5-isoquinolinesulfonamide, a Newly Synthesized Protein Kinase ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 5, Ieaue of March 10,pp. 2922-2926.1985 Printed in U.S.A.

N-(Z-Aminoethyl)-5-isoquinolinesulfonamide, a Newly Synthesized Protein Kinase Inhibitor, Functions as a Ligand in Affinity Chromatography PURIFICATION OF Ca2+-ACTIVATED, PHOSPHOLIPID-DEPENDENT AND OTHERPROTEIN

KINASES*

(Received for publication, August 23,1984)

Masaki Inagaki,Masato Watanabe, and Hiroyoshi HidakaS From the Department of Phnrmncology, Mie University School of Medicine, Edobushi,Tsu 514, Japan

We designed a simple procedure for thepurification activated by the Ca2+-calmodulincomplex (4,5),and another of Ca2+-activated,phospholipid-dependent protein ki- species of Ca2+-dependent protein kinase, which requires nase (protein kinaseC) from rabbit brain,using affin- phospholipid as a cofactor, was described by Takai et al. (6). ity chromatography with a new affinity adsorbent. The However, the precise relationship between these two types of adsorbent wassynthesized by attaching theamino res- Ca’+-dependent protein phosphorylations has notbeen deteridue of N-(2-aminoethyl)-S-isoquinolinesulfonamide mined. (H-9) to cyanogen bromide-activated Sepharose. H-9 Protein kinase C activity levels are exceedinglyhigh in is a potent competitive inhibitor of protein kinase C, various tissues, as compared to CAMP-dependent and cGMPcGMP-, and CAMP-dependent protein kinase with re- dependent protein kinases (7, 8). Although the widespread spect to ATP and exhibits inhibition constants of 18, and extensive activity of protein kinase C has been demon0.87, and 1.9 PM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochernis- strated, its physiological function and exact kinetics have not t r y , 23, 6036). A 960-fold purification was achieved been elucidated. A simple procedure for purification of the in the two-step procedure, which entailed DEAE-cel- protein kinase C would lead to a better understanding of this lulose and the affinity chromatography. The resultantenzyme and ita functions. Neuroleptics (9, 10) and naphthalenesulfonamides (11, 12) preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis underconditions have an inhibitory effect on both Ca2+-activated,phosphoof denaturation withsodium dodecyl sulfate. The affin- lipid-dependent phosphorylation and Ca2+-dependent, cality of protein kinase for C theH-9-Sepharose was high, modulin-dependent protein phosphorylation. These inhibiand the enzyme could not be eluted either by a high tors, however, bind to cofactors of both kinases, such as concentration of sodium chloride or by 40% glycerol. calmodulin or phosphatidylserine, through hydrophobic interThe protein kinaseC could be eluted fromH-9-Sepha- actions (13-15). Thus, compounds interacting directly with rose by the buffer containingboth 0.2 M NaCl and 20% each kinase are required to elucidate the physiological roleof glycerol, thereby suggestingthat thebinding between the protein kinase C system. We have recently synthesized protein kinase C and H-9-Sepharose was due to both novel protein kinase inhibitors, isoquinolinesulfonamides hydrophobic and electrostatic interactions. H-9 cou- which bind directly to protein kinase C, CAMP-dependent, pled to Sepharose retained both cyclic nucleotide-de- and cGMP-dependent protein kinases, with different affinipendent protein kinases and protein kinase C, and ties (1). these enzymes could be eluted separatelyby the buffer We describe herein application of one isoquinolinesulfoncontaining L-arginine, a potent inhibitorof these three amide, H-9, for the separation of these three multifunctional kinases. The novel aspects of these three multifunc- protein kinases by affinity chromatography and for purificational protein kinases can thus be investigated using tion of protein kinase C. isoquinolinesulfonamide derivatives. EXPERIMENTALPROCEDURES

Materials-Cyanogen bromide-activated Sepharose 4Bwas obtained from Pharmacia FineChemicals, Sweden. H-9 was synthesized Many diverse biological processes are regulated by the according to methods of Hidaka et al. (1). Histone III-S andhistone concentration of intracellular calcium (2). Although the exact H2B were purchased from Sigma. Phosphatidylserine (pig liver) was mechanism by which calcium ion exerts its influence is un- purchased from Serdary Research Laboratories, Inc. Chloroform was known, Ca2+-dependentprotein phosphorylation is one of the removed from this phospholipid by a stream of nitrogen, and the major general mechanisms by which intracellular events in phospholipid was sonicated in water for 1min to produce a suspension of 0.5 mg/ml. [-y-”PIATP was obtained form Amersham, England. mammalian tissues are controlled by external physiological The partially purified holoenzyme of CAMP-dependentprotein kinase stimuli (3).Tissues containat least two types of Ca2+-depend- I (second DEAE step) was prepared from rabbit skeletal muscle, by ent phosphorylations of protein. Some protein kinases are the method of Beavo et al. (16). cGMP-dependent protein kinase from pig lung was partially purified by the method of Kuo and * This work was supported in part by grants from the Ministry of Greengard (17). Coupling of the Ligands to the Sephrose-The coupling of H-9 to Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. cyanogen bromide-activated Sepharose 4B wascarried out as follows: This article must therefore be hereby marked “udvertisemnt” in H-9 (36 pmol) was dissolvedin 6 ml of 50 mM borate buffer (pH 8.0) containing 0.5 M NaCl and added to 3 ml of settled cyanogen bromideaccordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom all correspondence and reprint requests should be activated Sepharose 4B. After a 2-h incubation at 30 “C, the gel was washed with 30 ml of 50 mM borate buffer (pH 8.0) containing 0.5 M addressed.

2922

Affinity Purification of Ca2+-activated, Phospholipid-dependent Protein Kinase NaCl and was finally resuspended in6 ml of 1.0 M ethanolamine (pH 8.0). The mixturewas again incubated for 8 h at room temperature. The resin was then washed with 30 ml of distilled water, followed by 200 ml of 25 mM Tris-HC1 (pH7.0) containing 2 mM EGTA,' 0.001% leupepsin, and 50mM 2-mercaptoethanol (BufferA). The amountof H-9 coupledto cyanogen bromide-activated Sepharose 4Bwas determined by absorbance at 214 nm in the supernatant of the reaction mixture. The amountof H-9 coupled to Sepharose was 10-12 pmol/ ml. Analytical Procedures-Sodium dodecyl sulfate-polyacrylamidegel electrophoresis was carried in 7.5% gels, as describedbyLaemmli (18) containing 0.1% sodium dodecyl sulfate. Samples to be applied were treated with 1%sodium dodecyl sulfate and 1%P-mercaptoethanol for 2 min in a boiling water bath. Molecular weights of proteins were estimated on the gels using myosin (200,000), @-galactosidase (116,250),phosphorylase b (92,500), bovine serum albumin (66,200), and ovalbumin (45,000) as standards. Activity of protein kinase C was determined inthe absence or presence of CaZ+and phosphatidylserine. Enzyme Assay and Determinations-CAMP-dependent protein kinase activity was assayed in a reaction mixture containing,in a final volume of 0.2 ml, 50 mM Tris-HC1(pH7.0), 10 mM magnesium acetate, 2 mM EGTA, 1 p~ CAMPor absence of CAMP, 3.3-20 p~ [y-3zP]ATP(4 X lo6 cpm), 0.5 p~ of the enzyme,and 100 pg of histone H2B. cGMP-dependent protein kinase activity was assayed in a reaction mixture containing, ina final volume of 0.2 ml, 50 mM Tris-HC1 (pH 7.0), 50 p~ magnesium acetate, 2 mM EGTA, 1 p~ cGMP or absence of cGMP, 3.3-20 p~ [T-~'P]ATP(4 X lo6 cpm), 100 p~ histone H2B, and 2.4 pg of the enzyme. Protein kinase C activity was assayed in a reactionmixturecontaining, in a final volume of 0.2 ml,50 mM Tris-HC1(pH 7.0), 10 mM magnesium acetate, 0.5 mM calcium chlorideor 1 mM EGTA, 10 pg of phosphatidylserine, 3.3-20 p~ [y-"P]ATP (4 X 106 cpm), 100 pg of Histone 111-S,and 0.05 pg of the enzyme. The incubation was carried out at 30 "C for 5 min and terminated by the addition of 1 ml of ice-cold 20% trichloroacetic acid following additionof 500 pg of bovine serum albumin as a carrier protein. The related mixturewas centrifuged at 3000 rpm for 15 min, the pellet was resuspended in ice-cold 10%

trichloroaceticacidsolutions,andthecentrifugation-resuspension cycle was repeated three times. The final pellet was dissolved in 1ml of 1 N NaOH, and radioactivitywas measured by liquid scintillation counter.

Protein was determined by the method of Lowry et al. (19). RESULTS

Purification of Protein Kinase-Isoquinolinesulfonamides were found to inhibit several protein kinases and todifferent extents (1).Among these compounds, H-9 possesses a free amino group. Immobilized H-9 was prepared by direct coupling to Sepharose by using the cyanogen bromide method. The amount of bound H-9 was 10-12 pmol/ml wet polymer. H-9 proved to be a potent inhibitor of protein kinase C, CAMP-dependent, and cGMP-dependentprotein kinases. The Ki values of H-9 for protein kinase C, CAMP-dependent and cGMP-dependent protein kinases were 18, 0.87, and 1.9 p ~respectively , (1).Kinetic analysis by the double-reciprocal curve revealed that the inhibition of protein kinase C from rabbit brain produced by H-9 was competitive with respect to ATP and noncompetitive with respect to phosphate acceptor. Essentially, the same results were obtained with other protein kinases. Protein kinase C has been recently purified from bovine heart (20), pig spleen (21), and rat brain (22, 23). However, the yield is very low as theenzyme is unstable. We, therefore, attempted to purify protein kinase C by using an H-9-Sepharose gel. One rabbit brain was homogenized in a Potter-Elvehjem Teflon-glass homogenizer with 85 ml of0.25 M sucrose in

2923

Buffer A. The homogenate was centrifuged for 60 min at 100,000 X g. The soluble supernatant, used as the crude extract (90 ml, 997.7 mg of protein), was applied to a DEAE column (20 X 1.5 cm) equilibrated with Buffer A. The column was washed with 300 ml of the same solution. The enzyme was eluted from the column by application of a 400-ml linear concentration gradient of NaCl (0.0-0.4 M) in Buffer A at a flow rate of 30 ml/h. Fractions of 3.5 ml were collected.Each fraction was assayed for protein kinase in the absence or presence of Ca2+.The elution profile obtained was essentially the same as described earlier (22, 24). The DEAE fraction (52.1 mg protein/42 ml) was applied to a H-9-Sepharose column (0.6 X 3 cm) equilibrated with Buffer A containing 0.1 M NaCl, and the column was eluted with 50 ml of Buffer A containing 1%Triton X-100, followed by 40 ml of borate buffer (pH 9.8), 40 ml of Buffer A containing 2 mM ATP, and, finally, 150 ml of Buffer A containing 1 M NaC1. Fig. 1 shows a typical elution profile of protein kinase C. About 98% of the total protein and less than 2% of the protein kinase C activity in the DEAE preparations were not absorbed to the gel and wererecovered in the flow-through fraction. The protein kinase C activity was eluted with buffer containing Larginine, in a linear gradient concentration (0.0-1.5 M) and at the flow rate of 15 ml/h (Fig. 1).When fractions of 2 ml were collected, a major peak of protein kinase C activity appeared in Fractions 21 through 37 (Fig. 1). The purified preparation (Fraction 29 through 37) was apparently homogeneous, and a major protein band was detected upon SDSpolyacrylamide gel electrophoresis, as shown in Fig. 2. A M, of 84,000 (average of four determinations) was obtained, based upon its electrophoretic mobility on SDS-polyacrylamide gel. Fractions 29 through 37 were pooled (0.197 mgof protein) and used for the subsequent studies. Analytical polyacrylamide gel electrophoresis of the enzyme from the affinity step (Fractions 29 through 37) also yielded a major protein band corresponding to the enzyme activity (data not shown). The purified enzyme could be stored in a plastic tube in the presence of0.7-1.0 M L-arginine. When the L-arginine was removed from the enzyme solution, the enzyme activity decreased by half within 20 min at 4 "C. Properties of the purified preparation of protein kinase C were as previously described (6, 22). A typical purification is summarized in Table I.

Fractlon n.0. (4ml/ono tub.)

Fractionn.0.(2ml/ono

tub.)

FIG. 1. Elution profile of protein kinase C from a H-9-bound Sepharose. The DEAE fraction was mixed with 5 ml of a Sepharose gel uncoupled with H-9, and after 30 min, this mixture was centrifuged. The supernatant was applied to H-9-Sepharose column (3 X 0.6 cm) equilibratedwithBuffer A containing 0.1 M NaCl. The washing buffers are indicated by arrows. ArrowI , Buffer A containing with 1%Triton X-100; arrow 2, boratebuffer(pH9.8); arrow 3, Buffer A containing with 2 mM ATP; arrow 4, Buffer A containing with 1 M NaCl. The elution buffer contained L-arginine with linear ' The abbreviations used are: EGTA, ethyleneglycol bis(@-amino- gradient concentration (0.0-1.5 M). Protein kinase activitywas meaethyl ether)-N,N,N',N'-tetraacetic acid; SDS, sodium dodecyl sul- sured in the absence (0)or presence of Caz+and phosphatidylserine fate. (0).

Affinity Purification of Ca2+-actiuated,Phospholipid-dependent Protein Kinase

2924

l\

4

5

I

o

1

I

I

I

1

0.2

0.4

0.6

0.8

1.0

Relative mobility

FIG. 2. Molecular weight determination by SDS-polyacryl-

amide gel electrophoresis. SDS-polyacrylamidegel electrophoresis of protein kinase C. About 9 pg of protein were applied to a 7.5% polyacrylamide gel in the presence of 0.1% SDS.Estimation of molecularweight of protein kinase C bySDS-polyacrylamide gel electrophoresis. The markers used are: 1, myosin (M.200,000); 2 , p galactosidase (M, 116,250);3, phosphorylase b (M.92,500); 4, bovine serum albumin (M, 66,200);and 5,ovalbumin (M. 45,000). Thearrow indicates the positionof protein kinase C. Binding Mechanism of Protein Kinase C to H-9 Coupled to Sephurose-ATP was tested for its ability to elute the enzyme from a H-9-bound Sepharose, since H-9 proved to be a competitive inhibitor of protein kinase C, with respect to ATP. A peak of enzyme activity was eluted by 30 mM ATP. Kinetic analysis of the effect of H-9 on protein kinase C activity and the elution profile of protein kinase C from a H-9-bound Sepharose with buffer containing 30 mM ATP strongly suggest that thebinding site of H-9 on protein kinase C is at the dinucleotide fold. Essentially, the same results were obtained with cyclic nucleotide-dependent protein kinases (data not shown). In case of affinity chromatography, buffers of high ionic strength can prevent ion-exchange interactions (25). When examining the nature of binding of protein kinase C to the gel, we observed that this enzyme could not be eluted merely with high salt concentrations. Glycerol can be considered a hydrophobic solvent since it has a dielectric constant of 42.5 (26). This was also less effective for elution of the enzyme from the column than was arginine. The buffer in the presence of smaller amounts of both substances (0.2 M NaCl and 20% glycerol) eluted effectively protein kinase C from a H-9-bound Sepharose (data notshown). Thus, itappears that thebinding of protein kinase C to a H-9-boundSepharose is linked to the interdependence of salt and glycerol and, presumably, the mutually reinforcing effect of hydrophobic and electrostatic forces. Protein Kinase C, CAMP-dependent, and cGMP-dependent Protein Kinases by a H-9-bound Sephurose-We also compared the effect of pH on the elution of CAMP-dependent (2nd DEAE step)(16), cGMP-dependent protein kinases (DEAE step) (17), and protein kinase C (DEAE step) (24) from aH-9-boundSepharose column (Fig. 3). Thethree partially purified kinases were combined so that the same

number of units of each (activity, 7 nmol of P”/min) was applied to theSepharose column. The interaction of the three enzymes with this adsorbent was apparently pH-dependent only at values exceeding 8.5. In the case of CAMP-dependent protein kinase, elution was achieved at a pH of 9.7 (Fig. 3), whereas protein kinase C and cGMP-dependent protein kinase activities were not eluted by this pH. The latter two enzymes could be resolved at a pH value of 11.0. However, the three protein kinases were not completely separated by DEAE column chromatography under these conditions. Having achieved a satisfactory purification of protein kinase C by using the L-arginine elution technique, we used this technique to examine the properties of CAMP-dependent, cGMP-dependent protein kinases, and C kinases; gradient elution with L-arginine was carried out to assess the magnitude of the affinity of the gels for the three multifunctional protein kinases used. After applying the same enzyme activity of each (activity, 7 nmol P3+/min)toa H-9-Sepharose column, a gradient elution with 0.0-1.0 M NaCl was carried out. Activity in theeffluents was nil.Elution with a gradient concentration of L-arginine (0.0-1.5 M) in the same buffer subsequently resolved the three protein kinases, as shown in Fig. 3. The retention of protein kinases with a H-9-Sepharose gel depended on the sensitivity of each protein kinase for inhibition by L-arginine (Fig. 3). It is of interest that the inhibitory effects of H-9 on each enzyme activity were not related to retention of the enzymes. Therefore, these three enzymes can be separated and partially purified from a crude extract, in a single-step procedure, using H-9-bound Sepharose. DISCUSSION

Affinity chromatography, in other words biospecificadsorption, has in recent years received considerable attention, as reflected by the number of surveys (27-31). Dramatic enzyme purifications have been observed using this technique, yet the method of achieving the very specificity that has contributed to theextraordinary success of affinity chromatography somewhat hampers the wider application of the method. Thus, in most cases, and for each individual enzyme, a different specific ligand has to be chosen and bound to the matrix. In purification of various protein kinases (32, 33), affinity columns have been prepared from adenine nucleotide derivatives; however, a general ligand such as anadenine nucleotide can bind to a large number of proteins. Partial specificity in purification which occurs in the binding step canbe enhanced by use of a specific elution technique. In the present work, we used our newly synthesized protein kinase inhibitor H-9 as ligand a for the separation of three types of protein kinases and for the purification of protein kinase C, since H-9 is a selective inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C, among various protein kinases and ATPases (1). There are satisfactory methods for the purification of protein kinase C from a variety of sources (20-23), yet none of these methods equals the ease, rapidity, and high recovery of the procedure described in thepresent paper. The key to this purificaton procedure is the use of an affinity column com-

TABLEI Purification of Ca2+-actiuated, phospholipid-dependent proteinkinose from rabbit brain Fraction

Supernatant fluid 0.13 DEAE-eluted H-9 affinity-Sepharose 4B-eluted

Volume ml

90 42 18

Protein Specific activity n m l P3+/min/mg

1.3 126.9

w

Activity p m l P3+/min

Purification -fold

Yield %

997.7 52.1 0.197

129,340.6 66,500.0 25,000.0

1 9.6 959.2

100 51 19

Affinity Purification

of ea2+-activated,Phospholipid-dependent Protein Kinase

2925

Fraction no(lml/one tube) FIG. 3. Chromatography of three multifunctional protein kinaseson a H-9-Sepharosecolumn. A H9-Sepharose column (2 X 0.6 cm) was equilibrated with Buffer A. Protein kinases were added to the column and buffer (20 ml) was applied. A , the eluting medium was initially Buffer A and was changed to 25 mM borate buffer (pH 9.8),indicated by arrow 1 and 25 mM NaHCOs/NaOH buffer (pH l l . O ) , indicated by arrow 2. B, the column was washed with buffer (20 A linear gradient of 0.0-1.5 M L-arginine was applied). Enzymic activities were determined as described under "Experimental Procedures." 0, CAMP-dependent protein kinase; 0, cGMPdependent proteinkinase; A, protein kinase C. C, 0,CAMP-dependentprotein kinase; 0,cGMP-dependent protein kinase; A, protein kinase C was assayed as described under "Experimental Procedures" with various concentrations of L-arginine.

posed of H-9-bound Sepharose and the use of L-arginine as an elutant. It is of interest that protein kinase C adsorbed on H-9bound Sepharose can be eluted with L-arginine. This phenomenon may be a nonspecific interaction of an ion-exchange type, or a hydrophobic type, or even a specific interaction. Neither a high concentration of sodium chloride nor 40% glycerol sewed as an elutant,whereas buffer in the presence of both substances effectively eluted protein kinase C from a H-9-bound Sepharose. In view of the observations, L-arginine may cause elution, by a similar mechanism. The enzymes prepared by the method described here shows no detectable impurity on sodium dodecyl sulfate-acrylamide gel electrophoresis (in affinity step, Fraction 29-37). However, sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified fraction followedby silver staining revealed several minor bands, suggesting that the preparation thus purified is not 100% homogenous. The enzyme obtained by this procedure did not retain full activity for even 1day at 4 "C, despite attempts to stabilize it in either glycerol (20%), sucrose (25%), 2-mercaptoethanol (50 mM), or dithiothreitol (5 mM). Only L-arginine (0.7-1.0 M ) offered full protection for several days. However, the Ca2+ sensitivity of the enzyme was decreased in the presence of Larginine. In this purification procedure, the contaminating protein kinase activity ( i e . CAMP-dependent protein kinase activity) was effectively resolved by the effect of pH (pH 9.7). The large capacity of our newly synthesized protein kinase inhibitor H-9-bound Sepharose for thesethree multifunctional protein kinases suggests that H-9 affinity chromatography should be most useful to economize established purification procedures for protein kinases. Acknowledgment-We thank M. Ohara of Kyushu University for critical readings of the manuscript.

REFERENCES 1. Hidaka, H.,Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984)Biochemistry, 23,5036 2. Rasmussen, H., and Goodman, D.B. P. (1977)Physiol. Reu. 57,421 3. Rasmussen, H. (1983)in Calciumand Cell Function Vol. IV,p. 2,Academic Press, New York 4. Yamauchi, T., and Fujisawa, H. (1979)Biochem. Biophys. Res. Commun. 90, 1172 5. Dabmwska, R., Aromatorio, D., Sherry, J. M.F., and Hartahorne, D. J. (1977)Biqckm. Bio hys Res Commun. 78,1263 6. Taka, Y.,Klshlmoto, Iwasa,'Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979)J.Biol. Chem. 254,3692 7. Mlnakuchi, R., Takai, Y.,Yu,B., and Nishizuka, Y. (1981)J. Biochem. (Tokyo)89.1651 8. Kuo, J. F., Andersson, R. G. G., Wise, B. C., Mackerlova, L., Salomonsson, I., Brackett, N. L., Katoh, N., Shoji, M., and Wrenn, R. W. (1980)P m . Natl. Acad. Sci. U.S.A. 77, 7039 9. Levin, R. M., and Weiss, B. (1977)Mol. Pharmacol. 13,690 10. Schatzman, R. C.,Wise, B. C., and Kuo, J. F. (1981)Biochem. Biophys. Res. Commun. 98,669 11. Hidaka, H., Asano, M., Iwadare, S., Mataumoto, I., Totauka, T., and Aoki, N. (1978)J. Pharmacol. Er Ther. 207,s 12. Tanaka, T., Ohmura, T., dmakado, T., and Hidaka, H. (1982) Mol. Pharmacol. 22,408 13. LaPorte, D.C., Wierman, B. M., and Storm, D.R. (1980)Biochemistry 19, 3814 14. Tanaka, T., and Hidaka, H. (1980)J. Biol. Chem. 255,11078 15. Inagaki, M., Naka, N.,Nozawa,Y., and Hidaka, H. (1983)FEBS Lett. 151,67 16. Beavo, J. A,, Bechtel, P. J., andKrebs, E. G. (1974)Methods Enzymol. 38, 299 17. K i o J . F and Green a d , P. (1974)Methods Enzymol. 38,329 18. Laer$mli,'b. K. (19707Nature (Lord.)227, 680 19. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem. 193,265 20. Wise, B. C., Ra nor, R. L., and Kuo, J. F. (1982)J. Biol. Chem. 257,8481 21. Schatzman, R. Raynor, R. L., Fritz, R. B.,and Kuo, J. F. (1983)Biochem. J. 209.435 22. Kikkawa,'U., Takai, Y., Minakuchi, R., Inohara, S., and Nishizuka, Y. (1982)J. Biol. Chem. 257,13341 23. LePeuch, C. J., Ballester, R., and Rosen, 0.M. (1983)Pmc. NatL Acad. Sei. U. S.A . BO. M.58 24. Inoue, KishimoG,-A., Takai, Y., and Nishizuka, Y. (1977)J. Biol. Chem. 252, 7610 25. Lauuus, L. H., Lee, C. Y., and Wermuth, B. (1976)Anal. Biochem. 74,138 26. Hofstee, B. H. J. (1973)Biuchem. Bio hys Res Cornmun.,SO, 751 27. Cuatrecasas, P., and Anfinsen, C. B. 6971)Annu. Rev. Bwchem. 40,259 28. Cuatrecases, P., and Anfinsen, C. B. (1971)Methods Enzymol. 22,191 29. Friedberg, F. (1971)Chromatogr. Rev. 14,121 30. Feinstein, G. (1971)Naturwissenscha ten 58,389 31. Mosbach, K., Gullford, H., Ohlsson, #t.,and Scott, M. (1972)Biuchem. J. 127,625 32. Lee, C.-Y., and Kaplan, N. 0.(1976)J.Macromol. Sei. Chem. A 10,15 33. Ta lor, S. S., Lee, C.-Y., Swain, L., and Stafford, P. H. (1976)Anal. h m h e r n . 76,45

i.,

e.,

M.: