THE JOURNAL OF BIOLOGICAL CHEMISTRY Cc) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 7, Issue of March 5, pp. 4228-4236, 1991 Printed in U S A .

A 57-kDa Phosphatidylinositol-specific Phospholipase C from Bovine Brain*

(Received for publication, September 5, 1990)

Thomas A. Tompkins and Mario A. MoscarelloS From the Department o j Biochemistry, The Hospital for Sick Children, Toronto, Ontario M5G 1x8, Canada

A phosphatidylinositol-specific phospholipase C (PI- PLC) has been isolated from bovine brain (purification factor of 5.6 X lo4). By sodium dodecyl sulfate-poly- acrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the opti- mum ca2+ requirement was 4 mM, and K,,, was 260 pM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10” M, and the K,,, was reduced to 90 p ~ . Lipid specificity studies showed that equal amounts of inositol phosphate and diacyl- glycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was re- leased from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidyletha- nolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the ab- sence of a phospholipase D activity in the purified enzyme.

Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and %fold. Histone had a small effect only, whereas bovine serum albumin and cyto- chrome C had no effect. Phosphorylation of MBP re- duced the stimulatory effect, Protein-protein interac- tions between MBP and PI-PLC have been demon- strated both immunologically and by sucrose density gradients. A stoichiometry of 1:l has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic meth- ods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.

In an agonist-stimulated cell, the hydrolysis of phosphati- dylinositol 4,5-bisphosphate (PIP,)’ by a specific phospholi-

* This work was supported by Program Grant PG 11124 from the Medical Research Council of Canada. We thank the Beef Terminal for generous supplies of fresh bovine brain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

istry, The Hospital for Sick Children, 555 University Ave., Toronto, $ To whom correspondence should be addressed Dept. of Biochem-

Ontario M5G 1x8, Canada. I The abbreviations used are: PIP2, phosphatidylinositol 4,5-bis-

phosphate; PI, phosphatidylinositol; IP, inositol phosphate; PI-PLC, phosphatidylinositol-specific phospholipase C; MES, 4-morpholine- ethanesulfonic acid; HEPES, N-2-hydroxyethylpiperazine-N’-2-eth- anesulfonic acid EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; TBS, Tris-buffered saline; DAG, diacylglycerol; PA, phospha- tidic acid PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; MBP, myelin basic protein; ELISA, enzyme-linked immunosorbent assay; C-1, C-2, C-3, (2-4, and C-8, components 1, 2, 3, 4, and 8 from MBP, respectively; GTPyS, guanosine 5’-O-(thiotriphosphate).

pase C is a well documented process (for reviews, see Refs. 1 and 2). The result of this hydrolysis is the production of two second messengers: inositol 1,4,5-trisphosphate and diacyl- glycerol (DAG). The inositol 1,4,5-trisphosphate interacts with a receptor on the surface of a specialized component of the endoplasmic reticulum to mobilize intracellular calcium stores before being rapidly turned over through a complex metabolic pathway to inositol (3). The DAG can activate a calcium-dependent protein kinase, protein kinase C (4), or it can be deacylated, and the resulting free arachidonic acid can be used in eicosanoid biosynthesis. Through this mechanism, PIP, hydrolysis is linked to activation of cellular functions such as cell growth and differentiation (5).

Several phosphatidylinositol-specific phospholipase C (PI- PLC) isozymes, which vary with respect to molecular weight and enzymatic activity, have been purified from a wide range of mammalian cells. In a recent review, Rhee et al. (6) grouped the isozymes together in a designated nomenclature system (a, p, y, 6 , 6 ) . At least six PI-PLC isozymes have been described in mammalian brain (three P’s, y, 6, e ) . Two of these are brain-specific, whereas the others are more ubiquitous in nature (7-9). The localization of the PI-PLC isozymes to various cerebral regions and neuronal nuclei in the brain has been demonstrated using immunohistochemical methods and differential mRNA in situ hybridization studies (10, 11).

In brain, phosphatidylinositol lipids have been shown to be predominantly localized to the myelin fraction (12) and ac- count for approximately 1% of the total lipid of this membrane (13). The rapid incorporation of :<‘P into myelin phosphati- dylinositol lipids using i n vivo radiolabeling demonstrated that the phosphatidylinositol lipids were rapidly turned over in myelin (14). Early studies of PI-PLC in rat myelin char- acterized the enzymatic activity during myelin development, but the enzyme was not purified (15).

Myelin is no longer considered simply an inert insulating sheath, but is viewed as a dynamic membrane system con- taining a number of enzymes (16) that are responsible for metabolic reactions in a changing environment. Of particular interest in the present study is recent evidence that supports the existence of a second messenger system in myelin. For example, muscarinic receptors have been demonstrated on the surface of myelin that stimulate PI hydrolysis (17, 18). Stimulation of PI-PLC activity in isolated human myelin by GTP supported the existence of a second messenger system in this membrane (19). In addition, we previously presented evidence that myelin basic protein (MBP) can fulfill several of the criteria of a GTP-binding protein; that is, it bound GTP at a specific site near the N terminus and was ADP- ribosylated by cholera toxin (20). As an extension of these studies, we have also observed and partially isolated a calcium- dependent protein kinase C from white matter.’ Along with

J. Ramwani, and M. A. Moscarello, unpublished observation.

4228

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Bovine Brain Phosphatidylinositol-specific Phospholipase C

an additional phosphatidylinositol kinase directly involved in the PIP, turnover (21), most of the components of a putative signal transduction system have been demonstrated to exist in myelin.

We now report the isolation of a 57-kDa PI-PLC from bovine brain that has an amino acid composition similar to the 65-kDa PI-PLC from sheep seminal vesicles reported by Hofmann and Majerus (7). An interesting effect of myelin basic protein has been studied in some detail, suggesting specific interaction between this membrane protein and PI- PLC. Although the mechanism of this stimulation remains speculative, the data demonstrate that MBP is an important component of the GTP-induced stimulation of PI-PLC activ- ity in the myelin membrane system (19).

EXPERIMENTAL PROCEDURES

Materials

L-a-1-Stearoyl-2-arachidonoyl [arachidonoyl-l-‘“Clphosphatidyl- choline (specific activity, 60 mCi/mmol), L-a-1-palmitoyl-2-arachi- donoyl [arachidonoyl-1-“Clphosphatidylethanolamine (specific activ- ity, 60 mCi/mmol), L-cu-l-stearoyl-2-arachidonoyl [arachidonoyl-l- “C]phosphatidylinositol (specific activity, 30 mCi/mmol), L-(Y-[myo- inositol-2-3H]phosphatidylinositol (specific activity, 10 mCi/mmol), L-a-[myo-inositol-2-3H]phosphatidylinositol-4,5-bisphosphate (spe- cific activity, 2.5 mCi/mmol), and [choline-methyl-“Clsphingo- myelin (specific activity, 60 mCi/mmol) were obtained from Du Pont- New England Nuclear. The [sHIPI and [3H]PIP, had a mixed com- position at the sn-1 and sn-2 positions, with mainly stearoyl and arachidonoyl at these respective positions. EGTA, 1,2- and 1,3- diolein, and nonradioactive phosphatidylinositol 4,5-bisphosphate were purchased from Sigma. All other lipids and phospholipids were from Avanti Polar Lipids, Inc. The column-packing DE-52 pre- swollen anion exchange cellulose and phenyl-Sepharose CL-4B were purchased from Whatman and Pharmacia LKB Biotechnology Inc., respectively. MBP was prepared by the method of Lowden et al. (22). The charge isomers were isolated on CM-52 columns as described recently (23).

IODO-GEN was purchased from Pierce Chemical Co., and sodium [“‘Iliodide (2 mCi/l.P nmol of iodine) was obtained from Amersham Corp. Cathepsin D was purchased from Boehringer Mannheim. Rab- bit anti-bovine myelin basic protein IgG antibodies were a gift from Dr. J Boggs of the Research Institute, Hospital for Sick Children, Toronto.

Assay of PI-specific Phospholipase C Activity

Phospholipase C activity was monitored by the release of [2-“H] inositol 1-monophosphate from [inositol-2-3H]phosphatidylinositol as described by Low and Weglicki (24). For quick assays of column fractions or to study the effect of various factors (ions, pH, and other proteins) on phospholipase C activity, approximately 2 x 10’ dpm of [2-RH]PJ (specific activity, 500 dpm/nmol) were incubated for 20 min at 37 “C with 25 mM HEPES, pH 7.0, 4.0 mM CaCl*, 0.1% sodium deoxycholate, and the sample (5-20 pg of protein) in a final volume of 400 ~1. All tubes were sonicated for 60 s in a Bransonic 220 bath sonifier before the addition of the enzyme. The reaction was termi- nated by the addition of 1.5 ml of ice-cold 2:l methanol/chloroform (v/v) followed by an additional 0.5 ml of chloroform and 0.5 ml of 1 N HCl, vortexed, and centrifuged at 1,500 rpm for 10 min to separate the phases. A 0.5-ml aliquot of the upper aqueous phase, containing the water-soluble [2-3H]inositol 1-monophosphate, was removed for liquid scintillation counting.

To prepare the enzyme balance sheet, approximately 8 x lo4 dpm of [2-3H]PI (specific activity, 200 dpm/nmol) was incubated for 60 min as mentioned above. These conditions were also used to deter- mine the enzyme specificity and to generate substrate kinetic curves,

Purification Procedure

Preparation of Brain Extract (Fig. I&The extraction procedure followed was similar to that described by Ryu et al. (8) except that 0.5% Nonidet P-40 was included in the homogenization buffer. Two bovine brains were obtained fresh from a local slaughterhouse; the brain stem, cerebellum, and meninges were removed. The brains were homogenized with 1 liter of buffer A, which contained 20 mM Tris-

4229

Homogenats (H)

* 6,) supernstant Pellet (P,)

adJust pti to 5 0 with ecetlc acid

A (S2) SuPwllatant 2 Pllllst 2 (P,)

& (S3) Supematant 3 Pellet 3 (Pg)

& (S4) supomatant 4 Pallet 4(P4)

I dialyze against 25 mFl MES, pH 5 5

DE-52 column (2x90 cm)

I

dialyze against 25 mfi HEPES, pH 7 01 1 2 M KCI

Phenyl SepharOSe HIC COlUmn (I 5 X 16 Cm)

FIG. 1. Flow diagram of purification of bovine brain PI- specific phospholipase C. HZC, hydrophobic interaction chroma- tography.

0.6

0.0 0 50 .75 1.0 1.35 ,..5P 1.75

Elulbn Volume (L)

I 225 - I

0

FIG. 2. DE-52 column chromatography. Supernatant Sq was dialyzed against 25 mM MES buffer, pH 5.5, and applied to a 2 X 90- cm column of DE-52. Elution was done with a gradient of O-225 mM KCl. The ApRo and enzyme activity of each fraction were determined as described under “Experimental Procedures.” PLC act., PLC activ- ity.

HCl, pH 7.2, 5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, and 0.5% Nonidet P-40. The homogenate was centrifuged at 13,000 x g for 30 min, yielding a soluble fraction (S,) and a pellet (PI). The soluble fraction was decanted, adjusted to pH 5.0 with glacial acetic acid, and stirred for 30 min at 4 “C. The resulting suspension was centrifuged at 13,000 x g for 30 min, and the precipitate (PZ) was solubilized in 500 ml of buffer B (50 mM Tris-HCl, pH 7.4, 5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol). The solubilized P, was centrifuged again at 13,000 X g for 30 min to remove insoluble materials. The supernatant (S3) was centrifuged at 150,000 X g for 45 min to clarify it.

Anion Exchange Chromatography (Fig. 2)-The clarified superna- tant (S,) from above was dialyzed against 25 mM MES, pH 5.5, and was applied to a DE-52 cellulose column (2 X 90 cm), which had been equilibrated with the same buffer. After a 300-ml wash with the equilibrating buffer, the column was eluted with a gradient from 0 to 225 mM KCl. On each fraction, of approximately 10 ml, the absorb- ance at 280 nm, the conductivity, and the phospholipase C activity were determined.

Hydrophobic Interaction Chromatography (Fig. 3)-The pooled ac- tive fractions from the anion exchange chromatography were dialyzed overnight against 25 mM HEPES, pH 7.0, containing 1.2 M KCl. The dialyzed sample was applied at a flow rate of 1.5 ml/min to a phenyl- Sepharose CL-4B column (1.5 x 16 cm), which had been equilibrated with 25 mM HEPES, pH 7.0, containing 1.2 M KCl. The column was washed with 60 ml of the equilibrating buffer and then eluted stepwise using 60 ml of 25 mM HEPES, pH 7.0, containing 0.6 M KC1 followed by 100 ml of 25 mM HEPES, pH 7.0, without KCl. The fractions containing the enzyme activity were pooled (140-170 ml, Fig. 3) and concentrated using an Amicon Centriprep (molecular weight cut off,

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4230 Bovine Brain Phosphatidylinositol-specific Phospholipase C

b 0.6, I I

I

Q E

U

I -1.2

-0.0 5 'L

s

- 0.0

Elution volume (a) FIG. 3. Phenyl-Sepharose column chromatography. The en-

zyme fractions from the DE-52 column were dialyzed against 25 mM HEPES buffer, pH 7.0, containing 1.2 M KC1 and applied at 1.5 ml/ min to a phenyl-Sepharose CL-4B column, 1.5 X 16 cm. The column was eluted stepwise with 25 mM HEPES containing 0.6 M KC1 followed by 25 mM HEPES. The A280 and enzyme activity were determined on each fraction, as described under "Experimental Pro- cedures.''

10,000). After this step, the purification factor was approximately IO4 (Table I) using [2-3H]PI as substrate and 5.6 X lo4 when PIP, was the substrate.

Amino Acid and Sequence Analyses Prior to acid hydrolysis, performic acid oxidation converted cystine

to cysteic acid as follows. 35 pg of phospholipase C were precipitated with trichloroacetic acid and then incubated at 0 "C with 25 pl of 98% formic acid and 5 p1 of 30% hydrogen peroxide for 2 h. The sample was lyophilized in a Speed-Vac to remove reagents, washed with water, and lyophilized again. This product was hydrolyzed in 6 N HC1 for 24 h, derivatized, and analyzed on a Water's Pic0 Tag amino acid analysis system (Waters Chromatography Division, Mil- lipore, Ltd.).

Tryptophan was quantitated on the Pic0 Tag system after meth- anesulfonic acid hydrolysis (25). For this hydrolysis, 2.3 fig of the enzyme were dried, and 20 p1 of 4 M methanesulfonic acid containing 0.2% (w/v) tryptamine hydrochloride and 100 p1 of filtered water were added. The vial was sealed, and the sample was hydrolyzed at 110 "C for 24 h. After cooling, 22 p1 of 4 M KOH were added to neutralize the acid. The sample was then dried under vacuum, washed with methanol/water/triethylamine (2:2:1), dried, and derivatized with phenylisothiocyanate. The derivatized sample was filtered before injection onto the column.

The N-terminal analysis was attempted by application of 500 pmol performic acid-oxidized protein to the automated Beckman 890 MSJ sequenator. Cathepsin D digestion was done as follows. 150 pg of bovine PI-PLC were reduced with 7.5 pl of 2-mercaptoethanol in 0.3 ml of 0.5 M MES buffer, pH 8.0, under nitrogen for 1 h at 37 "C, followed by alkylation with an excess of iodoacetamide (36 mg). After dialysis, cathepsin D (1:100, w/w) in 50 mM ammonium acetate buffer, pH 3.5, was added and incubated for 12 h at 37 "C. The digest was lyophilized and sequenced for 10 cycles in a Porton model 2090 gas phase sequencer with on-line microbore HPLC. Phosphorus was determined by the method of Bartlett (26). SDS-polyacrylamide gels were run by the method of Laemmli (27).

Enzyme Specificity To determine the specificity of the enzyme for PI, several phos-

pholipids, including phosphatidylcholine (PC), phosphatidylethanol- amine (PE), sphingomyelin (SM), phosphatidylinositol (PI), and PIP, were tested.

The assay conditions were as follows. Approximately 400 nmol (specific activity, 200 dpm/nmol) of each of [ara~hidonoyl-l-'~C]PC, [arachidonoyl-l-"C]PE, [choline-methyl-"CISM, [arachidonoyl-l- "CIPI, [ino~itol-2-~H]PI, or [inosit01-2-~H]PIP2 were incubated sep- arately for 60 min at 37 "C with or without enzyme in the presence of 4 mM Ca2' except when PIP2 was used in which case the Ca" concentration was lo" M. The aqueous phase was removed and counted. A 100-p1 aliquot of the lower organic phase was removed, dried, and counted by liquid scintillation. The remaining organic phase (900 pl) was dried down and resuspended in 100 pl of chloro- form. One-half of this volume was spotted onto a Silica Gel 60 plate

and developed for neutral lipids (1,2-DAG, 1,3-DAG) using diethyl ether/hexane/acetic acid (70:301) (7). The remaining 50 pl were spotted onto a second Silica Gel 60 plate presprayed with 1 mM Na+/ EDTA, pH 5.5, and developed for phospholipids (PI, PIP2, PA, PC, PE, SM, lyophospholipids, and free fatty acid) using chloroform/ methanol/acetic acid/H20 (65:502:5) (28). The spots were visualized by iodine vapor and then scraped off the plate for liquid scintillation counting. Standards included 1,2-diolein, 1,3-diolein, phosphatidic acid, free arachidonic acid, and lyso-PC.

The water-soluble product was identified as inositol phosphate by elution from a Dowex 1 X 8 column (0.5 X 4 cm), as outlined by Irvine (28). Free inositol was found in the unbound fraction. Succes- sive washes with 0.1 M formic acid, 0.2 M ammonium formate; 0.1 M formic acid, 0.4 M ammonium formate; and 0.1 M formic acid, 1.0 M ammonium formate (6 ml each) eluted IP, inositol 4,5-bisphosphate, and inositol 1,4,5-trisphophate, respectively. Fraction volumes of 2 ml were collected and counted.

Effect of Proteins and Peptides on PI-specific Phospholipase C Activity

Increasing concentrations of bovine MBP, the charge isomers of bovine MBP, several MBP peptides, bovine serum albumin (BSA), cytochrome c, and histone were incubated with the PI in the presence or absence of PI-PLC. After the reaction was terminated, an aliquot of the aqueous phase was removed for liquid scintillation counting.

The peptides included several synthetic and enzymatically gener- ated peptides from a cathepsin D digest of component 1 (C-1) of MBP. The synthetic peptide consisting of residues 114-122 contain- ing the single tryptophanyl residue in MBP was purchased from Sigma. Synthetic peptides 24-33 (ARZ5HGFLPR3lHR); 24-33 in which RZ5 and R31 were replaced by citrulline, generating the sequence found in component 8 (C-8) of MBP; and peptides 157-170 (GGRDSRSGSPMARR), representing the C-terminal peptide in C- 1, were prepared in our biotechnology service. Peptide 1-116 was prepared by BNPS-skatole cleavage at the single tryptophanyl residue (29) which represents the N-terminal two-thirds of the molecule. Peptides 1-44, 45-89, and 90-170 were prepared by cathepsin D digestion of the two Phe-Phe linkages in MBP (23). Briefly, 30 pg of cathepsin D were incubated with 3 mg of C-1 in 200 pl of 50 mM ammonium acetate, pH 3.5, at 37 "C for 18 h. The peptides were separated on a PLRP-S 300 column from Polymer Laboratories Inc. by HPLC for 30 min with an acetonitrile 5-35% gradient in 0.05% trifluoroacetic acid. The peptides were identified by amino acid anal- yses as described above.

Enzyme-linked Immunosorbent Assay (ELISA) Aliquots of 100 pl of a 5-pg/ml PI-PLC solution were placed into

wells of a microtitration plate and incubated overnight a t 4 "C, and the liquid was removed. The coated wells and an equal number of blank wells were blocked with 5% milk powder in Tris-buffered saline (TBS) for 1 h at 22 "C. To each well, 100 p1 of MBP solutions (0-2.0 mM in 5% milk powder in TBS) were added. After shaking for 1 h at 22 "C, the liquid was removed and 100 pl of anti-bovine MBP IgG (0.05 mg/ml in 5% milk powder in TBS) was added and incubated for 1 h at 22 "C with shaking. After washing with 0.05% Tween 20 in TBS, followed by TBS, 50 p1 of the secondary antibody (goat anti- rabbit IgG conjugated to alkaline phosphatase (Bio-Rad), at a dilution of 1:2000 in 5% milk powder in TBS) was added and incubated for 1 h at 22 "C. A Bio-Rad alkaline phosphatase substrate kit was used to detect bound secondary antibody, 100 p1 of colorimetric solution was added to the wells, and the plate was incubated at 37 'C. After 1 h, the absorbance at 410 nm was read on a Dynatech microplate reader.

Dot Blot Assay Aliquots of 100 p1 of the PI-PLC solution used in the ELISA above

were blotted on nitrocellulose paper in a Bio-Rad dot blot apparatus. Other solutions such as that used for blocking, MBP of different concentrations, and primary and secondary antibodies were the same as described for the ELISA. Detection was carried out by adding 10 ml of developing solution containing 20 pl of nitro blue tetrazolium (50 mg/ml in 70% dimethylformamide) and 20 pl of bromochloroin- dolyl phosphate (50 mg/ml in 100% dimethylformamide) in 100 mM Tris-HC1 buffer, pH 9.2, containing 0.5 mM MgC1, and 100 mM NaC1. The reaction product was detected on the LKB Ultroscan XL.

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Bovine Brain Phosphatidylinositol-specific Phospholipase C 4231

Sucrose Density Gradients

Sucrose gradients (2.5-15% sucrose in 25 mM HEPES buffer, pH 7.0, containing 4 mM CaCI,) were prepared with a Biocomp Gradient Master 105. To the top of each tube, 300 p1 of sample were carefully layered, and the gradients were centrifuged at 40,000 rpm for 18 h at 4 “C in a SW 55 Ti rotor. A total of 40 fractions of 200 pl each were collected from each tube. The refractive index was determined on each fraction. The absorbance at 280 nm was used to detect the proteins after dilutions of the 2OO-pl fractions to 1 ml with water. The gradients were calibrated with proteins cytochrome c (12.5 kDa), chymotrypsinogen A (25.7 kDa), hemoglobin (62 kDa), bovine serum albumin (66.2 kDa), collagenase (105 kDa), and lactate dehydrogenase (144 kDa). When radioactive samples were used, each of the gradient fractions was counted in a LKB 1282 Compugamma Counter.

Protein Iodination

The protein iodination procedure was a modification of that of Markwell and Fox (30). Briefly, 0.6 pCi of [‘251]sodium iodide were incubated for 15 min with 200 pg of PI-PLC or C-1 from MBP in 200 pl of 0.1 M Na2HP04, pH 7.0, containing 0.02% sodium azide with 20 p1 of IODO-GEN solution (2 mg/ml chloroform) at 22 “C. The lz5I- labeled protein was separated from reagents by gel filtration on a Sephadex G-25 column equilibrated in 0.1 M Na,HP04, pH 7.0, containing 0.02% sodium azide. Fractions (0.5 ml) were counted m a LKB 1282 Compugamma Counter. The proteins that eluted in the void volume were dialyzed against water and lyophilized.

Other Methods

Protein concentration was determined using the Peterson assay (31) with BSA as a standard. SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (27) using 12.5% acrylamide in a Hoeffer mini-gel apparatus. The proteins were stained with Coo- massie Blue.

RESULTS

Purification of PI-specific Phospholipase C The enzyme purification data is summarized in Table I.

The final purification represented a 16.4% yield of activity and a lo4 purification factor that compared favorably with those in the literature, e.g. Ryu et al. (8) reported a purification factor of 6,800-fold. The purification factor reported in this paper was obtained with PI (to conserve cost) as a substrate and not phosphatidylinositol 4,5-bisphosphate, the preferred substrate for this enzyme. When PIP2 was used, a purification factor of 5.6 X lo4 was obtained. This purification factor is similar to that reported for the turkey erythrocyte enzyme (4.8 X lo4; specific activity, -10 pmol/min/mg of protein) by Morris et al. (32). The purity of the enzyme was monitored by SDS-polyacrylamide gels (Fig. 4, lane 2). One major band was seen at M, 57,000 under nonreducing conditions and

increased to M, 65,000-70,000 under reducing conditions (not shown). An enzyme of similar relative molecular weight was isolated from human myelin (Fig. 4, lane 1 ), demonstrating the myelin localization of this enzyme.

Primary Structure Amino Acid Analyses-The amino acid composition was

determined after acid hydrolysis, as described under “Exper- imental Procedures.” Tryptophan analyses were carried out separately after methanesulfonic acid hydrolysis. These anal- yses are reported in Table I1 for seven independent analyses, each from a different enzyme preparation. Also reproduced in the table is the composition reported by Hofmann and Ma- jerus (7) for their ram seminal vesicle enzyme. The composi- tion obtained was similar to that reported previously by Hofmann and Majerus (7) and Bennett et al. (33). The Hof- mann PI-PLC was a 65-kDa enzyme isolated from ram sem- inal vesicles, whereas the Bennett PI-PLC was a 57-kDa enzyme isolated from guinea pig uterus. Both have subse- quently been detected in sheep and rat brain, respectively, by immunologic methods (10, 34).

Partial Sequence-N-terminal sequence analysis showed that the enzyme failed to sequence; we concluded that the N terminus was blocked.

An investigation of the sequence deduced from the cDNA by Bennett et al. (33) for the enzyme from guinea pig uterus revealed the presence of two Phe-Phe linkages at residues 52- 53 and 160-161. Assuming considerable sequence homology among the enzymes, we digested our PI-PLC with cathepsin D. Cathepsin D digestion at these sites would yield three peptides consisting of residues 1-52, 53-160, and 161-504. Since the N terminus was blocked, only sequences from 53- 160 and 161-504 would be detected. After 10 cycles of sequenc- ing, we obtained two sequences, FGPWCXXXKR, cor- responding to the N terminus of peptide 53-160 (FAPWCGHCKR), and FRNLXXNGHX, corresponding to the N terminus of peptide 161-504 (FRDLFSDGHS). These data demonstrate that the bovine brain enzyme contains two Phe-Phe linkages and considerable sequence homology with the guinea pig uterus PI-PLC (33).

Characterization of the Enzyme In order to optimize the conditions of assay of the purified

enzyme for subsequent studies with myelin basic protein, we investigated time, pH, and calcium ion concentration.

Time Course-The time course for I P release from PI is

TABLE I Purification of phosphatidylinositol-specific phospholipase C from bovine brain

volume conc. protein activity activity factor Sample“ Total Protein Total Specific Total Purification

ml w / m l mg nrnol/min/mg nmollmin 76

610 H 1,000 36.5 36,500 0.18 6,570 100 1 S, 29.7 18,100 0.26 4,710 71.7 Pl 890 43 10,900 0.23 2,520 38.3

1.4

S2 500 12.3 6,150 1.3

0.19 P, 250 30.3

1,170 7,560

17.8 1.1 0.29 2,190 33.4

S3 246 32 7,870 0.31 2,440 37.1 1.6

P3 28 24.7 690 0.23 159 2.4 1.7

s4 190 16 3,040 0.24 730 11.1 1.3

p4 45 31 1,390 0.28 391 6 1.3

300 0.51 127.5 15.7 2,000 30.5 87.2 1.6

Phenyl-Sepharose 200 0.003 0.62 1,754 1,080 16.4 9,740b DE-52

a H, homogenate; S, soluble fraction; P, pellet. * By using the preferred substrate, phosphatidylinositol 4,5-bisphosphate, the purification factor increased 5.7

times to approximately 55,700.

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Bovine Brain Phosphatidylinositol-specific Phospholipase C 4233

I

zooF 100 4 - a .. 2 0 * 1 6 8 1 0 , -

s I W 0 0 1 2 3 4 5 6

concentration PI (mu)

FIG. 8. K, and V,, of bovine brain PI-PLC with PI as substrate. The incubation mixture contained 25 mM HEPES, 0.1% sodium deoxycholate, 4.0 mM CaC12, various concentrations of [2-3H] PI, 500 dpm/nmol specific activity, and enzyme in a final volume of 400 PI. After 60 min of incubation, acidified methanol/chloroform was added, the mixture was centrifuged at 1,500 rpm for 10 min, and the disintegrations/min in the aqueous phase were determined. Inset shows data plotted by the Lineweaver-Burk method.

TABLE 111 Bovine brain PI-PLC phospholipid specificity

Approximately 8 X lo‘ dpm of each phospholipid (specific activity, 200 dpm/nmol) was incubated in 25 mM HEPES, pH 7.0,0.1% sodium deoxycholate, 4 mM Caz+ (with PIP, as substrate, lo” M Ca2+ was used). The reaction was terminated by addition of acidified methanol/ chloroform. The phases were separated, and aliquots were counted. The organic phase was applied to a Silica Gel 60 TLC plate, and 1,2- DAG was separated from other components by diethyl ether/hexane/ acetic acid (70:30:1). The TLC plate was scraped, and the radioactivity of each component was measured.

Lipid“ DAG Head group

nmol PC PE SM PI PIP7 143

1.8 0.0

3.3 23.7 26.6

a PE, [arachid~noyl-l-’~C]phosphatidylethanolamine; PC, [arachi- don~yl-l-~~C]phosphatidylcholine; PI, [in0sitol-2-~H]phosphatidyli- nositol; PI, [ara~hidonoyl-l-~~C]phosphatidylinositol; PIPZ, [inositol- 2-3H]phosphatidylinositol 4,5-bisphosphate; SM, [choline-methyl- “C]sphingomyelin.

7.0, 0.1% sodium deoxycholate and 4 mM Ca2+ for 1 h at 37 “C. The results are summarized in Table 111. The radioac- tivity in both aqueous and organic phases was measured. Only PI and PIP, liberated significant radioactivity into the aqueous phase. The aqueous phase was then applied to a Dowex 1- X 8 column and eluted with 0.1 M formic acid as described under “Experimental Procedures.” Since all the radioactivity bound to the resin, we concluded that the prod- uct was IP and not inositol, which rules out a phospholipase D activity in our preparation.

The organic-soluble product of hydrolysis (DAG) was iden- tified by TLC for neutral lipids on a 20 X 20-cm Silica Gel 60 TLC plate and run in the solvent system diethyl ether/ hexane/acetic acid at 70:30:1 (see “Experimental Proce- dures”). Another portion of the chloroform phase was spotted on a Silica Gel 60 TLC plate (20 x 20 cm) that had been presprayed with 1 mM EGTA, pH 5.5, and run in a solvent system to separate phospholipids (chloroform/methanol/ace- tic acid/H20 at 65:50:2:5).

The neutral lipid system separated 1,2-DAG, 1,3-DAG, and free fatty acid, whereas the phospholipids remained at the origin. After development, the areas corresponding to the

DAG and free fatty acids were scraped and counted. The release of [ara~hidonoyl-l-’~C]DAG from radiolabeled PE, PC, and PI was monitored in this way. For PC and PE, 1,2-[14C] DAG was slightly above background, whereas much higher values were observed using PI as the substrate. A small amount of [14C]methyl choline was released from SM. PIP, was the best substrate for the enzyme. Since it was not labeled in the DAG moiety, we were unable to measure this portion of the molecule. From these data, we concluded that our phospholipase C was PI-specific. Since radioactivity was not detected in PA with any of the phospholipids used, we con- cluded that our purified enzyme did not contain phospholipase D activity which supports of the above mentioned data in which all the radioactivity on the inositol ring was bound to Dowex 1- X 8 as inositol phosphate.

The Effect of Added Proteins on PI-PLC Activity

Several proteins have been reported to stimulate the activ- ity of PI-PLC. These include histone, BSA, cytochrome c, and hemoglobin (8, 36). The nature of the stimulation is not understood. Of these, histone had the greatest stimulatory effect, which led Fukui et al. (36) to postulate a regulatory role for basic proteins. None of these studies explored the effects of MBP.

The effects of various concentrations of the four proteins cytochrome c, histone, BSA, and MBP on the activity of bovine brain PI-PLC is shown in Fig. 9. A small stimulation was observed with histone at low concentrations. No stimu- lation was observed with BSA or cytochrome c. The stimula- tion observed with bovine MBP was noteworthy in that the activity increased more than 250% in the presence of 1.5 ~ L M MBP, with PI as substrate.

Although the MBP used in the above experiments migrated as a single band on SDS-polyacrylamide gel electrophoresis with a M, of 20,000, at alkaline pH (10.6) in urea, it can be resolved into several charge isomers, which is the result of charge microheterogeneity. Several of these charge isomers have been isolated and studied extensively in recent years (see Moscarello (16) for a review). The most cationic of the charge isomers has been called C-1 and the least cationic has been called C-8. C-1 is considered to be the least modified of the components, whereas C-8 is extensively modified. The modifications include phosphorylation and the replacement of several arginine residues by citrulline (23). Each of the charge isomers has a distinct function in maintaining the

“ 1 . - 0.0 1 .o 2.0 3.0 4.0 5.0

. . .

Protein (pM)

FIG. 9. The effect of proteins on PI-PLC activity. The incu- bation mixture contained 25 mM HEPES buffer, pH 7.0,0.1% sodium deoxycholate, 4.0 mM CaC& plus various concentrations of the differ- ent proteins in a final volume of 400 PI. After addition of acidified methanol/chloroform and separation of the phases as described under “Experimental Procedures,” the radioactivity released into the aqueous phase was determined. 0, bovine MBP; 0, BSA; ., histone (111); 0, cytochrome c.

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4234 Bovine Brain Phosphatidylinositol-specific Phospholipase C

integrity of the myelin membrane by virtue of very different abilities to induce the formation of compact multilayers de- scribed earlier (38). In Fig. 10, the effect of C-l and C-8 on the activity of PI-PLC activity is shown. Whereas C-1 was effective at stimulating the PI-PLC activity, C-8 had no effect. The other charge isomers, C-2, C-3, C-4, etc., stimulated the PI-PLC activity but were slightly less effective than C-1. Therefore, in addition to their distinct abilities to organize the lipid bilayer, the different charge isomers of MBP may have unique roles in this enzymatic reaction in myelin. The net positive charge on C-1 was decreased by phosphorylation. This phosphorylated C-1 had only a small stimulatory effect on PI-PLC (Fig. 11).

Interaction between MBP and PI-PLC Interaction between the two proteins was demonstrated in

several ways. ELISA-When PI-PLC was immobilized to the wells in the

ELISA assay and reacted with C-1 of MBP followed by anti- bovine MBP IgG as described under “Experimental Proce- dures,” binding was concentration-dependent with maximal binding of antibody between 1.0-2.0 MM MBP (Fig. 12A).

Dot Blot-When antibody binding was studied by dot blot analysis as described under “Experimental Procedures,” max- imal binding of antibody occurred between 1.5 and 2.0 MM MBP (Fig. 12B).

Sucrose Density Gradient Analysis-A sucrose gradient

0.0 0.5 1 .o 1.5 2.0

MBP CM) FIG. 10. Effect of charge isomers of MBP on bovine brain

PI-PLC. The incubation mixture contained 25 mM HEPES buffer, pH 7.0, 0.1% sodium deoxycholate, 4.0 mM CaClz plus various con- centrations of bovine MBP in a final volume of 400 pl. After addition of acidified methanol/chloroform as described under “Experimental Procedures,” the radioactivity released in the aqueous phase was measured. X, whole M B P 0, C-1; 0, C-2; ., C-3; 0, C-8.

0.0 1.0 2.0 3.0 4.0 5.0

Protein Conc. (elm)

FIG. 11. The effect of phosphorylated MBP on bovine brain PI-PLC. Bovine MBP was modified by phosphorylation using a crude myelin protein kinase C preparation. The phosphorylated pro- tein was then incubated with bovine PI-PLC under conditions out- lined under “Experimental Procedures.” 0, bovine MBP; 0, phos- phorylated MBP.

E

5 n

0

0

A E 2, 1 0.6 -I 1

P I 1 CM) t W OIM) FIG. 12. Immunologic demonstration of protein-protein in-

teractions. Anti-bovine MBP IgG was used to detect C-1 bound to PI-PLC in an ELISA ( A ) or on a dot blot ( B ) as described under “Experimental Procedures.” 0, total binding to PI-PLC; 0, nonspe- cific binding; x, specific C-1 binding to PI-PLC.

= 0 0 r

x, f

150

1 w N 50

0 0 10 20 30 40

FRACTION FRACTION

FIG. 13. Protein-protein interactions. Preparation of sucrose gradient (2.5-15%) and sample centrifugation are outlined under “Experimental Procedures.” A, standard curve was obtained for pro- teins in the sucrose gradient. I, cytochrome c (12.5 kDa); 2, chymo- trypsinogen A (25 kDa); 3, hemoglobin (62 kDa); 4, bovine serum

ase (144 kDa). 0, Pi-PLC (57 kDa); ., C-1 (18.5 kDa). B, PI-PLC albumin (66.2 kDa); 5, collagenase (105 kDa); 6 , lactate dehydrogen-

addition to ‘2SI-labeled C-1 shifted the lZ5I peak from fraction 33 to fraction 22 (M, 68,000 f 4,000). The data suggest a 1:l stoichiometric ratio for PI-PLC to C-1. 0, 1251-labeled C-1 only; 0, lZ5I-labeled C-1 with PI-PLC present.

(2.5-15%) was prepared, and several proteins of known mo- lecular weight were applied. These included cytochrome c (12.5 kDa), chymotrypsinogen A (25.7 kDa), hemoglobin (62 kDa), bovine serum albumin (66.2 kDa), collagenase (105 kDa), and lactate dehydrogenase (144 kDa). A standard curve was obtained (Fig. 13A). C-1 of MBP (18.5 kDa) remained near the top of the gradient. In fact, its behavior was anom- alous because it remained above cytochrome c. This behavior is readily explained by the open conformation of this mem- brane protein, the result of total absence of cysteinyl residues (16). PI-PLC migrated just above hemoglobin in keeping with its M, of 57,000.

To determine if interaction between C-1 of MBP and PI- PLC had occurred, C-1 was iodinated as described under “Experimental Procedures” and the lZ51-labeled C-1 was ap- plied to a sucrose gradient; the majority of the radioactivity was recovered in fractions 31-32 near the top of the gradient. When lZ51-labeled C-1 of MBP was combined with PI-PLC, the peak of radioactivity was found in fractions 21 and 22. Although some trailing of radioactivity occurred, very little was found in fractions 31-33. In both experiments in which lZ51-labeled C-1 was used, some trailing of radioactivity was observed in fractions 25 and 26, which we attribute to dimers of MBP and which the protein is known to form (16). From the standard curve (Fig. 13A), the size of the MBP.PI-PLC complex of 68,000 f 4,000 (three independent experiments),

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Bovine Brain Phosphatidylinositol-specific Phospholipase C 4235

in keeping with a 1:l molar ratio. No increase in size of the ‘”I-labeled PI-PLC was observed when histone was used instead of MBP, suggesting the interaction between PI-PLC and C-1 of MBP was specific (histone failed to stimulate the activity of this enzyme (see Fig. 9)).

MBP Sites Involved in Interactions with PI-PLC Attempts to cross-link MBP and PI-PLC with cross-linking

reagents resulted in the formation of aggregates of high mo- lecular weight, which were poorly resolved. An alternative method to study interaction was to study the effects of various MBP peptides on the activity of the enzyme. For this purpose, a number of peptides were prepared by chemical and enzy- matic cleavages, and several small peptides were synthesized. These are listed in Table IV. Peptide 45-89 generated by cathepsin D digestion of C-1, synthetic peptide 157-170, and synthetic peptide 24-33 in which 2 arginyl residues were replaced by citrullines had no effect on enzyme activity. Peptides 1-44 from the cathepsin D digest and synthetic peptides 24-33 and 114-122 were 78-85% as effective as C-1 in stimulating the activity of the PI-PLC. The lower activity of peptide 1-116 was probably related to the harsh conditions of the BNPS-skatole reaction. These results suggest that two sites on MBP, one near the N terminus and the other near the center of the molecule, are most effective in the stimula- tion of PI-PLC and probably represent the binding sites of MBP to the enzyme.

DISCUSSION

A PI-PLC has been purified 5.6 X 104-fold from bovine brain. On SDS-polyacrylamide gel electrophoresis it migrated with a M, of 57,000. Since we isolated a similar enzyme from freshly prepared normal human myelin, localization of the enzyme to this membrane system is suggested. The amino acid composition of our enzyme was similar to that of Hof- mann and Majerus (7) from ram seminal vesicules, and partial sequence data showed considerable homology with the guinea pig uterus enzyme (33), suggesting that the enzyme is highly conserved in different tissues. Analyses for amino and neutral sugars were negative, and the enzyme was not phosphorylated.

In a recent report by Fukui e t al. (36), histone was shown to stimulate their PI-PLC activity. We have shown that MBP, a basic protein found naturally in myelin, stimulates our

TABLE IV Effect of MBP peptides on PI-PLC activity

Peptide 1-116 was prepared from MBP by BNPS-skatole cleavage at the single Trp residue. Peptides 1-44, 45-89, and 90-170 were prepared by cathepsin D digestion at the two Phe-Phe linkages (residues 44-45 and 89-90). Peptides 24-33, 114-122, and 157-170 were synthetic peptides. Peptide 24-33 in which Arg at positions 25 and 31 were replaced by citrulline is also a synthetic peptide. The sequences of the synthetic peptides are given under “Experimental Procedures.”

Residues relative to C-1 % stimulation Generated by

1-170 1-116 1-44

45-89 90-170 24-33 24-33 (Cit)b

114-122

100 66 85

NS” 85 83

NS 78

No cleavage BNPS-skatole Cathepsin D Cathepsin D Cathepsin D Synthetic Synthetic Svnthetic

157-170 NS Synthetic NS, no stimulation of PI-PLC activity.

* Peptide 24-33 in which citrulline replaced Arg at positions 25 and 31, generating the sequence in C-8 of MBP.

purified PI-PLC 2-3-fold. This effect is specific to MBP, since the other major myelin protein the proteolipids (lipo- philin in the human) failed to stimulate the PI-PLC activity. When phosphorylation of MBP with protein kinase C from white matter was carried out, it was only marginally effective, stimulating PI-PLC slightly above the activity observed in the absence of MBP, which demonstrates that the stimulatory effect was essentially abolished by phosphorylation of MBP.

The 18.5-kDa isoform of MBP represents the major isoform in both human and bovine species. This isoform has been shown to have considerable charge microheterogeneity. Thus, at alkaline pH, several of these charge isomers can be sepa- rated on CM-52 columns (23). The most cationic, C-l , elutes last from the column, whereas the least cationic fails to adsorb. This latter material (C-8), has been shown to contain citrulline, replacing arginine a t selected sites (23). When its ability to stimulate PI-PLC activity was tested, it failed to stimulate the activity. Although these are both basic proteins, C-8 is much less cationic than C-1 and less cationic than the phosphorylated MBP.

The mechanism through which the activation of PI-PLC occurs has been shown to involve protein-protein interactions between MBP and PI-PLC. Interaction between the two proteins has been demonstrated by immunologic method and by sucrose density gradient centrifugation. A 1:l stoichiome- try has been suggested by this method. With a number of peptides isolated from chemical and enzymic cleavages of MBP and synthetic peptides corresponding to MBP se- quences, interaction has been demonstrated between peptides involving residues 24-33 and 114-122 of the sequence. The C-terminal portion of the molecule was not involved in the interaction since peptide 157-170 did not stimulate the en- zyme. When the arginyl residues of peptide 24-33 at positions 25 and 31 were replaced by citrullines, this latter peptide was unable to stimulate the enzyme. These studies suggest that MBP plays a functional role in myelin in regulating PI-PLC activity and, furthermore, that the individual charge isomers may each have distinct functional roles.

This data support the earlier data suggesting that some of the charge isomers of MBP are involved in a putative signal transduction system in myelin. C-1 was ADP-ribosylated by cholera toxin in the presence of NAD, whereas C-8 could not be ADP-ribosylated (39). C-1 was shown to bind 1 mol of GTP at a specific site in the N terminus of the molecule (20). Taken together with the present data in which C-1 was shown to stimulate PI-PLC, a central role for C-1 in a signal trans- duction system in myelin is emerging.

In earlier structural studies (38), we reported that C-1 induced the formation of compact multilayers, whereas C-8 was unable to organize the lipid in model systems as deter- mined by liquid x-ray diffraction studies. In a developmental study in the human, C-1 could not be detected before 2 years of age in the MBP fraction isolated from brain. Instead, all the MBP could be accounted for by C-8.3 Thus, the differential effects of C-1 and C-8 in the compaction of myelin and in the effects on the activity of PI-PLC demonstrate clearly that microheterogeneity of MBP is not merely a curiosity but has important implications for both the structure and function of myelin. At the present time, a definitive statement cannot be made concerning the roles of net charge on the molecule since, in addition to differing significantly in charge, C-1 and C-8 have been shown to contain differing amounts of @-structure (40). Furthermore, phosphorylation of C-1 with protein kinase C has been shown to increase the amount of @-structure,

M. A. Moscarello, J. McLaurin, and D. D. Wood, submitted for publication.

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4236 Bovine Brain Phosphatidylin

approaching the value obtained for C-8 ( i e . about 40%). The failure of phosphorylated C-1 to stimulate the PI-PLC activity suggests a mechanism of control of this important enzymatic activity involving both charge and conformational factors. Further studies are required to determine the roles played by each of these factors.

Although an effect of MBP on the highly purified PI-PLC has been demonstrated, addition of GTPyS did not affect the activity. In an earlier study (19), we demonstrated that addi- tion of GTPyS to isolated myelin membranes stimulated the hydrolysis of PIP, by 500%, suggesting that other factors are involved in the MBP effect in the myelin membrane. The nature of these other factors is being explored.

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T A Tompkins and M A MoscarelloA 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain.

1991, 266:4228-4236.J. Biol. Chem. 

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