Purification and Properties of Membrane and Cytosolic ...

THEJOURNAL

OF

Vol . 266, No. 18, Issue of June 25, pp , 11495-11501,1991 Printed in U.S.A.

BIOLOGICAL CHEMISTRY

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Properties of Membrane and Cytosolic Phosphatidylinositol-specific Phospholipases C from Human Spleen* (Received for publication, November 2, 1990)

Garbiiie Roy, Luisa M. Villar, Iciar Lazaro, Miguel Gonzalez, Alfred0 Bootello, Pedro Gonzalez-PorqueS From the Servicio de Znmunologia, Hospital Ramon y Cajal, Carretera de Colmenar K m 9.1, Madrid 28034, Spain

Two phosphatidylinositol-specific phospholipases C (PI-PLC) have been purified from human spleen. PIPLC, represents the main activity detected in the membrane, while PI-PLC, is the main activity present in the cytoplasm. PI-PLC, can be resolved into two peaks of activity of high M, (60,000-70,000) and low M. (16,000-18,000). High salt concentration ((NH4)2S04,2 M) dissociates the high M , form yielding the low molecular form and increasing the specific activity. The same effect of dissociation and potentiation of the activity is observed when membranes solubilized by n-octyl glucoside are subjected to the high voltage conditions of an isoelectric focusing run. The purified Pi-PLC, has a M , of about 18,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration and a basic PI (9.09.2). Purified PI-PLC, has a M, of 57,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration) and a slightly acid PI (6.2). Other characteristics of both enzymes, such as cations dependence, substrate specificity, optimum pH, and kinetic parameters, are also discussed.

thus implying the presence of a regulatory component blocking PI-PLC activity. Other authorshave reported the presence of the same PI-PLC in soluble and particulate forms as a possible mechanism of regulation for its activity, as has been described for protein kinase C (Nishizuka, 1984, 1986; Lee and Bell, 1986). Such PI-PLC found both in particulate and soluble forms have been reported for bovine brain (Lee et al., 1987) and guinea pig uterus (Bennet and Crooke, 1987) but have not been observed in human platelets (Baldassare et al., 1989; Banno et al., 1987; Banno and Nozawa, 1987) or pig lymphocytes (Carter and Smith,1987). Inthis paper, we report the presence in human spleen membranes of a basic, low molecular weight PI-PLC, which has not been described before. The enzyme shows very little activity when associated to another membrane component, not yet characterized, and high activity after dissociation from it. We also report that the main activity present in the cytoplasm from human spleen has physicochemical characteristics different from the membrane-bound enzyme. These results imply that both enzymes play different roles and are subjected to different modes of regulation for their activity. MATERIALS AND METHODS~

PI-PLC’ is a key component of the signal transducing system activated by the effect of different stimuli in a wide variety of cells (Berridge, 1987; Hirasawa and Nishizuka, 1985; Hokin, 1985). Although many efforts have been made in order to obtain a clear picture of the sequence of events between the recognition of the agonist and phospholipid breakdown, the triggering mechanism for PI-PLC remains to be elucidated. Recently, the involvement of a proteintyrosine kinase in the system as one of the early events in the activation of PI-PLC (Nishibe et al., 1990; Wahl et al., 1988) or the possible implication of G proteins regulating the system (Banno et al., 1986, 1987; Banno and Nozawa, 1987; Wahl et al., 1988) has been postulated. Both models require that PIPLC, in the cell resting state, should be in a inactive form,

* This work was supported by a research grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social and Caja de Ahorros de Madrid. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertkement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence and reprint requests should be addressed. The abbreviations used are: PI-PLC, phosphatidylinositol-specific phopholipases C; PI-PLC,, activity detected in the membrane; PI-PLC,, activity detected in the cytoplasm; PC-PLC, phosphatidylcholine-specific phospholipases C; HPLC, high performance liquid chromatography; FPLC,fastprotein liquid chromatography; IEF, isoelectric focusing; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

RESULTSANDDISCUSSION

Detection of Membrane-associated PI-PLC Two methods wereemployed in order to ascertain the presence of PI-PLC associated to theparticulate fraction. Sepharose 2B Gel Filtration of Crude Membranes-Fig. 1 shows that around 10-15% of the total PI-PLC detected in crude membranes is recovered in the void volume where the membrane marker alkaline phosphatase is also present. A PC-PLC is also detected although thisenzyme wasnot further investigated. The majority of the PI-PLC detected in the crude membrane corresponds to cytoplasmic PI-PLC, and can be separated from the membrane-bound PI-PLC,. Successive Washes of Membranes-Fig. 2 shows the effect of successive washes on the distribution of PI-PLC between the membrane fraction andthe 100,000 X g supernatant. After the third wash, the supernatant obtained has no detectable PI-PLC, implying that the membrane PI-PLC is not a contaminant from the cytoplasmic PI-PLC. Detergent Solubilization of PI-PLC, The following detergents were found to inhibit the assay for PI-PLC, at a concentration above 0.01%: Triton X-100, ‘Portions of this paper (including “Materialsand Methods,” Tables 1 and 2, and Miniprint Figs. 1-3) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

11495

Phospholipases C from Human Spleen

11496

Nonidet P-40, Lubrol, Brij 30, and sodium dodecyl sulfate (SDS), andwere therefore excluded from solubilization studies; n-octyl glucoside or sodium cholate did not interfere with the PI-PLC, assay up to a concentration of 0.2% and were considered as good solubilizing agents for the enzyme with a maximum of effectivity at 1%final concentration. In all cases, the buffer used was 0.01 M Tris-HC1, pH 7.8. Purification of Membrane PI-PLC (PI-PLC,) Two procedures have been used for the purification of PIPLC, depending on the detergent utilized for the solubilization (see "Miniprint"). As will be discussed later, the enzyme solubilized with sodium cholate and the enzyme solubilized with octyl glucoside share properties such as isoelectric point or molecular weight, but differ in the kinetic properties and calcium dependence, most probably due to the influence of detergents on the binding of different metals. Purification of Cytoplasmic PI-PLC (PI-PLC,) The purification procedure of PI-PLC, is shown in the Miniprint. Although two PI-PLC were detected, only one of them (M, = 56,000), which represents the predominant form of PI-PLC, in all the preparations so far tested, was investigated.

Characteristics of PI-PLC, and PI-PLC, Molecular Weight of PI-PLC,-One of the most relevant features of PI-PLC, is the shift in the molecularweight observed by gel filtration when the enzyme is subjected to different treatments. As shown in Fig. 3A, PI-PLC, solubilized with sodium cholate behaves as a 60-70-kDa protein during the early stages of purification. When a hydrophobic chromatography step on octyl-Sepharose was introduced in the purification procedure (high salt concentration ((NH4)*SO4,2 M) is required in order to increase the hydrophobicity and thus thebinding of the protein to the column), the material desorbed by decreasing the ionic strength (10 mM Tris-HC1, pH 7.4) yields two peaks of activity upon gel filtration (60-70 kDa and 15-20 kDa) (Fig. 3B). If the high molecular weight peak is rechromatographed on octyl-Sepharose and thematerial desorbed analyzed by gel filtration, the results obtained are shown in Fig. 3C, where it can be observed that all the activity has shifted to low molecular weight. We interpret this result dissociation as of the PI-PLC, from some other membrane component to which it is bound by ionic forces. This interpretation is reinforced by the fact that the total activity recovered in the hydrophobic chromatography step is 5-10 times higher than the startingactivity, implying c.p.rn In on0

A 280nm

diacyigiyceroi(DAG) in c.p.m 1400 I

1

am

A

A 215nrn

\now

at-o T

800 400 200 0

0

10

20

50

40

30

em

fractions

FIG. 1. Gel filtration on Sepharose 2B.Sample: 15 ml(30 mg/ ml) of crude membranes obtained as described in the Miniprint. Column: Sepharose 2B (50 X 5 cm) equilibrated in 10 mM Tris-HC1, pH 7.8. Flow rate, 50 ml/h; fractions, 10 ml. Protein/A2m nm, -; alkaline pho~phatase/A,,,~", - - -; PI-PLC/cpm, A-A, PC-PLC/ cpm, W.

L/

PI-PLC ~

~~

120

il

4m 100

80 o

x)

20

30

40

w

rn

70

m

fractlon number 80

40

20

0

wash 1membranes wash

2

wash 3

FIG. 2. Distribution of PI-PLC activity between membrane fraction and 100,000 X g supernatant. Sample: crude membranes (see Miniprint). The membranes are subjected to successive washes with 10 mM Tris-HC1, pH 7.8, by centrifugation at 100,000 X g. The resulting pellets are resuspended in the same volume of buffer as the initial supernatant. PI-PLC activity was measured in each pellet and supernatant.

FIG. 3. A, PI-PLC, gel filtration on Sephacryl. Sample: 2 ml of PI-PLC, after hydroxyapatite chromatography, concentrated by lyophilization. Column: Sephacryl S-200 (80 X 2.5 cm) equilibrated in 10 mM Tris-HC1, 0.15 M NaC1, 0.2% Cholate, pH 7.5. Flow rate, 30 ml/h; 5-ml fractions. A215nm, -; PI-PLC/cpm, 0-0. B, PI-PLC, gel filtration on Sephacryl S-200. Sample: 2 mlof PI-PLC, after hydrophobic chromatography on octyl-Sepharose (Miniprint, Fig. 1B), concentrated by ultrafiltration. Column: Sephacryl S-200, in the same condition as in A . A215 ", -, PI-PLC/cpm, 0-0. C, rechromatography of the high molecular weight PI-PLC,. Sample: pooled fractions (peak I, B ) are adjusted to 2 M (NHdzSOI and applied to a column of octyl-Sepharose (2 X 0.5 cm) equilibrated in 2 M (NH,),SO,, 10 mM phosphate buffer, pH 7.0, and eluted in a batch with 10 mM phosphate buffer, pH 7.0. The material obtained is filtered through a Sephacryl S-200 column in the same conditions as in A. DAG, diacylglycerol.

Phospholipases Human C from

Spleen

11497

a regulatory role for the component bound to PI-PLC,. We have failed to reconstitute the high molecular weight complex by combining different fractions from the Sephacryl S-200 or to reproduce the phenomenon of dissociation of the active 18kDa PI-PLC, by incubation with GTP, ATP, or 7-thio-GTP of crude membranes or a t different stages of the purification procedure. Other authors(Margolis et al., 1989; Meisenhelder 600 et al., 1989; Nishibe et al., 1989, 1990) have described the phosphorylation of PI-PLC, as a mechanism of regulation of the enzyme. However, we have been unable to confirm this point. Another explanationfor the appearance of the 18-kDa PI-PLC, could be proteolysis in this particular step of the purification. This possibility is difficult to prove because the presence of protease inhibitors such as phenylmethylsulfonyl 0 6 10 15 20 26 fluoride and chelating agents inhibit the assay for PI-PLC,. Gel slice o.p.8. In DAG . 1000 For thisreason, we tried toreproduce the phenomenon under D ..........-.............,.. a different set of conditions. Fig. 4 shows the gel filtration of the enzyme solubilized with n-octyl glucoside and partially 800. purified by preparative isoelectrofocusing. As shown in the Miniprint, when the octyl glucoside-solubilized enzyme is 600 ' subjected to the effect of pH and voltage of the isoelectric -8. PI-PLC focusing run, the total activity recovered is around 10 times ...... DH 400 higher than in the startingmaterial, and themolecular weight of the PI-PLC, obtained by gelfiltration is again in therange of 15,000-20,000. These results confirm those obtained when 200 the enzyme is solubilized with sodium cholate and point toward a regulatory mechanism of the PI-PLC, by association-dissociation with other membrane components. 0 10 20 30 40 60 Molecular Weight of PI-PLC,-As shown in the Miniprint, fractions the purified cytoplasmic PI-PLC, behaves as a monomeric enzyme with a molecular weight of about 56,000. Isoelectric Point of PI-PLC,-During the early stages of purification, PI-PLC, solubilized with sodium cholate binds weakly to DEAE-cellulose, behaving as a slightly acidic protein. However, when dissociation has taken place following the octyl-Sepharose chromatography, the PI-PLC, (18 kDa) obtained does not bind to DEAE-cellulose (data not shown) and, when subjected to analytical electrofocusing (Fig. 5A), the main activity is located at a PI of about 9.0-9.5. If PIPLC, is solubilized with octyl glucoside, the enzyme does not bind toDEAE-cellulose and is recovered from the preparative FIG. 5. A, agarose IEF of PI-PLC, activity solubilized with sodium isoelectrofocusing step in a broad peak with PI 7.5-9.5 (Fig. cholate. Sample: peak active fractions eluted at low molelular weight 2, Miniprint). Further purification of the PI-PLC, solubilized from a Sephacryl S-200 column (Fig. lC, Miniprint) were concenwith octyl glucoside shows a PI of 9.0-9.2 when subjected to trated on a Centricon-10 and electrofocused on a pH gradient 3-10. The gel wascut into3-mm slices, and PI-PLC, activity was evaluated preparative chromatofocusing (Fig. 5B). Theseresults demonstrate that the active PI-PLC, (18 incubating each gel fragment directly in the assay mixture buffer '

1260

c.p.m. In DAQ

A 280nm

1000

0.00

760 0.04

600 0.02

260 0 0

10

20

30

40

time elution (mln)

FIG. 4. Gel filtration on TSK-3000 SW. Sample: PI-PLC, activity solubilized with octyl glucoside and focused in a preparative IEF at a basic PI (procedure 2, Miniprint) concentrated by ultrafiltration on a Centricon-10. Column: TSK-3000 SW (300 X 7.5 mm) equilibrated in 0.2 M phosphate buffer, 0.1% octyl glucoside, pH 7.5. Flow rate, 0.5 ml/min; 0.5-ml fractions. A ~ w", -; PI-PLC, A-A.

overnight. B, chromatofocusing on Mono-P HR 5/20. Sample: PIPLC, activity eluted at low molecular weight in Fig. 4. This material was dialyzed against 0.075 mM Tris-HCI, 0.1% octyl glucoside, pH 9.5, and absorbed onto a Mono-P HR 5/20 column equilibrated in the same buffer. The elution was performed in a pH gradient between 9.5 and 6, by means of Polybuffer 96 solution (diluted 1/10) and adjusted to pH6.0 with 1N HCL. C, agarose IEF of PI-PLC,. Sample: PI-PLC, from step 5 (Miniprint) was concentrated by ultrafiltration and electrofocused on a pH gradient 3-10, 90 min at 5 watts (constant).

kDa) behaves as a basic protein with a PI of about 9.0-9.5. The different behavior of the enzyme solubilized with sodium cholate or octyl glucoside on the first DEAE-cellulose chromatography can be explained by the fact that, in the case of solubilization with sodium cholate, the detergent bound to the protein increases the negative charges of the protein while octyl glucoside does not modify the charge characteristics of the enzyme. Isoelectric Point of PI-PLC,-Fig. 5C shows the analytical isoelectrofocusing of the purified material (see Miniprint) which inSDS-PAGE gives a single band of56kDa. The

Phospholipases Human C from

11498

protein moves as a doublet with a PI of 6.0-6.2 Substrate Specificity-Both PI-PLC, and PI-PLC, were specific for phosphatidylinositol andcould utilize phosphatidylinositol ( [2-'4C]linoleoyl) andphosphatidylinositol ( [2-14C] arachidonoyl) as substrates. Less than 5 % activity was found when phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid was used asa substrate. The productof the reaction was analyzed by high performance thin layer chromatography (HPTLC) and found to be in both cases diacylglycerol. No phospholipase AP activity was found in the purified enzymes. pH Optimum of PI-PLC,-As shown in Fig. 6A, PI-PLC, solubilized with sodium cholate exhibits a p H optimum of about 7.0 while the P1-PLC, solubilized with octyl glucoside has a pH optimum of about 8.0 (Fig. 623). This difference may reflect a differential effect of detergents on the stateof ionization of the enzyme or the interactionof the detergent with the buffer used in the study. pH Optimum of PI-PLC,-As shown in Fig. 6C, PI-PLC, activity has an optimump H of about 7.0. c.p.rn In DAG

700,

I

ooo 'I

7 1

01

Spleen

pH Stability-Both PI-PLC, and PI-PLC, arefairly stable proteins when incubated for short periods of time (30 min) to a wide range of pH. PI-PLC, shows a fall of about 20-30% activity below pH 5.0 and is stable to alkaline pH up to 11. We haveobserved duringtheearlystages of purification (before dissociation has occurred) a n increase in the activity when extracts are incubated at pH above 9, thus indicating that under these conditionsa partial dissociation does occur. CationDependence of PI-PLC,-Ca2+ requirement is a general property of mammalian PI-PLC. In general, if phosphatidylinositoldiphosphateisusedassubstrate, a lower concentration isneeded than with phosphatidylinositol phosphate or PI (Banno et al., 1987; Banno and Nozawa, 1987; Homma et al., 1988; Kamishaka et al., 1986; Rebecchi and Rosen, 1987; Ryu etal., 1987a, 1987b),but exceptions are also described presenting optimum activities with micromolarCa2+ al., 1988; concentrationsandPI as substrate(Hommaet Nakanishi etal., 1988). Fig. 7A shows the effect of increasing concentrations of Ca2+and M g + in the assay of PI-PLC, solubilized with sodium cholate. As can be observed, the enzymedoes not show any dependence on the presence of these cations. However,theinhibition by EDTAcanbereverted upon the addition of increasing concentrationsof Ca2+and Mg2+ in the assay (Fig. 7B). If the enzyme has been solubilized and purified with octyl glucoside (Fig. 7C), both Mg2+ and Ca2+ can activate the enzyme, reaching a maximum of activity at a concentration of 1 mM ca2+,50 p M Mg", or 0.1 mM Mn2+. These results indicate that care should be taken when analyzing the characteristics of an enzyme assayed in the presence of detergents. Ionic detergents can bind ions from the buffer or during the solubilization procedure changing the charge characteristics of the enzyme, thus affecting the interaction between the enzyme, substrate, and cofactors needed for the reaction. Cation Dependence of PI-PLC,-As shown in Fig. 70, PIPLC, presents a total dependence on the presenceof Ca2+ in the assay,which cannot be substituted by otherdivalent cations (M$+, Mn", Zn2+). An optimumconcentration of Ca2+has beenfoundbetween 0.5 and 1 mM, and higher concentrations are inhibitory. The need for this nonphysiological concentration of Ca2+for the enzyme to be activewith PI as substrate has also been reported by other authors for other PI-PLC obtained from different sources (Carter and Smith, 1987; ManneandKung, 1987; Bennetand Crook, 1987; Wilson et al., 1984; Fukui et al., 1988). Kinetic Parameters-The K, for PI-PLC, was found tobe about 5 p ~ while , a K,,, of 1.5 p~ was obtained for PI-PLC, (data not shown). Effect of -SH Reagents onPI-PLC,and PI-PLC,-SH reagents(dithiothreitol, 5,5'-dithiobis(2-nitrobenzoic acid, iodoacetamide) did notshow a significant effecton the activity of PI-PLC, or PI-PLC, when assayed in the range 10-3-10-6 M.

-

3

CONCLUDING REMARKS 4

6

8

7

8

0

1

0

1

1

pH

FIG. 6. pH optimum. A , PI-PLC, activity solubilized with sodium cholate. Sample: PI-PLC, activity from Sepbacryl S-200 (Miniprint). B, PI-PLC, solubilized with octyl glucoside. Sample: PIPLC,, activity from preparative isoelectrofocusing (Miniprint). C, PIPLC, was obtained from step 3 (Miniprint). Assay conditions: 0.25% deoxycholate was not included in thebuffer assay mixture because of its insolubility below pH 7.0. All buffers were used at a final concentration of 50 mM. Other conditions were as described under "Methods."

One of the main purposes of our work was to ascertain if the PI-PLC detected in the membrane fraction and the PIPLC detected in the cytoplasm were the sameenzyme, active both in soluble and particulate forms as has been described for PI-PLC in bovine brain (Lee et al., 1987) and human platelet (Bennet andCrooke, 1987). Our results do not exclude the possibility that in human spleen minor activitiesof PI-PLC could work both in soluble and particulate forms. We have detected, both in membrane and cytoplasm, other activities thathave not yet been inves-

Phospholipases Spleen Human C from

11499

c.p.m in DAG 760 600

400

600

300

200 260 100

0

0.OlmM OmM

O.lmM 0.06mM

0.6mM

1mM

c.p.m in DAG 3000

2600

2000

1600

1000

600

T

0

5m

1Om 5 0 ~ MO.1mM 0.5mM 1rnM 5rnM 10rnM

FIG. 7. Effect of metals on PI-PLC activity. A, PI-PLC, activity solubilized with sodium cholate (enzyme sample as in Fig. 6A); B, recovery of the PI-PLC, activity by increasing concentrations of Ca2+ and M e , after inhibition with chelating agents (0.5 mM EDTA). C, PI-PLC, activity solubilized with octyl glucoside (enzyme sample as in Fig. 6B; D,PI-PLC, activity. Sample: PI-PLC, obtained from step 5 (Miniprint).

tigated and purified. However, insofar as the main activities after the stimulation of the T cell receptor (Koretzky et al., 1990) in T lymphocytes deficient intyrosinephosphatase present in membranes and cytoplasm are concerned, they clearly demonstratethatbothactivitiesare different. PI- CD45. Another possibility to be considered is the existence of PIPLC, is a low molecularweight (18,000) basic protein (PI which dissociates to active 9.0-9.5) that seems to be associated a regulatory to component PLC, asaninactivetetramer by ionic interactions which can be dissociated to a regulatory monomer upon treatment withhigh salt concentration or the component by ionic interactions which can be dissociatedby effect of high voltage and pHof the isoelectric focusing run. high salt concentrations or by the effect of high voltage and REFERENCES p H of an isoelectric focusingrun. On the other hand, PI-PLC, Baldassare, J. J., Henderson, P. A,, and Fisher,G . J. (1989) Biochembehaves as a 56-kDa monomeric protein, which is slightly istry 28, 6010-6016 acidic (PI6.2) and which seems tobe regulated by the presence Banno, Y., and Nozawa, Y. (1987) Biochem. J . 248,95-101 of calcium in the reaction as has been described for other PI- Banno, Y., Nakashima, S., and Nozawa, Y.(1986) Biochem. Biophys. PLC (Hoffman and Majerus,1982; Kimura, 1987). Res. Commun. 136,713-721 The fact that PI-PLC,, when isolated in the high molecular Banno, Y., Nagao, S., Katada, T., Nagata, K., Ui, M., and Nozawa, Y.(1987) Biochem. Biophys. Res. Commun. 146,861-869 weight form (60,000-70,000) has very little activity and can be enhanced by dissociation suggests that the membrane PI- Bennett, C. F., and Crooke, S . T. (1987) J . Bid. Chem. 262, 1378913797 PLC is bound to a regulatory component which releases the Berridge, M. J. (1987) Annu. Reu. Biochem. 56, 159-193 active enzyme ( M , = 18,000) when the charge interactions Carter, H. R., and Smith, A. D. (1987) Biochem. J . 244,639-645 are disrupted. Thepossibility of a release mechanism involv- Fukui, T., Lutz, R. J., and Lowestein, J. M. (1988) J. Biol. Chem. ing phosphorylation/dephosphorylation of the enzyme cannot 263,17730-17737 Hirasawa, K., and Nishizuka, Y. (1985) Annu. Reu. Pharmacol. Torbe excluded. PI-PLC phosphorylated in tyrosine has been icol. 25, 147-170 described by several authors (Meisenhelderet al., 1989; NishHoffman, S. L., andMajerus,P. W. (1982) J . Biol. Chem. 257, ibe et al., 1990) and very recently it has been shown that 14359-14364 Genistein(aninhibitor of tyrosineproteinkinase) blocks Hokin, L. E. (1985) Annu. Reu. Biochem. 54, 205-235 phospholipid metabolism in lymphocytes stimulated by anti- Homma, Y., Imaki, J., Nakanishi, O., and Takanawa, T. (1988) J . CD:, (Mustelin et al., 1990). A similar phenomenon isobserved Biol. Chem. 263,6592-6598

11500


Kamishaka, Y., Toyoshima, S., and Osawa, T. (1986) Arch. Biochem. Bioohvs. 249., 669-578 --Katsimka, M , Gupta, C., and Goldman, A. S. (1986) Anal. Biochem. 154,676-681 Kimura, Y. (1987) J. Membr. Biol. 9 6 , 187-191 Koretzky, G. A., Picus, J., Thomas, M. L., and Weiss, A. (1990) Nature 346,66-68 Kuntz, F. (1973) Biochim. Biophys. Acta 2 9 6 , 331-334 Lee, K. Y., Ryu, S. H., Choi, W. C., and Rhee, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,5540-5544 Lee, M.-H., and Bell, R. M. (1986) J. Biol. Chem. 2 6 1 , 14867-14870 Manne, V., and Kung, H. F. (1987) Biochem. J . 2 4 3 , 763-771 Margolis, B., Rhee, S. G., Felder, S., Mervic, M., Lyall, R., Levitzki, A., Ullrich, A., Zilberstein, A., and Schlessinger, J. (1989) Cell 57, ~

1101-1107

~~"

Meisenhelder, J., Suh, P. G., Rhee, S. G., and Hunter, T. (1989) Cell 57, 1109-1122 Mustelin, T., Coggeshall, K. M., Isakov, N., and Altman, A. (1990) Science 247, 1584-1589 Nakanishi, O., Homma, Y., Kawasaki, H., Emori, I., Suzuki, K., and Takenawa, Y. (1988) Biochem. J. 256,453-459

Nishibe, S., Wahl, M. I., Rhee, S. G., andCarpenter, G . (1989) J. Biol. Chem. 264,10335-10338 Nishibe, S., Wahl, M. I., Wedegaertner, P. B., Kim, J. J., Rhee, S. G., and Carpenter,G. (1990) Proc. Natl. Acad. Sci. U. S. A. 8 7 , 424-428 Nishizuka, Y. (1984) Nature 308, 693-698 Nishizuka, Y. (1986) Science 233,305-312 Rebecchi, M. J., and Rosen, 0.M. (1987) J. Biol. Chem. 2 6 2 , 1252612532 Reinhold, J. G. (1983) in Methods of Clinical Chemistry (Seligron, D., ed) Vol. 1,p. 88, New York Academy Press, New York Ryu, S. H., Suh, P. G., Cho, K. S., Lee, K. Y., and Rhee, S. G. (1987a) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 6649-6653 Ryu, S. H., Cho, K. S., Lee, K.-Y., Suh, P. G., and Rhee, S. G. (198713) J. Biol. Chem. 2 6 2 , 12511-12518 Wahl, M. I., Daniel, T. O., and Carpenter, G. (1988) Science 241, 968-970 Wang, P., Toyoshima, S., and Osawa, T. (1988) J. Biochem. ( Tokyo) 103, 137-142 Wilson, D. B., Bross, Y. E., Hoffman, S. L., and Majerus, P. W. (1984) J. Biol. Chem. 259, 11718-11724

SUPPLBIIBNTARY UATERIAL ?C PURIFICATION AND PROPERTIES OF PI-PIC ACTIVITIES FROU HmvLN SPLEEN AUTH0RS:Garbiae Roy, Luisa 1. Villar,Iciar IAraro. Uiguel Gonzalez. Alfrado MATERIALS

llETnODS

PI-PIC assay W ~ Ba modification Of the assay described by Kataumate et a1.,1986 for phospholipase and is based on the different solubility of the substrate (phosphatidylinositol) and the product (diacylglyoerol) in hexane, and is performed an follows:

+

-.-

TO each tube, lpl (0.01Wi) Of the labelled Substrate Was added and the solvent (CHCl~ClZ,OH 1:l v/v)evaporated by flushing a stream of N Then 5pl of methanol were added to each tube in order to obtain a better di&ibution of the phospholipid at the bottom Of the tube. We have found that a oonsentration of methanol Up to 25 per Cent v/v is not inhibitory and produoes .Or0 reproducible result-. Finally 25pl Of the incubation mix (HCl-Ris 100mM pH 7.8, LWX 0 . 5 Cacl. 21wl and 20ul of the aamnle at the amronriate dilution were added and

*.

TABLE 1 PURfFICATION OF PI-PIC. SPLEEN I(EIIQRANES)

step

P In , , order t e iOfn increasing . sensibility

the following methods hava been used in thie rrtudy. Biuret (Reinhold, 1983). absorbanae at 280 nm, absorbance at 215 m , and flUOr'BSCBI1CBe m i s d o n at 340 nm after excitation at 280 nm.

Spleen wan minoed and homogenized with 10 times (veight/volume) of H C 1 - R h lorn PN 7.5 at 4.C in a s o m a 1 1 m i u i x e r (output 4) for 2 minutes in intervals of 30 seconds pulse followed by 1 minute pause in an ice bath. The h o w e n a t e was then filtered through a cbeeae cloth and subjected to 15 minutes Centrifugation at 4.0009, followed by 15 Din. Dit 1O.OOOg and finally for l hour at 100.0009. The pellet obtained between 10.0009 and 1OO.OOOg was considered cruds membrane and was washed 2-3 times w i t h the same buffer by centrifugation. A yield of about 70-90 mg of membrane protein (biuret) was obtained rrom 10 g of tissue.

SOWBILZZEO WITH S O D N W CHOLATE (PROU 400 Ul O? HmvLN

Total protein r, unit./%

Total nativity units*.

Specific aotivity

Yhld

*

Sodium Cholate extract

212

DEAB-Cellulose

50

22,500

450

100

Hydroxyapatite

31

30,133

1.000

134

m t y l sepharose

6

244,285

41,000

1,085

sophacry1 8-200: pool (60-64) pool (65-68)

0.12

65,089 34,151

562,000

289 152

0.04

885,000


11501

TABLE 2 PURIFICATION OF MEMBRANE PI-PIC SOWBILIZED (from 300 nq of human spleen membranes) Total protein Am

step Octy1 glucoside extract

200

DEIIEcellulose break

19

ROTOFOR

*,**.

WITHW

Y L

Specific actlvity UnIts/Am

Total activity Units**

4,608

240

3.6 54,000 As described in Table I.

15,000

GWCOSIDE Yield 1

100 1.100

PI-PIC wIzels