supernatant (cytosol fraction) is withdrawn, and the pellet (membrane fraction) is resuspended in a small volume of buffer A [20 mM Tris-HCl, pH 7.4, 20 mM EGTA, 1 mMEDTA, 1 mM dithiothreitol (DTT), and 0.5 mM PMSF] and stored at - 8 0 °.
Assay for Phosphoinositide-Specific Phospholipase C Activity PI-PLC activity is assayed by measuring the formation of radioactive inositol phosphates from 250/zM [3H]PI [15,000 disintegrations per minute (dpm)] or 200/zM [3H]PIP2 (20,000 dpm) prepared from [inositol-2-3H(N)] phosphatidylinositol (16.6 Ci/mmol) or [ i n o s i t o l - 2 - 3 H ( N ) ] p h o s p h a t i d y l inositol 4,5-bisphosphate (6.7 Ci/mmol) (New England Nuclear, Boston, MA), as described previously.4 A s s a y M i x t u r e . The final concentrations of components of the mixture (total volume 50/zl) are as follows: 1. 250/~M PI containing [3H]PI (15,000 dpm) CaC1z, 2 mM Tris-maleate buffer, 25 mM, pH 5.5 KCI, 80 mM 2. 250 brM PI containing [3H]PI (15,000 dpm) CaC12, 2 mM Tris-maleate buffer, 25 mM, pH 7.0 Sodium deoxycholate, 1 mg/ml KCI, 80 mM 3. 200/zM PIPz containing [3H]PIP2 (20,000 dpm) or PIPJphosphatidylethanolamine (PE), 40/200 /zM containing [3H]PIP2 (15,000 dpm) CaCI2/EGTA, 10/xM (free Ca 2÷ concentration) Tris-maleate buffer, 25 mM, pH 6.5 3 S. E. Rittenhouse-Simmons, J. Clin. Invest. 8, 580 (1979). 4 y . Banno and Y. Nozawa, Biochem. J. 248, 95 (1987).
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PHOSPHOLIPASE C
[50]
Sodium deoxycholate, 1 mg/ml KCI, 80 mM Procedure. Free Ca 2+ concentrations are adjusted to the desired levels using Ca 2÷/EGTA buffers containing 2 mM EGTA final concentration and the appropriate amount of CaCI 2.5 To prepare substrates for kinetics study, [3H]PI or [3H]PIP2 dissolved in chloroform, enough for several assays, is kept under a stream of nitrogen, and the residues are dissolved in distilled water in a sonicator bath. Over the course of purification steps on various columns, PLC activity is assayed using [3H]PI as substrate at pH 5.5 (assay mixture 1) or at pH 7.0 (assay mixture 2) and with [3H]PIP2/PE (1:5 molar ratio) as substrate (assay mixture 3). Small unilamellar vesicles of [3H]PIP2/PE are prepared as follows: the lipids are dispersed into the assay buffer by vigorous vortexing and then sonicated for 2 min to yield 2 nmol of [3H]PIP2 (15,000 dpm) with 10 nmol of PE/assay. The assay is initiated by the addition of enzyme solution. After incubation for 10 min at 37°, the reaction is terminated by addition of 0.25 ml of chloroform/methanol/concentrated HC1 (100:100:0.6, by volume) and 0. I ml of a 5 mM EGTA, 1 N HCI solution. The mixtures are vortex-mixed and then centrifuged for 10 min at 2000 g at 25°. A 0.2-ml portion of the upper aqueous phase is carefully transferred to a vial, mixed with 6 ml of scintillation fluid, and the radioactivity determined in a liquid scintillation counter (Beckman LS-9000). The water-soluble reaction products from incubation of the purified PLCs with [3H]PI and [3H]PIP2 are analyzed by chromatography on a column of Dowex AG1-X8 according to the method of Downes and Michell. 6More than 90% of the product derived from [3H]PI and [3H]PIP 2hydrolysis corresponds to the carrier inositol monophosphate and inositol 1,4,5-trisphosphate, respectively.
Purification of Membrane-Bound Phospholipase C The study of distribution of PIP2-hydrolyzing activity in human platelets has shown that 20% of the total activity of the homogenate is associated with the particulate fraction. 4 The frozen platelet membrane fractions are thawed, resuspended in buffer A, and spun down at 105,000 g for 60 min. The washed pellet is resuspended in 2 M KC1 in buffer A and stirred for 2 hr at 4 °. Buffer A is then added to adjust the KC1 concentration to 1 M, and the suspension is centrifuged at 105,000 g for 60 min. The residual pellet is suspended in buffer A and extracted with an equal volume of 5 j. Raaflaub, Methods Biochem. Anal. 3, 301 0950). 6 C. P. Downes and R. H. Michell, Biochem. J. 198, 133 (1981).
[50]
521
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS Ht~m~a~le~
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l
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FIG. 1. Purification of phosphatidylinositol-specific phospholipases C from human platelets.
buffer A containing 2% sodium cholate and 200 mM NaC1, pH 7.4. After stirring for 2 hr at 4 °, the suspension is sedimented by centrifugation at 105,000 g for 60 min to obtain the cholate extract for the subsequent purification process (Fig. I). About one-fourth of the particulate-associated [3H]PIP2-hydrolyzing activity is released by extraction with 2 M KC1. When the residual pellet obtained after the repeated KC1 extraction is treated with 1% sodium cholate and 0.1 M NaC1, nearly 80% of the [3H]PIP2-hydrolyzing activity and about 40% of the total membrane protein are released. Fast Q-Sepharose Column Chromatography. The cholate extract is dialyzed overnight against buffer B containing 20 mM Tris-HC1, pH 7.4, 1 mM DTT, I mM EGTA, 0.5 mM PMSF, 0.1 M NaCI, and 0.5% sodium cholate. The dialyzed solution is applied onto a Fast Q-Sepharose column
522
PHOSPHOLIPASEC
[50]
(6.0 × 25 cm) equilibrated with buffer B. After washing the column with 500 ml of the same buffer to remove unbound protein, elution is performed with a linear concentration gradient of NaCI from 0.1 to 0.5 M in 2000 ml of buffer B. The majority of PLC activity for [3H]PI and [3H]PIP2 is eluted between 0.2 and 0.3 M NaC1. The active fractions are pooled and dialyzed overnight against buffer C (20 mM Tris-HC1, pH 7.4, 1 mM EDTA, 0.1 M NaCI, 0.5 M PMSF, 1 mM DTT, and 0.5% sodium cholate). Heparin-Sepharose Column Chromatography. The dialyzate from the Fast Q-Sepharose step is applied to a heparin-Sepharose column (3 × 10 cm) equilibrated with buffer C, and the column is first washed with the same buffer and then eluted with a 1000-ml NaC1 linear gradient (0.1-0.7 M) in buffer C. The two activity peaks, mPLC-I and mPLC-II, are eluted at 0.35-0.45 and 0.5-0.6 M NaC1, respectively. The fractions containing mPLC-! and mPLC-II are pooled separately and concentrated in an AmP con (Danvers, MA) filtration apparatus (YM10 membrane). Ultrogel AcA 44 Column Chromatography. The concentrated samples (mPLC-I and mPLC-II) obtained as above are separately applied to Ultrogel AcA 44 columns (2.5 × 90 cm) equilibrated with buffer C containing 0.3 M NaC1. The active fractions are pooled and concentrated as above. The concentrated mPLC-I and mPLC-II solutions are diluted one-tenth with salt-free buffer D (20 mMTris-HCl, pH 7.4, 1 mMDTT, 1 mMEGTA, and 10% glycerol). Mono Q HPLC. Aliquots of mPLC-I and mPLC-II obtained from the previous step are applied separately to Mono Q columns (HR 10/10) equilibrated with buffer D. The proteins are eluted at a flow rate of 2 ml/ min by successive application of increasing NaCI gradients from 0 to 0.3 M for 5 min, from 0.3 to 0.5 M for 30 min, and from 0.5 to 0.6 M for 5 min using a fast liquid chromatography. The fractions exhibiting PLC activity are pooled and concentrated in a Centricon microconcentrator (Amicon). Superose 6-12 Combination HPLC. The mPLC-I and mPLC-II (0.1 ml) solutions pooled from the Mono Q column step are separately applied to Superose 6-12 combination columns (HR 10/30) connected in series and equilibrated with buffer C containing 0.3 M NaC1. Elution is carried out with the same buffer at a flow rate of 0.4 ml/min, and 0.2-ml fractions are collected for assay of activity. Superose 12 HPLC. The mPLC-II solution obtained from the Superose 6-12 combination column is applied to a Superose 12 column (HR 10/30) equilibrated with buffer C containing 0.15 M NaC1. The column is eluted with the same buffer at a flow rate of 0.4 ml/min, and 0.1-ml fractions are collected. The active fractions are pooled and concentrated for rechromatography on the same column, producing a nearly symmetric activity peak.
[50]
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS
523
Purification of Cytosolic Phospholipase C PI-hydrolyzing activity (98% at pH 5.5 and 90% at pH 7.0) and PIP 2hydrolyzing activity (80%) are found to locate in the cytosolic fraction of human platelets. The cytosolic fraction (1500 ml) is dialyzed against buffer A (20 mM Tris-HCl, pH 7.4, 5 mM EGTA, I mM EDTA, 0.5 mM PMSF, 1 mM DTT, and 10% glycerol) for subsequent purification (Fig. 1). Fast Q-Sepharose. The supernatant obtained after centrifugation of the dialyzate is loaded onto a Fast Q-Sepharose column (6.0 × 20 cm) equilibrated with buffer A. After washing with the same buffer, elution is performed with a linear concentration gradient from 0.1 to 0.4 M NaC1 in buffer A. When assayed for [3H]PI hydrolysis at pH 5.5 and [3H]PIP2 hydrolysis at pH 6.5, the two activity peaks are eluted at between 0.15 and 0.28 and between 0.3 and 0.4 M NaCI. The first peak fraction (peak I) exhibits major [3H]PI-hydrolyzing activity at pH 5.5, and the second activity peak (peak II) shows preferential hydrolysis for [3H]PIP2. The activity for [3H]PI hydrolysis at pH 7.0 coincides with the peak II fraction. The fractions containing peak I and peak II are pooled separately and concentrated in an Amicon concentrator (YM 10 membrane). The concentrated enzyme solutions are dialyzed against buffer B (20 mM Tris-HC1, pH 7.4, 1 mM EDTA, 0.1 mM PMSF, 1 mM DTT, and 10% glycerol). Heparin-Sepharose Column Chromatography. The dialyzates of peak I and peak II from the Fast Q-Sepharose column steps are separately applied to heparin-Sepharose columns (3 x 15 cm) equilibrated with buffer B. The column is first washed with the same buffer, and then elution is performed with a linear gradient ranging from 0.1 to 0.7 M NaCI in buffer B. The [3H]PI-hydrolyzing activity of peak I is eluted in a single peak between 0.2 and 0.3 M NaC1. The [3H]PIP2-hydrolyzing activity of peak II is resolved into three activity peaks (IIa, IIb, IIc) with linear gradient of NaCI (0.1-0.7 M). The first activity peak (IIa) elutes at between 0.2 and 0.35 M, the second (IIb) between 0.35 and 0.55 M, and the third (IIc) between 0.55 and 0.65 M are pooled separately. The ratios of total [3H]PIP2-hydrolyzing activity of peaks IIa, IIb, and IIc are 24.8, 49.6, and 25.6%, respectively. When the PLC activity of the three peaks is measured using [3H]PI as substrate at pH 7.0, activity is detected in both peak IIa and peak IIb, whereas [3H]PI-hydrolyzing activity is hardly detected in peak IIc. Peak I, designated cPLC-I, with high [3H]PI-hydrolyzing activity at 5.5 from the heparin-Sepharose column chromatography step is further purified by Mono Q HPLC, heparin-agarose HPLC, hydroxyapatite HPLC, and Mono S HPLC. The cPLC-I is eluted with 0.15-0.18 M NaC1 from Mono Q, with 0.3 M NaC1 frorn heparin-agarose, with 0.2 M NaC1
524
PHOSPHOLIPASEC
[50]
from Mono S, and with 0.23 M potassium phosphate buffer from the hydroxyapatite column. Peak IIc obtained by heparin-Sepharose column chromatography, which exhibits a preferential specificity for [3H]PIP2 hydrolysis, is further purified as follows. The activity is measured using [3H]PIP2/PE as substrate. Ultrogel AcA 44 Column Chromatography. The concentrated peak IIc solution is applied to an Ultrogel AcA 44 column (4.0 × 90 cm) equilibrated with buffer B containing 0.3 M NaC1. Active fractions are pooled. Phenyl-Sepharose HPLC. Solid KCI is added to the peak IIc solution from the Ultrogel AcA 44 column to give a concentration of 3 M. The enzyme solution is applied to an HPLC Phenyl-Sepharose column (HR 5/5) equilibrated with buffer B containing 3 M KC1. The proteins are eluted at a flow rate of 0.5 ml/min for 5 min, with a decreasing KC1 gradient from 3.0 to 1.8 M for 5 min, and with decreasing KC1 followed by a linear cholate gradient of 0-0.4% in buffer B for 30 min. The active fractions eluted between 0.15 and 0.25% cholate are pooled and diluted to one-fifth with buffer B. Mono Q HPLC. The enzyme solution from the Phenyl-Sepharose column step is applied to a Mono Q column equilibrated with buffer B. The proteins are eluted at a flow rate of 1 ml/min with increasing NaCI from 0 to 0.2 M for 5 min, from 0.2 to 0.35 M for 30 rain, and from 0.35 to 0.7 M for 5 min. The fractions coinciding with PLC activity elute at about 0.3 M NaCI and are pooled and diluted to one-tenth with buffer C (20 mM Tris-HCl, pH 7.4, 1 mM DTT, 0.1 mM EDTA and 10% glycerol). Hydroxyapatite HCA-IO0 HPLC. An aliquot of peak IIc after the Mono Q column step is applied to a hydroxyapatite HCA-100 column (5 × 10 cm) equilibrated with buffer C. The proteins are eluted with a 30-ml linear gradient ranging from 50 to 400 mM potassium phosphate buffer, pH 7.4, at a flow rate of 0.5 ml/min. The activity fractions are pooled and concentrated in a Centricon microconcentrator (cPLC-IV). Peak IIa and peak IIb obtained from the heparin-Sepharose column chromatography step are further purified on Ultrogel AcA 44, the first Mono Q column, phenyl-Sepharose, hydroxyapatite, and the second Mono Q column. By the first mono Q HPLC, IIa is eluted with 0.2-0.28 M NaCI, and IIb is eluted with 0.3-0.4 M NaCI. Peak IIa is eluted with 0.3-0.37% cholate on Phenyl-Sepharose and with 0.25 M potassium phosphate buffer on hydroxyapatite. The elution profiles of peak IIb on these two columns are similar to those of peak IIa. By the second Mono Q column chromatography, IIa (designated as cPLC-II) and IIb (designated as cPLC-III) are eluted with 0.25 and 0.32 M NaCI, respectively.
[50]
PI-sPECIFIC PHOSPHOLIPASE C FROM PLATELETS
525
Properties There are more than four different forms of PLC in the cytosol 7-9 and two forms in the membrane fraction ~°,H of human platelets. The results of identification of human platelet PLC isoforms by antibodies against various types of soluble PLCs (brain PLC-fl, -T1, -8,12'13 uterus PLC-ct, 14 and PLC-y215'~6) indicate that human platelet cPLC-I is recognized by the antibody against PLC-Tz. On the other hand, platelet cPLC-II, -III, and mPLC-II preparations do not react with any other PLC antibodies. The catalytic properties of the platelet PLC isoforms are studied by using either [3H]PI or [3H]PIP2 as substrates. Hydrolysis of both PI and PIP 2 by platelet PLC isoforms is dependent on Ca 2+. However, at low Ca z+ concentrations (10/xM) [3H]PIP2 is the preferred substrate for platelet PLC isoforms. When assayed with [3H]PI as substrate at high calcium concentration (2 mM), cPLC-I activity is most active at pH 5.5; however, at neutral pH the order of specific activity is cPLC-II > cPLC-I > cPLCIII. On the other hand, cPLC-IV has very low activity for PI hydrolysis at both pH values. The order of specific activity is cPLC-IV > cPLC-III > cPLC-II > cPLC-I for [3H]PIP2 hydrolysis, indicating that cPLC-IV is the most specific for [3H]PIP2. The preference for [3H]PIP2 hydrolysis is also observed for the mPLC-I and mPLC-II preparations. The Km values for [3H]PI hydrolysis of cPLC-IV or mPLC-II are higher than those of cPLC-I or cPLC-II, namely, 0.7 and 0.5 mM for cPLC-IV and mPLC-II, and 0.1 and 0.08 mM for cPLC-I and cPLC-II, respectively. The effects of various metal ions on the PIP2-hydrolyzing activities of platelet PLC isoforms have been examined. Platelet cytosolic and membrane PLCs are inhibited by addition of 2 mM EGTA or 2 mM EDTA. The addition of Ca 2÷ (10/xM free) causes activation whereas Mg2÷ does 7 y. Banno, S. Nakashima, and Y. Nozawa, Biochem. Biophys. Res. Commun. 136, 713 (1986). s M. G. Low, R. C. Carroll, and A. C. Cox, Biochem. J. 243, 763 (1987). 9 V. Manne and H. F. Kung, Biochem. J. 243, 763 (1987). 10y . Banno, Y. Yada, and Y. Nozawa, J. Biol. Chem. 263, 11459 (1988). 11 j. j. Baldassare, P. A. Henderson, and G. J. Fisher, Biochemistry 28, 6010 (1989). 12 S. n . Ryu, P.-G. Suh, K. S. Cho, K. Y. Lee, and S. G. Rhee, J. Biol. Chem. 262, 12511 (1987). 13 S. H. Ryu, P.-G. Suh, K. S. Cho, K. Y. Lee, and S. G. Rhee, Proc. Natl. Acad. Sci. U.S.A. 84, 6649 (1987). 14 C. F. Bennett and S. T. Crooke, J. Biol. Chem. 262, 13789 (1987). 15 S. Ohta, A. Matsui, Y. Nozawa, and Y. Kagawa, FEBS Lett. 242, 31 (1988). 16y. Homma, T. Takenawa, Y. Emori, H. Sorimachi, and K. Suzuki, Biochem. Biophys. Res. Commun. 164, 406 (1989).
526
PHOSPHOLIPASEC
[51]
not. At low C a 2+ concentrations (10 -7 M) corresponding to the level in resting platelets, cPLC-IV and mPLC-II act to hydrolyze PIP 2 at 20-26% of maximum hydrolysis, but cPLC-I does not. Other metal ions (50/zM) such as Mn 2÷ , Fe 2÷ , and Cu 2÷ show some inhibition (15-20%). The inhibitory potency of Hg 2÷ differs among PLC isoforms; cPLC-I and mPLC-II are extremely sensitive to Hg 2÷ (•o.5 <1 /zM), whereas cPLC-IV is less sensitive to the ion (10.5 >5/zM).
[51] P u r i f i c a t i o n o f G u i n e a P i g U t e r u s P h o s p h o i n o s i t i d e Specific P h o s p h o l i p a s e C
By C. FRANK BENNETT, MICHAEL P. ANGIOLI, and STANLEY T. CROOKE
Introduction Hydrolysis of membrane phospholipids following occupancy of cell surface receptors is a common signal transduction pathway for a variety of agonists. Hydrolysis of phosphoinositides by phospholipase C (PLC) is well documented to be a major transduction mechanism for calciummobilizing agonists. Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generates two second messenger molecules, inositol 1,4,5-trisphosphate and diacylglycerol. 1,2Purification of the enzymes which hydrolyze the phosphodiester bond of phosphatidylinositol 4,5-bisphosphate (PI-PLC) from a variety of tissues has recently been accomplished. These studies have demonstrated that multiple forms of PIPLC exist within the same tissue or cell type. 3'4 Isolation of the cDNA clones for four PI-PLC isoenzymes demonstrated that the different forms of PI-PLC are distinct gene products with similar enzymatic activity (reviewed in Refs. 3 and 4). In a study of phospholipases in tissues which respond to peptidoleukotrienes, as part of our long-term studies on these agents, we determined that guinea pig uterus was a relatively rich source of phosphoinositidespecific phospholipase C and exhibited moderate levels of a phosphatidylethanolamine-preferring phospholipase A2 activity and low levels of phosphatidylcholine-specific phospholipase C activity. We subsequently puriI M. J. Berridge, Annu. Rev. Biochem. 56, 159 (1987). 2 U. K i k k a w a and Y. Nishizuka, Annu. Rev. Cell Biol. 2, 149 (1986). 3 S. G. Rhee, P.-G. Suh, S.-H. Ryu, and S. Y. Lee, Science 244, 546 (1989). 4 S. T. Crooke a n d C. F. Bennett, Cell Calcium 10, 309 (1989).
METHODS IN ENZYMOLOGY, VOL. 197
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