Proc. Natl. Acad. Sci. USAVol. 84, pp. 7046-7050, October 1987Biochemistry

The 87-kDa protein, a major specific substrate for protein kinase C:Purification from bovine brain and characterization

(phosphoprotein/phosphorylation/nervous system)

KATHERINE A. ALBERT, ANGUS C. NAIRN, AND PAUL GREENGARDLaboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399

Contributed by Paul Greengard, June 26, 1987

ABSTRACT The 87-kDa protein, a major specific sub-strate for protein kinase C, has been purified 500-fold toapparent homogeneity from bovine forebrain supernatant. Thepurification procedure included batch adsorption to DE-52(DEAE-cellulose), (NH4)2SO4 precipitation, and chromatogra-phy on DEAE-Sephacel, Bio-Gel HTP (hydroxylapatite), Seph-acryl S-400, and fast protein liquid chromatography ProRPC.The amino acid composition was notable for its high proportionof alanine (28.6 mol%) and its enrichment in glutamate/glutamine (18.1 mol%), glycine (12.6 mol%), and proline(11.3 mol%). The partial specific volume was 0.702 ml/g; theStokes radius and sedimentation coefficient were 85 A and 2.11S, respectively. Although the relative molecular mass of theprotein on NaDodSO4/8% PAGE was 87-90 kDa, the molec-ular mass as determined from the above values was 68 kDa. Thefrictional ratio was 3.2, and the axial ratio was 60, indicatingthat the 87-kDa protein is an extremely elongated monomer.The purified 87-kDa protein was phosphorylated by purifiedprotein kinase C to a stoichiometry of 2.2 mol of 32p per molof 87-kDa protein (calculated using a value of 68 kDa for themolecular mass). Phosphorylation was exclusively on serineresidues.

The 87-kDa protein was first described as a component of ratbrain synaptosomes (1), and we have since carried out anumber of additional studies of this protein. Phosphorylationof the 87-kDa protein was found to be increased (i) in intactrat brain synaptosomes by depolarization-induced calciuminflux (1, 2), (ii) in rat brain synaptosomes by phorbol estersthat are active in stimulating protein kinase C (2, 3), and (iii)in adrenal chromaffin cells by acetylcholine and phorbolesters (4). Increased phosphorylation of the 87-kDa proteinwas also shown to correlate with increased calcium-depen-dent, evoked neurotransmitter release in response to phorbolester treatment of rat brain synaptosomes (3). The 87-kDaprotein was shown to be phosphorylated by protein kinase Cin vitro in crude (1, 5-8) and purified (7) preparations frombrain (1, 5-8) and a number of other tissues (8). The 87-kDaprotein has been shown to have a widespread species, tissue,and subcellular distribution (8).The 87-kDa protein has also captured the interest of a

number of other research groups, especially those ofBlackshear (9-12), Rozengurt (13-17), Takai (18), and Hunt-er (19) (for review, see ref. 20). It has been widely used as amarker for the activation of protein kinase C in fibroblasts byphorbol esters (9-13, 16-19, 21-25), synthetic diacylglycerols(10, 14, 18), exogenous phospholipase C (13), serum (15, 21),transformation (24), and a number of peptides and growthfactors (10, 13, 17-19, 21, 22, 25). The 87-kDa protein has alsobeen shown to be phosphorylated in lymphocytes in responseto treatment with concanavalin A and calcium (26). Phos-

phorylation of the 87-kDa protein by protein kinase C in vitrohas been observed in nerve growth cones (27) and in responseto activation of protein kinase C by a protein modulator (28).The widespread distribution and prominent phosphoryl-

ation of the 87-kDa protein make it a likely mediator of amajor action of protein kinase C. As a further step towardscharacterization of the 87-kDa protein, we report here itspurification from bovine brain and its physicochemical prop-erties.

MATERIALS AND METHODSMaterials. Fresh calf brains were obtained from a local

slaughterhouse. Protein kinase C was purified to homogene-ity from bovine forebrain by modification of the procedure ofKikkawa et al. (29). Antiserum against the 87-kDa proteinwas prepared as described (8).Antifoam A, ATP, bovine serum albumin, CaC12, bovine

erythrocyte carbonic anhydrase, Coomassie brilliant blueR-250, bovine heart cytochrome c, dimethyl sulfoxide,dithioerythritol, 1,2-diolein, EGTA, H1 histone, Mr proteinstandards for PAGE, NaDodSO4, bovine brain L-a-phos-phatidyl-1-serine, and Tris were from Sigma. Ethylene gly-col, formic acid, glycerol, KP1, Mg(C2H302)2, NaCl, NaN3,and sucrose were from Fisher. DEAE-Sephacel, fast proteinliquid chromatography (FPLC) system and ProRPC 5/10column, gel filtration calibration kit for high-Mr proteins, andSephacryl S-400 Superfine were from Pharmacia.Other reagents were purchased from the following sources:

DE-52 DEAE-cellulose (Whatman); Bio-Gel HTP hydroxyl-apatite (Bio-Rad); EDTA and 2-mercaptoethanol (Baker);Ultrapure (NH4)2SO4 (Bethesda Research Laboratories);[y-32P]ATP and 251I-labeled protein A (New England Nucle-ar); antipain, chymostatin, leupeptin, and pepstatin (Chem-icon, El Segundo, CA); Trasylol (FBA Pharmaceuticals,New York); phenylmethylsulfonyl fluoride (Calbiochem-Behring); isopropyl alcohol (Burdick and Jackson, Muske-gon, MI); trifluoroacetic acid (CF3COOH) (Pierce); nitrocel-lulose (Schleicher & Schuell); and nonfat dry milk (Carna-tion).

Buffers. All buffers were cold (0-40C), and contained 14mM 2-mercaptoethanol, 0.02% NaN3, and the followingprotease inhibitors: 25,000 kallikrein inhibitor units ofTrasylol per liter of buffer, 100 AM phenylmethylsulfonylfluoride (plus 0.1% isopropyl alcohol to dissolve thephenylmethylsulfonyl fluoride), 0.001% leupeptin, 0.001%antipain, 0.0002% pepstatin, and 0.0017% chymostatin (plus0.1% dimethyl sulfoxide to dissolve the pepstatin and chymo-statin). Additional buffer components were as follows: bufferA: 10 mM Tris-HCl, pH 7.6/2 mM EDTA/0.5 mM EGTA;buffer B: 10 mM Tris HC1, pH 7.6/1 mM EDTA; buffer C:buffer B with 10% ethylene glycol; buffer D: 25 mM Tris HC1,pH 7.6/1 mM EDTA/300 mM NaCI/10% ethylene glycol.

Abbreviations: FPLC, fast protein liquid chromatography;CF3COOH, trifluoroacetic acid.

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Standard Phosphorylation Assay for the 87-kDa Protein.The standard reaction mixture (final volume 100 p.l) con-tained 20 mM Tris-HCI, pH 7.4/10 mM Mg(C2H302)2/1 mMEGTA/2 mM dithioerythritol/10 pug leupeptin/10 pug an-tipain/1.5 mM CaC12/5 tkg of phosphatidylserine/0.2 ttg ofdiolein/3 ng of protein kinase C (specific activity, 2 ,tmol of32p incorporated per min per mg of protein with H1 histoneas substrate), and an aliquot of sample containing the 87-kDaprotein. The reaction was initiated by the addition of [y-32P]ATP (final concentration 10 kLM; specific activity S x 106cpm/nmol) and was continued for 30 min at 30'C. Thereaction was terminated by the addition of 20 ,ul ofNaDodSO4-stop solution [20% (vol/vol) glycerol/10%(wt/vol) NaDodSO4/10% (vol/vol) 2-mercaptoethanol/0.25M Tris HCl, pH 6.7, containing a trace of Pyronin Y] andheating in a boiling water bath for 5 min. NaDodSO4/8%PAGE was performed according to the method of Laemmli(30). Gels were stained with Coomassie brilliant blue R-250[0.1% in 50% (vol/vol) methanol/10% (vol/vol) acetic acid]and destained in 30% methanol/10% acetic acid, or werestained with silver by the procedure of Poehling and Neuhoff(31), with the silver reagent of Ohsawa and Ebata (32). Gelswere dried onto filter paper in vacuo, and were subjected toautoradiography at room temperature or at -70°C (withDuPont Cronex Lightning Plus intensifying screens) usingKodak XAR-5 x-ray film. Bands containing 87-kDa proteinwere excised from the gels, and the radioactivity was quan-titated by liquid scintillation counting.

Immunoblotting of the 87-kDa Protein. Immediately follow-ing NaDodSO4/8% PAGE, proteins were transferred ontonitrocellulose sheets (pore size, 0.2 ,um) at 200 mA for 12 hrusing the buffer system of Towbin et al. (33). All immuno-blotting steps were performed at room temperature in blottingbuffer (34) [20 mM Tris HCl, pH 7.4/5% (wt/vol) nonfat drymilk/0.02% NaN3/0.01% Antifoam A] as follows: 1 hr inbuffer, 2 hr in buffer with a 1:200 dilution of 87-kDaantiserum, 3 x 20 min in buffer, 12 hr in buffer with 1251.labeled protein A (2000 cpm/10 ,ul buffer), and Sx 20 min inbuffer. The sheets were air dried and were subjected toautoradiography as described above. Bands containing 87-kDa protein were excised from the sheets, and the radioac-tivity was quantitated by counting in a Micromedic SystemsAutomatic gamma counter. Quantitative immunoblottingwas accomplished by including a standard curve of purified87-kDa protein (50-250 ng) on each gel.

Protein Determinations. Protein was determined by themethod of Peterson (35) using bovine serum albumin asstandard.Amino Acid Analysis. Purified 87-kDa protein was dialyzed

extensively against H20. Amino acid analysis was done byDonna Atherton of The Rockefeller University Protein Se-quencing Facility as follows: 2 and 4 ,ug of protein werehydrolyzed for 10, 24, and 72 hr each. Amino acids werederivatized, separated, and identified as described (36). Fordetermination of cysteine, 10 jig of 87-kDa protein wassubjected to performic acid oxidation (37) followed by aminoacid analysis. A blank contained 10 ,ug of 87-kDa proteintreated with formic acid only, followed by amino acidanalysis.

Determination of Stokes Radius by Gel Filtration. Gelfiltration was by the method ofWhittaker (38) in a column (2.5x 90 cm) of Sephacryl S-400 previously equilibrated withbuffer D. The column was calibrated with the followingstandards: bovine thyroid thyroglobulin (85.0 A), horsespleen ferritin (61 A), bovine liver catalase (52.2 A), andrabbit muscle aldolase (48.1 A). Stokes radius of the 87-kDaprotein was determined according to the method of Laurentand Killander (39).

Determination of Sedimentation Coefficient by Linear Su-crose Density Gradient Ultracentrifugation. Ultracentrifuga-

tion was done at 45,000 rpm for 21 hr at 40C in a BeckmanL3-50 ultracentrifuge using an SW 50.1 rotor. Samples plusstandards (150 p.1) were layered onto 5-ml linear sucrosegradients (5-20%) that were made in 50 mM Tris HCI, pH7.6/0.2 mM EDTA/50 mM NaCI/14 mM 2-mercaptoethanol.The standard proteins were as follows: bovine serum albumin(4.3 S), bovine erythrocyte carbonic anhydrase (2.85 S), andbovine heart cytochrome c (1.83 S). After centrifugation,0.25-ml fractions were collected and analyzed by the standardphosphorylation assay followed by NaDodSO4/13% PAGEand protein staining. The sedimentation coefficient wasdetermined by the method of Martin and Ames (40).Time Course and Stoichiometry of Phosphorylation of the

87-kDa Protein. The standard phosphorylation assay wasperformed, except that the reaction mixture contained 1.5 Iugof purified 87-kDa protein, 80 ng of protein kinase C, and 200,uM [y-32P]ATP (specific activity 0.2 x 106 cpm/nmol). Thetubes were preincubated for 1 min at 30°C, and the reactionwas stopped after various times from 20 sec to 70 min. Thephosphorylated amino acid was identified as described (41,42).

RESULTSPurification of the 87-kDa Protein from Bovine Forebrain.

All purification steps were performed at 0-4°C. Each cen-trifugation was in a Sorvall RC-5B centrifuge, using a GSArotor.

Step 1. Homogenization: One kilogram of forebrain washomogenized in 4 vol of buffer A for 60 sec with a Polytrongrinder; the homogenate was then centrifuged for 45 min at27,000 x g, and the pH of the clear red supernatant wasadjusted from 6.8 to 7.6 with 1 M NaOH.

Step 2. DE-52 batch adsorption: The supernatant wasstirred for 1 hr with 1 liter ofDE-52 that had been equilibratedwith buffer A. The resin was washed on a Buchner funnelwith 2 liters of buffer A; then it was stirred for 1 hr in 1 literof buffer A plus 0.5 M NaCl. The suspension was filtered ona Buchner funnel followed by a 1-liter wash with bufferA plus0.5 M NaCl.

Step 3. (NH4)2SO4 precipitation: The pale yellow filtratewas taken to 30% saturation with solid (NH4)2SO4 and stirredfor 1 hr. The sample was then centrifuged for 30 min at 16,000x g, and the supernatant was taken to 60% saturation withsolid (NH4)2SO4 and stirred for 1 hr. After centrifugation for30 min at 16,000 x g, the pellet was resuspended in 250 ml ofbuffer B and dialyzed overnight against 2x 5.5 liters of bufferB.

Step 4. DEAE-Sephacel chromatography: After a 20-min,16,000 x g centrifugation to remove particulate impurities,the dialyzed sample was applied at 90 ml/hr to a DEAE-Sephacel column (2.5 x 25 cm) previously equilibrated withbuffer B. After a 125-ml wash with buffer B, the 87-kDaprotein was eluted with a 1-liter linear gradient of 0-500 mMNaCl in buffer C. The 87-kDa protein eluted in a broad peakbetween 150 and 250 mM, with the apex between 180 and 200mM NaCl.

Step 5. Bio-Gel HTP chromatography: The pooled sample(140 ml) from the DEAE-Sephacel column was applieddirectly at 120 ml/hr to a Bio-Gel HTP column (2.5 x 25 cm)previously equilibrated in buffer C. After washes with 125 mleach of buffer C, buffer C plus 1 M NaCl and 5 mM MgCl2,and buffer C, the 87-kDa protein was eluted with a 1-literlinear gradient of 0-350 mM KPi in buffer C. The 87-kDaprotein eluted in a sharp peak at 150 mM KP1.

Step 6. Sephacryl S-400 chromatography: The proteins inthe pooled Bio-Gel HTP sample (80 ml) were precipitatedwith (NH4)2SO4 (65% saturation) and resuspended in 5 ml ofbuffer D. The sample was applied to a Sephacryl S-400column (2.5 x 90 cm) previously equilibrated in buffer D, and

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the 87-kDa protein was eluted with buffer D. One major peakof 87-kDa protein was eluted at 300 ml, and one minor peak(about 30% of the total) was eluted at 360 ml. Furtherpurification was done on the major peak.

Step 7. FPLC ProRPC chromatography: The pooledsample (40 ml) from the Sephacryl S-400 column was dialyzedagainst 3 x 6 liters of 10 mM NH3HCO3 plus proteaseinhibitors. The sample was concentrated to 15 ml under N2 inan Amicon Ultrafiltration unit fitted with a PM 10 membrane.This sample was split into 5 x 3-ml aliquots. Each aliquot wasdiluted with 9 ml of H20/0.1% CF3COOH, filtered through aMillex-GV 0.22-,um filter unit (Millipore), and applied at 18ml/hr to an FPLC ProRPC 5/10 column previously equili-brated in H20/0.1% CF3COOH. After the column waswashed with 12 ml of H20/0.1% CF3COOH, the 87-kDaprotein was eluted at 12 ml/hr with a 40-ml linear gradient of0-50% isopropyl alcohol/0.1% CF3COOH. The pH of eachfraction was adjusted to -8 as it was collected. The 87-kDaprotein eluted as a pure protein in a sharp peak at about 23%isopropyl alcohol/0. 1% CF3COOH. The fractions containingthe purified 87-kDa protein were pooled, the volume wasreduced in a Speed Vac concentrator (Savant), the samplewas dialyzed extensively against a buffer of 20 mM Tris HCl,pH 7.6/14 mM 2-mercaptoethanol containing protease inhib-itors, and the final sample was stored at 0-40C. The purifiedsample was stable at 0-40C, whereas it was partially degradedafter any freeze/thaw procedure tested.The purification procedure for the 87-kDa protein is

summarized in Table 1. The protein was purified 500-foldfrom the crude supernatant. Fig. 1 shows the purified 87-kDaprotein after Coomassie blue staining (lane 1), and silverstaining (lane 2), and an autoradiogram of the protein afterphosphorylation by protein kinase C (lane 3). In each casethere was a single band which electrophoresed with anapparent molecular mass of 90 kDa on these NaDodSO4/8%polyacrylamide gels.We have also described a membrane-bound fraction of the

87-kDa protein (8). Upon extraction from the particulatefraction with a nonionic detergent such as 1% Triton X-100,the extracted 87-kDa protein behaved similarly to the solubleprotein on DE-52 and Bio-Gel HTP chromatography (K.A.A.and J. K. T. Wang, unpublished results). It will be of interestto compare further the soluble and membrane-bound 87-kDaprotein.Amino Acid Analysis. The amino acid composition of the

87-kDa protein is shown in Table 2. The composition wasremarkable for its very high content of alanine (28.6 mol%)and its high content ofglutamate/glutamine (18.1 mol%). Theprotein was also found to be rich in glycine (12.6 mol%) andin proline (11.3 mol%). The partial specific volume calculatedfrom the amino acid composition was 0.702 ml/g.Molecular Mass Determination. It was important to deter-

mine the molecular mass of the 87-kDa protein, because therelative molecular mass on NaDodSO4/PAGE has beenreported to vary with acrylamide concentration (11). TheStokes radius of the major peak on the Sephacryl S-400column was calculated to be 85 A (identical value for two

90- _m UMb

Origin

116- 97

67

- 43

1 2 3

FIG. 1. NaDodSO4/8% polyacrylamide gel stained with Coomas-sie brilliant blue (lane 1) showing the purified 87-kDa protein (4 ,ug)and an autoradiogram (lane 3) showing phosphorylation of the samesample by protein kinase C. The standard phosphorylation assay wasperformed, except that the reaction mixture contained 85 mMTris HCI and 4 ng of protein kinase C, and the reaction time was 2min. Another NaDodSO4/8% polyacrylamide gel stained with silver(lane 2) shows the purified 87-kDa protein (1 ,ug). Molecular sizemarkers are in kDa.

determinations) (Table 3), which for a typical globular proteinwould correspond to a molecular mass of 669 kDa. Thesedimentation coefficient of the major Sephacryl S-400 peakwas calculated to be 2.11 + 0.05 S (n = 3) (Table 3), whichfor a typical globular protein would correspond to a molec-ular mass of about 16 kDa. The Stokes radius, sedimentationcoefficient, and partial specific volume were used to calculatethe molecular mass of the 87-kDa protein according toequation 1 of Siegel and Monty (43); a molecular mass of 68kDa for a monomer was calculated (Table 3). The frictionalratio (f/fo) was calculated by equation 2 of Siegel and Monty(43) to be 3.2 (Table 3), which corresponds to an axial ratioof60 for a prolate ellipsoid (44) (Table 3). All ofthese data areconsistent with a very asymmetric, highly elongated tertiarystructure.The same properties were examined for the minor Seph-

acryl S-400 peak. The Stokes radius was calculated to be 56A (identical value for two determinations), which for a typicalglobular protein would correspond to a molecular mass ofabout 320 kDa. The sedimentation coefficient was calculatedto be 2.01 ± 0.25 S (n = 3), which for a typical globularprotein would correspond to a molecular mass of about 14kDa. Using these values, a molecular mass of 43 kDa for a

monomer was calculated. The frictional ratio was calculated

Table 1. Purification of the 87-kDa protein from 1 kg of bovine brain

Volume, Protein, 87-kDa protein,* Purity, Purification,Purification step ml mg mg % -fold Yield, %

1 27,000 x g supernatant 4,000 20,400 35 0.2 1 1002 DE-52 2,250 7,200 10 0.15 1 293 (NH4)2SO4 precipitation 250 3,500 6.3 0.2 1 184 DEAE-Sephacel 140 800 5.0 0.6 3 145 Bio-Gel HTP 80 33 1.6 5.0 25 4.66 Sephacryl S-400 40 8 0.4 5.0 25 1.17 FPLC, ProRPC 5/10 7.5 0.3 0.3 100 500 0.9

*Determined by quantitative immunoblotting.

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Table 2. Amino acid composition of the 87-kDa protein

Amino acid Mol %

AsxGIxSer*GlyHisArgThr*AlaProTyr

ValtMetCysIletLeutPheLysTrp

4.118.19.412.60.20.72.0

28.611.30.23.00.21.80.00.61.38.8NDt

*Obtained by extrapolation to zero time.tValue at 72 hr.:tND, not determined.

to be 2.45, which corresponds to an axial ratio of 33 for aprolate ellipsoid. These data are also consistent with anasymmetric, elongated tertiary structure.

Stoichiometry of Phosphorylation. The stoichiometry ofphosphorylation by protein kinase C of the purified 87-kDaprotein (using a value of 68 kDa for the molecular mass) wascalculated to be 2.2 mol Of 32p incorporated per mol of87-kDaprotein after 70 min (Table 3). The addition ofanother aliquotof kinase at 50 min did not increase significantly the stoichi-ometry of phosphorylation at 70 min. The only amino acidresidue phosphorylated by protein kinase C was serine (Table3).

DISCUSSIONThe 87-kDa protein, a major, specific substrate for proteinkinase C, was purified to apparent homogeneity from bovineforebrain supernatant (Table 1, Fig. 1). Preparation of anapparently homogeneous protein with only a 500-fold puri-fication indicated that the 87-kDa protein is highly abundantin brain, constituting 0.2% of total soluble protein. Thepurification procedure included batch adsorption to DE-52cellulose, (NH4)2SO4 precipitation, and chromatography onDEAE-Sephacel, Bio-Gel HTP, Sephacryl S-400, and FPLCProRPC (Table 1). The DE-52 and (NH4)2SO4 precipitationsteps did not result in any overall fold-purification but werenecessary for the efficient use of the DEAE-Sephacel step.The Sephacryl S-400 step removed certain minor impurities

that were not removed by the FPLC step. The FPLC ProRPCcolumn provided a high degree of purification (20-fold), butthe chromatography also exposed the protein to acid condi-tions (pH 2.5 for about 3 hr) and an organic solvent (up to 23%isopropyl alcohol). Therefore, each property of the 87-kDaprotein was examined with the sample considered to be mostappropriate. The Stokes radius and sedimentation coefficientwere determined on the Sephacryl S-400 peaks. At this stageof the preparation the sample had not been exposed to anydenaturing agents (heat, acid, detergents, or organic sol-vents), so that the values obtained should reflect the nativeconformation of the protein. Phosphorylation by proteinkinase C was examined with the FPLC-purified pool, becausethe purified preparation was free of endogenous kinaseactivities.The hydrodynamic properties of the 87-kDa protein were

as follows: for the major Sephacryl S-400 peak, a Stokesradius of 85 A, sedimentation coefficient of 2.11 S, andfrictional ratio of 3.2; for the minor Sephacryl S-400 peak, aStokes radius of 56 A, sedimentation coefficient of 2.05 S,and frictional ratio of 2.45. The molecular mass calculatedfrom these properties was about 68 kDa for the major peakand about 43 kDa for the minor peak, although they electro-phoresed with the same apparent molecular mass onNaDodSO4/PAGE. The significance of the minor peak isunclear. The sedimentation coefficient, phosphorylationcharacteristics, and behavior on other chromatographic sys-tems were similar for both Sephacryl S-400 peaks (presentwork; K.A.A., unpublished results). The two peaks possiblyrepresent subpopulations of the 87-kDa protein that havestructural differences. It is less likely that the major peakrepresents a dimer of87-kDa protein, whereas the minor peakrepresents a monomer. In this case one would expect a

significant difference in the sedimentation coefficient and a

greater difference in the calculated molecular mass. Thus,while the difference between the Sephacryl S-400 peaks maybe of interest, the majority of the 87-kDa protein behaved as

a very asymmetric, highly elongated monomer with a calcu-lated molecular mass of 68 kDa.The amino acid composition (Table 2) ofthe 87-kDa protein

was notable for a very high content of alanine (28.6 mol%)and a high content of glutamate/glutamine (18.1 mol%), themajority of which is likely to be glutamate, because the87-kDa protein has an acidic pI (multiple forms varying from4.4 to 4.9) (8). The protein was also found to be rich in glycine(12.6 mol%) and proline (11.3 mol%). Together, alanine,glutamate/glutamine, glycine, and proline comprised 70% ofthe protein.The purified 87-kDa protein was phosphorylated by protein

kinase C to a stoichiometry of2.2 mol of32P per mol of87-kDaprotein (calculated using a molecular mass of 68 kDa),

Table 3. Summary of physical and chemical properties of the 87-kDa protein

Property Method of determination Value

Molecular mass NaDodSO4/8% PAGE 87-90 kDaMolecular mass Stokes radius, sedimentation coefficient,

and partial specific volume 68 kDaStokes radius (a) Gel filtration 85 ASedimentation coefficient (s20.w) Sucrose density gradient centrifugation 2.11 SFrictional ratio (f/f0)* Stokes radius, partial specific volume

and molecular weight 3.2Axial ratiot Frictional ratio 60Amino acid composition High Ala/Glx/Gly/ProPartial specific volume (v) Amino acid composition 0.702 ml/gPhosphoamino acid Phosphorylation by protein kinase C SerineStoichiometry of phosphorylation Phosphorylation by protein kinase C 2.2

*Calculated according to Siegel and Monty (ref. 43).tValue for a prolate ellipsoid (ref. 44).

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exclusively on serine residues (Table 3). Thus there were twoor three serine residues phosphorylated by protein kinase C.Two other major endogenous substrates for protein kinase

C have been examined in detail. A platelet protein of Mr40,000 was first described by the laboratory of Nishizuka (forreview, see ref. 45), was purified by Imaoka et al. (46), andhas recently been identified by Connolly et al. (47) as theinositol trisphosphate 5'-phosphomonoesterase that hydro-lyzes inositol 1,4,5-trisphosphate. A brain protein, most oftenreferred to as B-50 (48), F1 (49), or GAP-43 (50), has beenpurified by Zwiers et al. (48), Chan et al. (49), and Benowitzet al. (51), and has been hypothesized to modulate the activityof phosphatidylinositol 4-phosphate kinase (52). Whether the87-kDa protein also has a function related to the modulationof phosphoinositide metabolism remains an interesting ques-tion.

We thank Dr. James Wang, Dr. S. Ivar Walaas, and Dr. AndrewCzernik for assistance and advice; and Dr. Perry Blackshear forhelpful discussions. This work was supported by U.S. Public HealthService Grant MH-39327.

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