Phospholipase C from human melanoma: purification and characterization of a phosphatidylinositol-selective enzyme

Biochimica et BiophysicaActa, 1076(199I) 209-214 © 1991 ElsevierSciencePublishersB.V.(BiomedicalDivision)0167.4838/91/$03.50 ADONIS 016748389100056X

209

BBAPRO33816

Phospholipase C from human melanoma: purification and characterization of a phosphatidylinositol-selective enzyme Frank W. Perrella, Rosemarie Jankewicz and Elizabeth A. Dandrow Medical ProductsDepartment, E.L DuPom de Nemours and Company. Glenolden, PA (U.S.A.)

(Received31 May 1990) (Revisedmanuscript received4 September1990)

Key words: PhospholipaseC; Human; Melanoma; Phosphatidylinositol Phospholipase C was purified from human melanoma grown as solid tumors in trade mice. The specific activity of the pure enzyme was approx. 100 pmol/min per rag; its apparent molecular mass was determined by sodium dedecyl sulfate polyacrylamide gel electroi~resis to be 150 kDa. The enzyme required calcium for activity and was activated by deoxycholate in the presence of the substrate phosphatidylinositol. The melanoma phospholipase C has a distinctly different substrate preference than those identified from normal tissues; it prefers phosphatidylinositol to phosphatidylinositol bisplmsl~te. The tumor enzyme was approx. 4-5-fold more active using phosphatidylinositol than phosphatldylinositol bisplmsl~te as the substrate.

Introduction The hydrolysis of phosphoinositides by phospholipase C (PLC) is known to be a key step in the .cgulation of hormone and growth factor related processes [1-4]. PLC is an enzyme that resides in both the cytosol and membrane fractions and catalyzes the hydrolysis of phosphoinositides to diacylglycerol and inositot phosphate(s) [5,6]. Reports of the purification of several forms of PLC have been published [5] using normal tissues, but no purification has been reported using tumor tissue. Many of these enzymes were purified using PI as the substrate, yet they showed a preference toward PlP2, We describe the purification and characterization of a PI-selective PLC from human melanoma.

Materials and Methods

Materials

Fast Q-Sepharose, Mono P HR 5 / 2 0 chromatofocusing column, Heparin Sepharose, Polybuffer 74 and DEAE-SPW column were from Pharmacia LKB Biotechnology (Uppsala, Sweden). The hydroxyapatite (HCA) column was purchased from Mitsui Toatsu Chemicals (Japan). Phosphatidylinositol was from Avanti Polar Lipids (Alabama, U.S.A.). [3H]PI and [3H]PIPz were purchased from Du Pont NEN Research Products (Boston, M.A, U.S,A.). All other chemicals were purchased from Sigma (Missouri, U.S.A.).

Animals

Abbreviations: PLC,phosphoinositidephospholipas¢C; Pl, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; InPs. inositol phosphate(s); IP3, 1,4.5.iaositol phosphate; DAG, diacylglycerol; SDS. sodium dodecyl sulfate; DOC, sodium deoxycholate; BSA, bovine serum albumin; PMSF. phen2dmethylsulfonylfluoride; HPLC, high-pressure liquid chromatography; pL isoelectrie point; K~pp,apparent K~; PAGE,polyactylam/degel electrophoresis. Correspondence: F.W. Perrella, Medical Products Department, E.I. DuPont de Nemours and Company. 500 South RidgewayAvenue, Glenolden. PA 19036,U.S.A.

Mice for xenografts were 6-week-old N u / N u Swiss weighing from 24 to 30 g and bred at Gienolden, Animals were maintained in a regulated fighting cycle (light 12 h, dark 12 h). The human melanoma line RPMI 7272, obtained from D,B. Rifldn (New York University Medical Center, N.Y.C., U.S.A.), was originally derived from a patient with malignant melanoma [71. The melanoma was introduced by s.c. implantation of cultured tumor cells in nude mice. The mice were killed by cervical dislocation and tumors (weighing 1 to 2 g) were rapidly removed and frozen at - 7 0 ° C in hexane on solid CO2.

210

PLC assay Colorimetric assay. PLC activity was measured by determining the formation of InPs from either PI or PIP2 using a modification of the method of Palmer [g]. All concentrations are final assay values unless stated otherwise. The assay was prepared in 96-well microtiter plates using a mixture of 25 mM imidazole buffer (pH 7.2) 100 mM KCI, 1 mM CaCl2, 0.8 mg/ml deoxycholate, 0-1.0 mM phospliatidylinositol or PIP2, and 1 to 40/~g of PLC protein fraction in a final volume of 0.1 nil. PI and PIPz were prepared as a 10 mM stock solution in deionized water by sonicating for 30 s. The enzyme reaction was started by adding the substrate and deoxychotate together, and then incubating for 15 rain at 37 o C. The calcium dependent PLC reaction was stopped by the addition of 8 mM EDTA and 80 mM Tris-HCI (pH 8.0). 10 units of alkaline pliosphatas¢ (Type II-T, Sigma, Missouri, U.S.A.) were added and incubated for an additional 30 min at 37°C to hydrolyze the InPs product of the reaction to inorganic phosphate(s). The phosphatase reaction was stopped by adding 3.7~ SDS and 29 mM EDTA (pH 4.0) to the assay mixture. Inorganic phosphate was quantitated by adding 36.5 mM ZnCI 2 and 5.5 raM ammonium molybdate. The mixture was incubated for an additional 20 rain at 37 °C before the absorbance was read speetrophotometrically at 360 nm in a Titertek Multiscan microtiter plate reader (Flow Laboratories, Virginia, U.S.A.) con.nected to an IBM PC. The rate of PI hydrolysis was linear at protein concentrations up to 30/tg per assay volume and for the first 30 rain of incubation. Inorganic phosphate and InPs hydrolyzed by alkaline phosphatase were used as standards in the assay. The nonspecific hydrolysis of PIP2 by alkaline phosphatase produced a background of inorganic phosphate in the assay of approx. 10~ during an incubation period of 30 min at 37 °C and pH 8.0.

Radiochemical assay The PI and PIP2 hydrolyzing activity of PLC was measured using [3H]Pl and [3H]PIP2 as the substrates in the presence of 0.08~ deoxycholate as described [6,12,17]. Assays were performed in a 100/tl reaction mixture containing 10000 cpm of [3H]PI or [3H]PIP2, 100 ~tM PI or PIP2, 0.8 mg/ml of DOC, 1 mM CaCI2, 0.15 rag/m1 of bovine serum albumin, 100 mM NaCI, 50 raM Hepes (pH 7.0) and a source of enzyme. After 10 rain at 37°C, the reaction was terminated by the addition of 0.5 ml of chloroform/methanol/concentrated HC! (100:100:0.6). The mixture was vortexed for 40 s and 0.15 nd of 1 M HC! containing 5 mM EDTA was added and the mixture was vortexed for 40 s once again. A 200 gl aliquot of the aqueous phase extract was removed for liquid scintillation counting.

Buffers Buffer A: 10 mM imidazole-HCl, 10 mM EDTA,

0.25 M sucrose (pH 7.2), buffer B: 10 mM imidazoleHCI, 1 mM EGTA, 1 mM DTr, 5~ glycerol, 55 ethylene glycol, 0.005~ Triton X-100 (pH 7.2); buffer C: buffer B plus 1 M NaCI, buffer D: 0.5 M ammonium carbonate-HCl (pH 8.0); buffer E: buffer B at pH 6.0; and buffer F: 10~ Polybuffer 74, 5~ glycerol, 5~ ethylene glycol, 1 mM DTI', 0.005% Triton X-100 (pH 4.0). PMSF (0.1 raM) and leupeptin (1/tM) were added to all buffers just before use.

Purification of PLC PLC was purified from the soluble fraction of human melanoma xenograft tumors. All operations, unless stated otherwise, were carried out at 0-4°C. A purification procedure starting from 20 g of melanoma is described below. Preparation of tissue extracts. Frozen tumors were cut into pieces and homogenized in 6.5 vol. of buffer A with a Tekmar ultrasonic tissue disruption homogenizer for 30 s at 20000 rpm. Large tissue debris was removed by low-speed centrifugation for 20 rain at 250 × g, The pellet was washed once with 6.5 vol. of buffer A and centrifuged as above. The two supernatants were pooled and centrifuged at 200000xg for 60 rain. The fatty layer was removed using cotton swabs. The supematant was dialyzed twice against 2 ! of buffer B (Spectra/Pot 7 m.w. cutoff 25000, Spectrum Medical Industries, Los Angeles, U.S.A.). Fast Q-Sepharose chromatography. The supernatant from the high-speed centrifugation step was loaded directly onto a Fast Q-Sepharose column (1.6 × 50 cm) equilibrated with buffer B. The flow rate was 2.5 ml/min and 12.5 ml fractions were collected. The column was washed with buffer B containing 0.1 M NaCI and PLC was eluted with two sequential linear NaCI gradients (10-40~, 40-100~ buffer C). The total volume of the gradient was 800 ml. Two peaks of enzyme activity were recovered, one at 0.30 M and the other at 0.35 M NaC1, respectively. Column fractions representing the first peak of PLC activity were combined and dialyzed against 2 1 of buffer B. Heparin-Sepharose chromatography. The dialyzed peak-one activity was loaded onto a heparin-Sepharose column (1.6 × 10 cm) equilibrated with buffer B. The flow rate was 1 ml/min and 4 ral fractions were collected. After washing the column with buffer B containing 0.18 M NaCI, two sequential linear gradients (1840~, 40-100% buffer C) were applied. The enzyme activity was eluted at 0.35 M NaCI. The total volume of the gradient was 240 nil. The column fractions representing the PLC activity were combined and dialyzed against 2 [ of buffer B minuq EGTA. Hydroxyapatite chromatography. The dialyzed enzyme activity was loaded onto a hydroxyapatite column (8 × 100 ram) equilibrated in deionized H20. The flow rate was 1 ml/min and 2 mI fractions were collected.

211 After washing the column with deionized H20, two sequential linear gradients (0-50~, 50-100~ buffer D) were applied. PLC activity was eluted from the column at 0.12 M NH4CO3. The total volume of the gradient was 135 ml. The active fractions were pooled and dialyzed against buffer E. Chromatofocusing. The dialyzed fractions containing PLC activity were loaded onto a Mono P HR 5/20 chromatofocusing column equilibrated in buffer E. The enzyme was eluted with 50 ml of buffer F (pH 4.0) to form a linear pH 6-4 gradient, at a flow rate of 0.5 ml/min. To each collected tube was added 0.1 ml of 1 M imidazole buffer (pH 7.5) to neutralize acidic fractions. The volume of each fraction was 1 ml. The enzyme activity was ehted at a pH of 4.4. DEAE.SPW chromatography. Chromatofocusing fractions containing the enzymatic activit)' were loaded onto a DEAE-SPW column (7.5 x 75 mm) equilibrated in buffer B. The ftow rate was 1 ml/min and 3 ml fractions were collected. The column was washed with buffer B and PLC was eluted by a linear gradient of 150 ml of buffer C. A single peak of activity was recovered at 0.17 M NaCI. The active fractions were combined and concentrated using Centricon 10 microconcentratots (Amicon, MA, U.S.A.). When analyzed by SDSPAGE, the enzyme was purified to apparent homogeneity using silver staining of protein. The enzyme was stored at 4°C in buffer B.

Units o/activity One unit of PLC activity is the amount of enzyme that catalyzes the formation of 1 p.mol of inositol 1phosphate per minute at 37°C in the standard assay system. Other methods Protein was determined by the method of Bradford [9] or a modified Lowry procedure in the presence of SDS [10] using bovine serum albumin as the standard. SDS-PAGE was performed according to Laemmli [11]. Results

Purification of PI.PLC The results of a typical purification are shown in Table I. The enzyme was purified several thousand-fold with a 2~ yield. We observed that dithiothreitol was necessary during the purification to stabilize the enzyme. The results of the purification as outlined in Table I were as follows. The soluble tissue extract, following high-speed centrifugation, was applied to a Fast Q-Sepharose column. The PLC activity eluted as two peaks from the column with a yidd for the first peak of 16~. The first peak was subjected to further purification on hepadn-Sepharose chromatography. One broad peak of activity was eluted at 0.35 M NaCt.

TABLE 1 Purificationof Pl.selectivePLC from human melanoma The PLC activityof each step was determinedusing 1 ram Pi as the substrate as describedin the Materialsand Methodssection. Protein concentrations were used for which the activity was linear with respect to the time of incubation.One unit is the amountof enzyme that hydrolyzcs I /~moi of PI per minute under standard assay conditions. Similar resultswereobtained from six other independent experiments. Purification step

Total Total Specific Purity protein activity activity (-fold) (mg) (~moi/ (Units/ min) rag) Supematant 1569 23.5 0.015 1.0 Fast QoSepharose 159 3.8 0.024 1.6 Heparin-Sepharose 15 3.9 0.26 17 Hydroxyapatite 1.4 0.93 0.66 44 Chromatofocusing 0A 0.46 1.15 76 DEAE-5PW 0.004 0.49 122 8166

Yield (%)

100 16 17 4 2 2

Chromatography on heparin-Sepharose was very effective for the purification, since the peak of PLC activity eluted after the bulk of the proteins. The active fractions were then applied to a hydroxyapatite column and the enzyme activity was resolved into one major and several minor peaks. The fractions that constituted the major peak were pooled and chromatographed on a chromatofocusing column. PLC activity was resolved to a single peak of activity eluting with a pl of 4.4. This was followed by anion-exchange HPLC on a DEAE5PW column where the enzyme eluted as a single peak of activity with apparent homogeneity. The specific activity of the purified enzyme was 122 units/rag of protein.

S,'ability The purified enzyme could be stored in 10 mM imidazole (pH 7.2), 1 mM DTr, 1 mM EGTA, 0.1 mM PMSF, 03 laM leupeptin, 5~ glycerol, 5~ ethylene glycol and 0.005~ Triton X-100 at 4°C for at least 2 weeks with no appreciable loss of activity.

Molecular mass The purified protein appeared as a single band of apparent molecular mass 150 kDa on SDS-PAGE (Fig. 1). When gel-permeation HPLC (Superose or TSK G3000 SW, Pharmacia LKB; Zorbax GF250, E.I. Du Pont) was used, either in the presence or absence of NaCI or D e c , no activity was recoverable (data not shown). The loss of activity appears to be due to the interaction of the protein with the gel matrix. Properties of the purified enzyme The activity of the enzyme increased proportionally with protein concentration under standard assay condi-

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Fig. I. SDS-PAGEof PLC. Samples from the peak activities of PLC from the six column chromatographic steps were run on SDS-PAGE (see Materials and Methods section). Protein bands were visualized by means of silver staining. Lane 1, crude soluble extract; lane 2, Fast Q-Sepharose chromatography;lane 3, heparin-Sepharo~ chromatography; lane 4, hydroxyapatite chromatography; lane 5, chromatofocusing chromatography;lane 6, DEAE-SPWchromatography;and std., molecular mass markers (170 kDa, reduced a2-macroglobulin; 97A kDa, phosphorylaseb; 55A kDa, glutamate dehydrogenase).The data shownare from one of several represemative independent experiments. lions. A linear relationship between enzyme activity and protein concentration was observed in the range of 14 to 56 ng protein. PI rather than PIP2 was the preferred substrate for this enzyme at the protein concentrations tested, showing activity enhancements of 3-6-fold over

that of PIP2. A 4-fold enhancement of PLC activity for PI over PIP2 was also observed when our standard assay buffer (see Materials and Methods section) ~,as re-

placed with 0.8 mg/ml DOC, 1 mM CaCI e, 0.15 mg/ml bovine serum albumin, 100 mM NaCI, and 50 mM Hepes (pH 7.0), a similar reaction buffer used by other workers [12,1"~] to assay PLC {data not shown). The purified enzyme had a dependence on Ca 2+ for activity. As shown in Fig. 2, PLC was 5-fotd more active using PI as a substrate than PIP2. This substrate preference was independent of the Ca 2+ concentration and peaked at about 3 raM. The enzyme showed little activity at Ca 2+ concentrations below 10 pM. The difference in substrate preference of the enzyme was also observed at earlier stages of the purification, . . . .

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Fig. 3. Effect of PI and PIPz on (A) human melanoma and (B) bovine brain PLC activity of heparin-Sepharose column fractions. The first PLC activity peak from Fast Q-Sepharose column chromatography was dialyzed to remove NaCI and applied to a heparln-~pharos¢ column as described in the Materials and Methods section. An aliquot of each cohv~ fr~_c~ionwas assayed for eozymeactivip/. The standard assay was used except that the substrate was either Pl or PIPz (1 raM). Each datum no~a, is Ibc mean of trip|icate determinations and is representative of results obtained in a secondexperiment; S.E,M. Iessthan 10 percent.

213 The activity of tumor PLC using either PI or PIP2 as the substrate was compared in the peak fractions following Fast Q-Sepharose and heparin-Sepharose column chromatography (Fig. 3A). The data for the melanoma enzyme show that PI rather than PIP2 was the preferred substrate under standard assay conditions, suggesting that a PI-selective form of PLC was a major component of the active peak. An identical analysis was performed with PLC from bovine brain prepared using the same purification steps and assay conditions as the tumor enzyme (Fig. 3B). In this instance, PIP2 was the preferred substrate for the bovine brain PLC, in agreement with previously pubhshed data [12]. This parallel comparison of the melanoma vs. brain PLC activities suggests further that the melanoma enzyme has a preference for Pl as the substrate. The analysis of the pH for PI-PLC showed a pH maximum for the substrate PI of approx. 7 when DOC (0.08%) was present (Fig. 4). This pH maximum may be the result of the PI/DOC 'particle' surface charge (pK, of DOC is approx. 5.5) and therefore may not represent the true pH optimum of the enzyme for its substrate. The pH optimum in the presence of DOC is intended to be used for comparison purposes with other published data on PLC where pH maxima were obtained under similar conditions [17,19]. DOC is known to enhance the activity of the substrate for the enzyme [12,17-19] and, in its absence, no pH optimum was observed in the pH range 5 to S. In contrast to Pl, PiP2 showed no pH optimum either in the presence or absence of DOC. The kinetic parameters of PLC substrate saturation data for PI and PIP2 were obtained by nonlinear regression using the EZ-FIT microcomputer program [13]. The Kapp value of both PI and PIP2 for PI-PLC, under standard assay conditions, was 200 #M (Fig. 5). These

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lag activities were measured using 0.1 to 0.7 mM substrate under standard assay conditions, The V~,~ values were determined by nonlinear regressionanalysisof the saturationdata usingthe EZ-FIT microcomputer program [13]. Each datum point is the mean of triplicate determinationsand is representativeof resultsobtainedin a secondexperiment;S.E.M.lessthan 10percent.

values were consistent with those from other workers when PLC was assayed in the presence of DOC [I7-19]. The apparent Km values were expressed as K,pp'S because micelles of DOC and Pl were used as the substrate for the enzyme. The surface concentration of PI in PI/DOC micelles does not represent the actual PI concentration recognized by the enzyme due to the effect of surface dilution on the kinetics. The apparent Vma~ values were calculated as 52 Fmol/min for Pl and 11 ~tmol/min for PIP2 hydrolyzed per mg protein; the ratio of Vm=~for PI and PlP2 was approx, 5 : 1. Similar results were obtained using the radiochemical substrates [3H]Pl and [3H]PIP2; a 4.4-fold ($.E.M. = 1.5, n = 4) increase in enzyme activity for Pl over that of PIP2 was observed when tumor PLC was assayed under conditions employed by other laboratories [6,12,17]. Furthermore, the enzyme was not active when either phosphatidylserine or phosphatidylcholine was used as a substrate, demonstrating the specificity of the enzyme for phosphoinositides (data not shown).

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the presenceor absenceof deoxycholateunder standard assaycondilions exceptthat the pH of the bufferwas varied.Eachdatumpoint is the meanof triplicate deterrnination~and is representativeof results obtainedin a secondexperiment; S.E.M.lessthan 10percent.

Discnssion Various forms of PLCs from different tissues have been reported [5]. These studies report that at least six distinct forms of this enzyme exist. The purifications of PLCs to date have been from normal differentiated tissue, i.e., brain. W¢ described here the purification of a PI-sclective PLC from human tumor tissue. To our knowledge, this is the first purification of a PLC from human melanoma. The PI-PLC from human melanoma exhibits some interesting properties with respect to its substrate preference. Our r,:~ults differ from those reported previously

214 for normal non-proliferating tissue; we fred that the tumor enzyme has a preference for the substrate P! rather than PIP2 over a Ca 2+ concentration range of 10-3000/LM. The relative amounts of DAG produced in the cell depend in part on the availability of precursor phospho'mos,.'fi~e Since at least 905[ of phosphoinosiddes in the cell are in the form of PI, a PLC that hydrolyzes preferentially this substrate could produce a sustained increase in DAG levels without mobilizing Ca 2+ via IP3. Protein Idnas¢ C ¢psilon is known to be calcium independent and therefore could be activated by this mechanism [14,16]. PLCs of molecular masses of 145 and 154 kDa have been purified from bovine brain [5,6,12]. Although the size of these isozymes (PLCs beta and gamma) are similar to that of the human tumor enzyme, these enzymes prefer PIP2 to PI as a substrate. In contrast to the PIPe selective PLCs, little data is available regarding enzymes that prefer PI [15]. Preliminary experiments, using immunoblots, suggest that the melanoma PI-PLC cross-reacts with polyclonal antibodies prepared against synthetic pepddes (amino acids 1272-1287) of PLCgamma t (data not shown). One possibility is that the melanoma PI-PLC is a variant of the gamma class of PLC's. In conclusion, several forms of PLC have been reported that hydrolyze phosphoinosifides. The individual isozymes of PLC may be regulated by fundamentally different mechanisms. It remains to be determined whether certain classes of PLCs are involved in different physiological events, i.e., cell growth vs. differentiation. It is possible that minor calls as well as normal proliferating tissues express a form of PLC that is either not expressed or expressed at very low levels in nonproliferating tissues. Further studies will be necessary to determine whether the enzyme described here is unique to tumor tissues.

A ~ t s We are grateful to Paul A. LaRocco for his technical assistance during the PLC purification studies and to Alan Tarver for his immuno-blot analysis of PLC.

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