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Fatty acid activation of protein kinase C: Dependence on diacylglycerol
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 825-829
Vol. 152, No. 2, 1988 April 29, 1988
FATTY ACID ACTIVATION OF PROTEIN KINASE C : DEPENDENCE ON DIACYLGLYCEROL
Veronik Verkest, Mark McArthur and Susan Hamilton
Department of Biochemistry, University of Queensland, St Lucia, Queensland, Australia 4067 Rece±ved March 9,
1988
Summary. The kinetics of activation of protein kinase C by oleic acid have been reinvestigated, using highly purified preparations of the rat brain and bovine spleen enzymes. Activation of both enzymes by oleic acid is enhanced dramatically by diolein, contrary, to previous reports. In the presence of 9.7 ~M diolein, the concentrations of oleic acid required for half-maximal activation are 5 p~M and 9 la/Vlfor the rat brain and bovine spleen enzymes respectively, indicating that the system is much more sensitive to activation by fatty acids than previously recognized. Both enzymes also exhibit a pronounced lag in the activation at low concentrations of oleic acid. The kinetics of activation are very similar to those reported by Hannun et al. (J. Biol. Chem 260, 1003910043), who characterized the activation of the rat brain enzyme by mixed micelles containing Triton X-100, phosphatidylsefine arid diolein. © 1988 A c a d e m l c Press, Inc.
Protein kinase C is one of a diverse group of enzymes which is activated by lipids. The activity of this important regulatory enzyme is enhanced more than 100-fold in the presence of phosphatidylserine and calcium ions, and the affinity of the enzyme for phospholipid and calcium is apparently increased by low concentrations of diacylglycerol. Several reports indicate that protein kinase C is also activated by unsaturated fatty acids. McPhail et al. (1) demonstrated that oleic, linoleic, linolenic and arachidonic acids activated a crude preparation of the
human neutrophil enzyme. Maximal activation required > 100 ~¢I fatty acid, and there was a further - 1.6-fold increase in activity in the presence of 2 I.tg/ml diolein. Saturated fatty acids were ineffective as activators. Horn et al. (2) found that a purified preparation of chick oviduct protein kinase C was similarly activated by unsaturated
fatty acids, in the presence of 0.1 p2¢I 12-O-tetradecanoylphorbol-13-acetate (TPA). The extent of activation in the absence of TPA was not reported. Recently Murakami and Routtenberg (3) and Murakami et al. (4) showed that a preparation of rat brain protein kinase C was also activated by unsaturated fatty acids. In the presence of 0.5 mM calcium ions, the concentration of oleic acid required for haif-maximal activation was 50 laM, and the activities in the presence of saturating concentrations of fatty acid and phosphatidylserine were similar. The effect of added diacylglycerol or phorbol esters on the activation by fatty acids was not reported. In this study, we have investigated in more detail the kinetics of activation of protein kinase C by oleic acid, with a view to clarifying in particular the effect of diacylglycerol on the system. These experiments were undertaken using highly purified preparations of both the rat brain and the bovine spleen enzyme. During the
825
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
course of the work, Nishizuka's group reported that three distinct forms of protein kinase C isolated from rat brain were activated to similar extents by oleic acid, but that the activation was not enhanced by diacylglycerol (5). EXPERIMENTAL SECTION All chemicals were Analytical Reagent Grade, and buffers were prepared in distilled, deionized water. Oleic acid was obtained from Sigma Chemical Co. and was dissolved in redistilled ethanol. [32P]ATP was supplied by Bresatec. Protein kinase C activity was assayed at 25 °C essentially as described by Le Peuch et al. (6). Test assays (0.2ml) contained 0.5 mM CaC12, 10 mM MgC12, 5 mM dithiothreitol, 60 I.tg/ml or 180 I.tg/ml phosphatidylserine, 6 ~tg/ml diolein, 50 IJ2¢I [32p]ATP ( - 4 x 106 dpm) and 1 mg/ml lysine-rich histone in 0.02 M Tris buffer, pH 7.5. Control assays contained 0.5 mM EGTA instead of calcium, and no lipid. Samples (20 I-tl) of each assay were removed every 15 s and applied to phosphocellulose paper which was then washed exhaustively. The bound radioactivity was quantified by liquid scintillation counting. The kinetics of activation by fatty acids were studied at 25 °C using a modification of the routine assay, in which phospholipid was replaced by an aliquot of fatty acid dissolved in ethanol. The final concentration of ethanol was <1% (v/v). Protein concentrations were measured by a modification of the Lowry method (7). SDS-Polyacrylamide gel electrophoresis was performed according to Laemmli (8). Protein kinase C was isolated from rat brain (45 g of tissue) and bovine spleen (1000 g of tissue) by a modification of the method of Kaqd~wa et al. (9). The procedure employs sequential chromatographies on DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose. A chromatography on DEAETrisacryl was included as a final step in the spleen enzyme isolation. The detailed procedure for isolation of the spleen enzyme will be reported elsewhere. The final preparations were substantially pare on SDS-polyacrylamide gel electrophoresis. The maximum specific activities of the preparations were 2888 units/mg (rat brain enzyme) and 5100 units/mg (bovin6 spleen enzyme), where 1 unit of activity is the amount of enzyme which catalyses the transfer of 1 nmol of phosphate into histone per min at 25 °C. The ratio of activities in test and control assays was >100 for both preparations. The yields of enzyme activity in the final preparations were ~ 12% for the rat brain enzyme and ~ 15% for the bovine spleen enzyme. RESULTS AND DISCUSSION The activation of rat brain and bovine spleen protein kinase C by oleic acid is shown in Fig. 1. The kinetics of activation of the two enzymes appear virtually identical. The maximum activity for each enzyme,
60
:q
20
->
o
i150
m E IO0
N III
50
j I
I
L °
q 20
I
I
IO
0 I ~ I
I
II
I
[Oleic
[ 40 or
6 0j
o I
•
P l2~o
q 40O
a c i d ] (,uM)
Figure 1. Activation of vat brain protein kinase C (above) and bovine spleen protein kinase C (below) by oleic acid, in the presence ( • ) and absence ( O ) of diolein (9.7 laJvl). ( • ), activity in the presence of saturating phosphatidylserineand diolein; ( A ), activity in the absence of added lipid or calcium ions. 826
Vol. 152, No. 2, 1988
o,,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.
.
~0
I
I
I
r_
|
i'ls
5 .4o
~
2oi
Q
OI 0
I I 1 2 10-.5 [Oleio
~ 3 aoid]
i 4 -1 (Z -11
Q
0
zxzxl
2
~ I
4
I
6
I
8
10
[Diolein] ( u r n )
q
lOO
Fzgure 2. Double reciprocal plots of the data from Figure 1. Activities were measured in the presence of 9.7 I.tM diolein. Above, rat brain protein kinase C; below, bovine spleen protein kinase C.
Figure 3. Effect of diolein on the activation of rat brain protein kinase C by 25 ~ phosphatidylserine ( • ), and diolein alone ( A ).
oleic acid ( • ), 180 I.tg/ml
measured in the presence of 9.7 ~M diolein, and > 20 ~M oleic acid, is the same, within experimental error as the value obtained in the presence of saturating concentrations of phosphatidylserine and diolein. The concentrations of oleic acid required for half-maximal activation (KDapp) in the presence of 9.7 ~
diolein are - 5 ~tM and ~ 9 ~tM
for the rat brain and bovine spleen enzymes respectively. Values of KDapp were estimated from Fig. 1 since the corresponding double reciprocal plots were nonlinear (Fig. 2). Both enzymes exhibit a pronounced lag in the activation at low concentrations of fatty acid, and some inhibition is evident at higher concentrations (_>_100 txM). No measureable activation of either enzyme is seen at < 100 I.tM oleic acid in the a b s e n c e of diolein. At higher concentrations of fatty acid (200 - 800 I.tM), a small increase in activity above the control activity is observed. However this is at most only 10% of the maximum activity measured in the presence of saturating concentration of oleic acid plus diolein. The effect of varying the diolein concentration on the activity of the rat brain enzyme in the presence of 25 ~tM oleic acid is shown in Fig. 3. It is evident that the activation by 25 jaM oleic acid is enhanced at least 50-fold in the presence o f > 9 ~
diolein.
These results differ in several important respects from those of other investigators. The most notable discrepancy is the observed effect of diolein on the activation. Seldguchi et al. (5) report no enhancement of the activation by fatty acids in the presence of diolein, and a relatively small effect ( - 1.6 fold enhancement) was noted by McPhail et al. (1). Further, the values of KDapp obtained in the present work are an order of magnitude lower 827
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
than previously reported. From the data of Sekiguchi et al. (5) values of KDa~ for the activation by oleic acid of three separate forms of rat brain protein kinase C may be estimated to be ~ 100 laM (type I enzyme) and > 200 t.tM (types II and III enzyme). Murakami and Routtenberg (3) cite a value of 50 ~tM for the rat brain enzyme. It should be noted that these values were all measured in the absence of diolein. Our results show that protein kinase C is much more sensitive to activation by fatty acids than was previously indicated, and that this activation is strongly dependent on diacylglycerols. Whether the activation by fatty acids has any physiological significance remains to be determined. We are unable at present to account for the differences between our results and those of other investigators. It is, however, tempting to speculate that a preparation of enzyme which contained some residual neutral lipid might be more susceptible to activation by fatty acids in the absence of added diacylglycerol than a lipid-free preparation. It is most unlikely that we have isolated a form of protein kinase C which is distinct from any of those isolated in other laboratories, because the method of isolation is very similar to that employed elsewhere (5), and the final recoveries of enzyme activity are relatively high. It may be relevant that the specific activities of our purified enzymes, measured at 25 °C, are consistently higher than the value of 624 units/mg (30 °C) cited by Murakami et al. (4), or the value of 820 units/mg (30 °C) reported by Nishizuka's (9) group for their preparations of the rat brain enzyme. In the final chromatography step of our rat brain enzyme isolation (threonine-Sepharose), partial separation of several forms of the enzyme was obtained.
While the present
experiments have not yet been undertaken using completely resolved forms of the rat brain enzyme, no differences have been detected amongst the partially resolved enzymes with respect to the activation by oleic acid and its dependence on diacylglycerol. It is not clear in the case of the spleen preparation whether we are dealing with a single form of the enzyme or multiple forms.
The critical micelle concentration of oleic acid has not yet been determined under the conditions of our assays. However literature values are higher by at least 100-fold than the KDaW values obtained in this work (10). It is reasonable to assume therefore that the effective activator in our experiments is the monomeric fatty acid. The results shown in Fig. 1 bear a striking similarity to those obtained by Hannun et al. (I1), who examined the activation of rat brain protein kinase C by mixed micelles of Triton X-100, phosphatidylserine and diolein. In this system, each enzyme molecule is able to interact with a limited number of phospholipid and diolein molecules, the number depending on the mole ratios of the micellar components. Under these conditions the activation by phosphatidylserine is almost completely dependent on the presence of diolein, and there is a pronounced lag in the activation at low concentrations of phosphatidylserine which is very similar to that seen in the present work. These results suggest that the effect of diacylglycerols on protein kinase C activity is much larger when the activator 828
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(phospholipid or fatty acid) is present as the monomeric species, and further, that more than one molecule of phospholipid or fatty acid may be involved in the activation of the enzyme. REFERENCES 1.
2. 3. 4. 5. 6.
7. 8.
9. 10. 11.
McPhail, L.C., Clayton, C.C., and Snyderman, R. (1984) Science 224, 622-624. Horn, F., Gschwendt, M., and Marks, F. (1985) Eur. J. Biochem. 148, 533-538. Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193. Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429. Sekiguchi, K., Tsukuda, M., Ogita, K., Kikkawa, U., and Nishizuka, Y. (1987) Biochem. Biophys. Res. Commun. 145, 797-802. Le Peuch, C.J., Ballester, R., and Rosen, O.M. (1983) Proc. Natl. Acad. Sci. USA. 80, 6858-6862. Lowry, O.H., Rosenbrough, N.J., Fan', A.L., and Rondall, R.J. (1951) J. Biol. Chem. 193. 265-275. Laemmli, U.K. (1970) Nature 227, 680. Kikkawa, U., Go, M., Koumoto, I., and Nishizuka, Y. (1986) Biochem. Biophys. Res. Commun. 135, 636643. Mukerjee, P., and Mysels, K.J. (1971) National Standard Reference Data Service Vol 36, National Bureau of Standards, Washington, D.C. tLannun, Y.A., Loomis, C.R., and Bell, R.M.J. Biol. Chem. 260, 10039-10043.
829
Vol. 152, No. 2, 1988 April 29, 1988
FATTY ACID ACTIVATION OF PROTEIN KINASE C : DEPENDENCE ON DIACYLGLYCEROL
Veronik Verkest, Mark McArthur and Susan Hamilton
Department of Biochemistry, University of Queensland, St Lucia, Queensland, Australia 4067 Rece±ved March 9,
1988
Summary. The kinetics of activation of protein kinase C by oleic acid have been reinvestigated, using highly purified preparations of the rat brain and bovine spleen enzymes. Activation of both enzymes by oleic acid is enhanced dramatically by diolein, contrary, to previous reports. In the presence of 9.7 ~M diolein, the concentrations of oleic acid required for half-maximal activation are 5 p~M and 9 la/Vlfor the rat brain and bovine spleen enzymes respectively, indicating that the system is much more sensitive to activation by fatty acids than previously recognized. Both enzymes also exhibit a pronounced lag in the activation at low concentrations of oleic acid. The kinetics of activation are very similar to those reported by Hannun et al. (J. Biol. Chem 260, 1003910043), who characterized the activation of the rat brain enzyme by mixed micelles containing Triton X-100, phosphatidylsefine arid diolein. © 1988 A c a d e m l c Press, Inc.
Protein kinase C is one of a diverse group of enzymes which is activated by lipids. The activity of this important regulatory enzyme is enhanced more than 100-fold in the presence of phosphatidylserine and calcium ions, and the affinity of the enzyme for phospholipid and calcium is apparently increased by low concentrations of diacylglycerol. Several reports indicate that protein kinase C is also activated by unsaturated fatty acids. McPhail et al. (1) demonstrated that oleic, linoleic, linolenic and arachidonic acids activated a crude preparation of the
human neutrophil enzyme. Maximal activation required > 100 ~¢I fatty acid, and there was a further - 1.6-fold increase in activity in the presence of 2 I.tg/ml diolein. Saturated fatty acids were ineffective as activators. Horn et al. (2) found that a purified preparation of chick oviduct protein kinase C was similarly activated by unsaturated
fatty acids, in the presence of 0.1 p2¢I 12-O-tetradecanoylphorbol-13-acetate (TPA). The extent of activation in the absence of TPA was not reported. Recently Murakami and Routtenberg (3) and Murakami et al. (4) showed that a preparation of rat brain protein kinase C was also activated by unsaturated fatty acids. In the presence of 0.5 mM calcium ions, the concentration of oleic acid required for haif-maximal activation was 50 laM, and the activities in the presence of saturating concentrations of fatty acid and phosphatidylserine were similar. The effect of added diacylglycerol or phorbol esters on the activation by fatty acids was not reported. In this study, we have investigated in more detail the kinetics of activation of protein kinase C by oleic acid, with a view to clarifying in particular the effect of diacylglycerol on the system. These experiments were undertaken using highly purified preparations of both the rat brain and the bovine spleen enzyme. During the
825
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
course of the work, Nishizuka's group reported that three distinct forms of protein kinase C isolated from rat brain were activated to similar extents by oleic acid, but that the activation was not enhanced by diacylglycerol (5). EXPERIMENTAL SECTION All chemicals were Analytical Reagent Grade, and buffers were prepared in distilled, deionized water. Oleic acid was obtained from Sigma Chemical Co. and was dissolved in redistilled ethanol. [32P]ATP was supplied by Bresatec. Protein kinase C activity was assayed at 25 °C essentially as described by Le Peuch et al. (6). Test assays (0.2ml) contained 0.5 mM CaC12, 10 mM MgC12, 5 mM dithiothreitol, 60 I.tg/ml or 180 I.tg/ml phosphatidylserine, 6 ~tg/ml diolein, 50 IJ2¢I [32p]ATP ( - 4 x 106 dpm) and 1 mg/ml lysine-rich histone in 0.02 M Tris buffer, pH 7.5. Control assays contained 0.5 mM EGTA instead of calcium, and no lipid. Samples (20 I-tl) of each assay were removed every 15 s and applied to phosphocellulose paper which was then washed exhaustively. The bound radioactivity was quantified by liquid scintillation counting. The kinetics of activation by fatty acids were studied at 25 °C using a modification of the routine assay, in which phospholipid was replaced by an aliquot of fatty acid dissolved in ethanol. The final concentration of ethanol was <1% (v/v). Protein concentrations were measured by a modification of the Lowry method (7). SDS-Polyacrylamide gel electrophoresis was performed according to Laemmli (8). Protein kinase C was isolated from rat brain (45 g of tissue) and bovine spleen (1000 g of tissue) by a modification of the method of Kaqd~wa et al. (9). The procedure employs sequential chromatographies on DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose. A chromatography on DEAETrisacryl was included as a final step in the spleen enzyme isolation. The detailed procedure for isolation of the spleen enzyme will be reported elsewhere. The final preparations were substantially pare on SDS-polyacrylamide gel electrophoresis. The maximum specific activities of the preparations were 2888 units/mg (rat brain enzyme) and 5100 units/mg (bovin6 spleen enzyme), where 1 unit of activity is the amount of enzyme which catalyses the transfer of 1 nmol of phosphate into histone per min at 25 °C. The ratio of activities in test and control assays was >100 for both preparations. The yields of enzyme activity in the final preparations were ~ 12% for the rat brain enzyme and ~ 15% for the bovine spleen enzyme. RESULTS AND DISCUSSION The activation of rat brain and bovine spleen protein kinase C by oleic acid is shown in Fig. 1. The kinetics of activation of the two enzymes appear virtually identical. The maximum activity for each enzyme,
60
:q
20
->
o
i150
m E IO0
N III
50
j I
I
L °
q 20
I
I
IO
0 I ~ I
I
II
I
[Oleic
[ 40 or
6 0j
o I
•
P l2~o
q 40O
a c i d ] (,uM)
Figure 1. Activation of vat brain protein kinase C (above) and bovine spleen protein kinase C (below) by oleic acid, in the presence ( • ) and absence ( O ) of diolein (9.7 laJvl). ( • ), activity in the presence of saturating phosphatidylserineand diolein; ( A ), activity in the absence of added lipid or calcium ions. 826
Vol. 152, No. 2, 1988
o,,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.
.
~0
I
I
I
r_
|
i'ls
5 .4o
~
2oi
Q
OI 0
I I 1 2 10-.5 [Oleio
~ 3 aoid]
i 4 -1 (Z -11
Q
0
zxzxl
2
~ I
4
I
6
I
8
10
[Diolein] ( u r n )
q
lOO
Fzgure 2. Double reciprocal plots of the data from Figure 1. Activities were measured in the presence of 9.7 I.tM diolein. Above, rat brain protein kinase C; below, bovine spleen protein kinase C.
Figure 3. Effect of diolein on the activation of rat brain protein kinase C by 25 ~ phosphatidylserine ( • ), and diolein alone ( A ).
oleic acid ( • ), 180 I.tg/ml
measured in the presence of 9.7 ~M diolein, and > 20 ~M oleic acid, is the same, within experimental error as the value obtained in the presence of saturating concentrations of phosphatidylserine and diolein. The concentrations of oleic acid required for half-maximal activation (KDapp) in the presence of 9.7 ~
diolein are - 5 ~tM and ~ 9 ~tM
for the rat brain and bovine spleen enzymes respectively. Values of KDapp were estimated from Fig. 1 since the corresponding double reciprocal plots were nonlinear (Fig. 2). Both enzymes exhibit a pronounced lag in the activation at low concentrations of fatty acid, and some inhibition is evident at higher concentrations (_>_100 txM). No measureable activation of either enzyme is seen at < 100 I.tM oleic acid in the a b s e n c e of diolein. At higher concentrations of fatty acid (200 - 800 I.tM), a small increase in activity above the control activity is observed. However this is at most only 10% of the maximum activity measured in the presence of saturating concentration of oleic acid plus diolein. The effect of varying the diolein concentration on the activity of the rat brain enzyme in the presence of 25 ~tM oleic acid is shown in Fig. 3. It is evident that the activation by 25 jaM oleic acid is enhanced at least 50-fold in the presence o f > 9 ~
diolein.
These results differ in several important respects from those of other investigators. The most notable discrepancy is the observed effect of diolein on the activation. Seldguchi et al. (5) report no enhancement of the activation by fatty acids in the presence of diolein, and a relatively small effect ( - 1.6 fold enhancement) was noted by McPhail et al. (1). Further, the values of KDapp obtained in the present work are an order of magnitude lower 827
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
than previously reported. From the data of Sekiguchi et al. (5) values of KDa~ for the activation by oleic acid of three separate forms of rat brain protein kinase C may be estimated to be ~ 100 laM (type I enzyme) and > 200 t.tM (types II and III enzyme). Murakami and Routtenberg (3) cite a value of 50 ~tM for the rat brain enzyme. It should be noted that these values were all measured in the absence of diolein. Our results show that protein kinase C is much more sensitive to activation by fatty acids than was previously indicated, and that this activation is strongly dependent on diacylglycerols. Whether the activation by fatty acids has any physiological significance remains to be determined. We are unable at present to account for the differences between our results and those of other investigators. It is, however, tempting to speculate that a preparation of enzyme which contained some residual neutral lipid might be more susceptible to activation by fatty acids in the absence of added diacylglycerol than a lipid-free preparation. It is most unlikely that we have isolated a form of protein kinase C which is distinct from any of those isolated in other laboratories, because the method of isolation is very similar to that employed elsewhere (5), and the final recoveries of enzyme activity are relatively high. It may be relevant that the specific activities of our purified enzymes, measured at 25 °C, are consistently higher than the value of 624 units/mg (30 °C) cited by Murakami et al. (4), or the value of 820 units/mg (30 °C) reported by Nishizuka's (9) group for their preparations of the rat brain enzyme. In the final chromatography step of our rat brain enzyme isolation (threonine-Sepharose), partial separation of several forms of the enzyme was obtained.
While the present
experiments have not yet been undertaken using completely resolved forms of the rat brain enzyme, no differences have been detected amongst the partially resolved enzymes with respect to the activation by oleic acid and its dependence on diacylglycerol. It is not clear in the case of the spleen preparation whether we are dealing with a single form of the enzyme or multiple forms.
The critical micelle concentration of oleic acid has not yet been determined under the conditions of our assays. However literature values are higher by at least 100-fold than the KDaW values obtained in this work (10). It is reasonable to assume therefore that the effective activator in our experiments is the monomeric fatty acid. The results shown in Fig. 1 bear a striking similarity to those obtained by Hannun et al. (I1), who examined the activation of rat brain protein kinase C by mixed micelles of Triton X-100, phosphatidylserine and diolein. In this system, each enzyme molecule is able to interact with a limited number of phospholipid and diolein molecules, the number depending on the mole ratios of the micellar components. Under these conditions the activation by phosphatidylserine is almost completely dependent on the presence of diolein, and there is a pronounced lag in the activation at low concentrations of phosphatidylserine which is very similar to that seen in the present work. These results suggest that the effect of diacylglycerols on protein kinase C activity is much larger when the activator 828
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(phospholipid or fatty acid) is present as the monomeric species, and further, that more than one molecule of phospholipid or fatty acid may be involved in the activation of the enzyme. REFERENCES 1.
2. 3. 4. 5. 6.
7. 8.
9. 10. 11.
McPhail, L.C., Clayton, C.C., and Snyderman, R. (1984) Science 224, 622-624. Horn, F., Gschwendt, M., and Marks, F. (1985) Eur. J. Biochem. 148, 533-538. Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193. Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429. Sekiguchi, K., Tsukuda, M., Ogita, K., Kikkawa, U., and Nishizuka, Y. (1987) Biochem. Biophys. Res. Commun. 145, 797-802. Le Peuch, C.J., Ballester, R., and Rosen, O.M. (1983) Proc. Natl. Acad. Sci. USA. 80, 6858-6862. Lowry, O.H., Rosenbrough, N.J., Fan', A.L., and Rondall, R.J. (1951) J. Biol. Chem. 193. 265-275. Laemmli, U.K. (1970) Nature 227, 680. Kikkawa, U., Go, M., Koumoto, I., and Nishizuka, Y. (1986) Biochem. Biophys. Res. Commun. 135, 636643. Mukerjee, P., and Mysels, K.J. (1971) National Standard Reference Data Service Vol 36, National Bureau of Standards, Washington, D.C. tLannun, Y.A., Loomis, C.R., and Bell, R.M.J. Biol. Chem. 260, 10039-10043.
829