ANALYTICAL
BIOCHEMISTRY
72, 593-599 (1976)
A Rapid and Sensitive Assay Method for Protein Kinase KUO-PING
HUANG
AND J. C. ROBINSON
Section on Developmental Enzymology, Laboratory of Biomedical Sciences. National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Mapland 20014 Received August 27. 1975; accepted January 30. 1976 A simplified assay method is described for the determination of protein kinase activity. Enzymatic activity is followed by measuring the incorporation of 32P from the terminal phosphoryl group of nucleoside triphosphates into protein substrate. Separation of the resulting 32P-labeled phosphoprotein from the unreacted 3ZP-labeled nucleoside triphosphate is achieved by chromatography of the reaction mixture on Gelman thin-layer chromatography medium for 5 min. The assay is shown to be more accurate than previous methods. Some properties of cyclic AMP-dependent and cyclic AMP-independent protein kinases from rabbit muscle are compared by using the assay method.
Since the discovery of cyclic AMP-dependent protein kinase (l), considerable effort has been focused on the study of protein kinases from different sources. This enzyme catalyzes the transfer of the terminal phosphoryl group of nucleoside triphosphates to serine and threonine residues in protein substrates. The method routinely used for the determination of protein kinase activity is based on the transfer of the 32Plabeled terminal phosphoryl group of a nucleoside triphosphate to protein substrate, the separation of the 32P-labeled protein from the unreacted 32P-labeled nucleoside triphosphate being achieved by precipitation of the protein with trichloroacetic acid. Removal of the contaminating 32Plabeled nucleoside triphosphate in the precipitated protein is achieved by repeated precipitation and washing (l-3), by prolonged washing of the precipitate on a filter disk (4,5), or by paper chromatography (6). All of these methods are relatively time-consuming. In this paper we describe a simple and accurate assay method for protein kinase. Separation of the labeled protein from unreacted nucleoside triphosphate is achieved by chromatography of the reaction mixture on Gelman chromatography medium (ITLC) for only 5 min. METHODS
Calf thymus histone, type II-A (histone mixture), type III-S (lysinerich fraction), and type VIII-S (arginine-rich fraction); protamine; phosvitin; ATP; GTP; trypsin; and subtilisin were purchased from Sigma Chemical Company. [y-32P]ATP and [-Y-~~P]GTP were obtained from 593 Copyright 0 1976 by Academic Pres\. Inc. All nghta of reproduction anany form rewrved.
594
HUANG
AND
ROBINSON
New England Nuclear Corporation. Vitamin-free casein (Nutritional Biochemicals) was suspended in water according to the method of Riemann et al. (4). Chromatographic sheet (ITLC type SG, 20 x 20 cm) was the product of Gelman Instrument Company. Cyclic AMP-dependent protein kinase from rabbit muscle was purified according to the method of Yamamuraet al. (7); the muscle protein kinase B, fraction was used. Cyclic AMP-independent phosvitin kinase was prepared from rabbit muscle by an isolation procedure which in essential details was identical to the method employed by Goldstein and Hasty (8) for the preparation of phosvitin kinase from rooster liver. Protein kinase activity was measured at 37°C in 0.1 ml of reaction mixture containing 25 mM /3-glycerophosphate, pH 7.0; 1 mM dithiothreitol; 0.5 mM EDTA; 1 mM magnesium acetate; 12.5 PM cyclic AMP; 0.3 mM ethylene glycol bis(P-amino-ethylether)-N,N’-tetraacetate; 0.07 mM [Y-~*P]ATP (400 to 1000 CPM/pmol); 200 pg of histone (for cyclic AMPdependent protein kinase) or 200 pg of phosvitin (for cyclic AMPindependent phosvitin kinase); and protein kinase. At timed intervals IO-p1 samples were spotted on the chromatographic strips (1.5 x 9.8 cm) at the origin (1.5 cm above the bottom edge) at a site which had previously received 15 ~1 of 20% TCA. Ascending chromatography was immediately carried out using a solvent system of 5% trichloroacetic acid containing 0.2 M KCI. An alternate procedure was used to stop the reaction by adding 10 ~1 of the reaction mixture to a test tube containing 20 ~1 of 25% acetic acid. A portion of the terminated reaction mixture was spotted on the chromatographic strip and developed immediately in the same solvent system. After developing for 5 min the strips were removed and dried with warm air. In this solvent system the reaction product (32P-labeled protein) remained at the origin, whereas [32P]ATP moved with the solvent front. A 2.5-cm segment of each strip, from 1.5 cm above to 1.0 cm below the starting line, was cut out and inserted into a scintillation vial. After adding 10 ml of Aquasol the radioactivity was measured in a scintillation counter. 32P-labeled proteins were prepared by incubating either histone or protamine with cyclic AMP-dependent protein kinase or by incubating either phosvitin or casein with cyclic AMP-independent phosvitin kinase in 0.3 ml of reaction mixture for 40 min. The reaction mixture which contained either histone, phosvitin, or casein was then applied to a Sephadex G-50 column (2.0 x 36 cm) previously equilibrated with 0.05 M tris-Cl buffer, pH 7.5, containing 1 mM dithiothreitol. The reaction mixture which contained protamine was applied to a Sephadex G-25 column (2.0 x 38 cm) previously equilibrated with 0.05 M tris-Cl buffer, pH 7.5, containing I mM dithiothreitol and 0.5 M KCl. Fractions of 3 ml were collected. The radioactive protein peak eluted at the void volume was pooled and concentrated in an Amicon ultrafiltration cell fitted with either a UM10 membrane (for histone, phosvitin, and casein) or a UM-05 membrane (for protamine).
ASSAY
METHOD
FOR PROTEIN
595
KlNASE
CPr/l PER SEGMENT 36503
43
111
83
137
272
12
20
27
16
ORIGIN
A
B
C
D
E
57600
1w
56904
177
1395
54
37
32903
9534
7587
F
G
H
FIG. 1. Chromatography of 32P-labeled nucleoside triphosphates and 3ZP-labeled protein on Gelman chromatography medium. After 5 min of chromatography in 5% trichloroacetic acid containing 0.2 M KCI, the strip was cut into one 25cm segment and six I-cm segments. Each segment was counted in scintillation counter. A, [Y-~*PJATP; B, [y-3ZP]GTP; C, [32P]phosvitin; D, [32P]histone II-A; E, [32P]casein; F, [32P]protamine; G, mixture of [32P]phosvitin and [yz2P]ATP; H, mixture of [32P]histone H-A and [Y-~~P]ATP. The radioactivity recovered from each strip accounted for the total amount of radioactivity applied.
RESULTS
AND DISCUSSION
The separation of radioactive ATP, GTP, phosvitin, histone, casein, and protamine on Gelman chromatography medium (ITLC) is shown in Fig. 1. Both ATP and GTP (Fig. 1, A and B) moved away from the origin, whereas phosvitin, histone, casein, and protamine (Fig. 1, C, D, E,
1
60 Time bin)
FIG. 2. Effect of incubation of [32P]histone with trypsin and subtilisin on the retention of radioactivity at the origin. To 120 pg of [3ZP]histone (25,000 cpm), either 2 fig of trypsin (- 0 -) or 30 pg of subtilisin (- 0 -) was added: Ten microliter samples were taken at timed intervals and chromatographed as described under Methods.
596
HUANG
AND ROBINSON
and F) remained at the origin. The recovery of 32P-labeled histone, phosvitin, and casein at the origin of the chromatography strip was quantitative, and the recovery of 32P-protamine was over 95%. Good separation between ATP and phosvitin (Fig. 1, G) and between ATP and histone (Fig. 1, H) was observed when the mixtures were chromatographed in 5% trichloroacetic acid containing 0.2 M KCl. A similar type of separation was observed between ATP and casein and between ATP and protamine. The separation was not affected by the addition of either bovine serum albumin (up to 50 pug) or ATP (up to 300 nmol) to each chromatographic strip. The quantitative retention of 3ZP-labeled protein at the origin on the ITLC strip after chromatography appears to be dependent upon both the precipitation of the protein by the solvent system and the adsorptive properties of the ITLC chromatographic medium. This view is based on the finding that 32P-labeled histone and protamine could not be quantitatively precipitated by 5% trichloroacetic acid containing 0.2 M KCI. Incubation of 32P-labeled histone with either trypsin or subtilisin progressively reduced the amount of radioactivity retained at the origin after chromatography (Fig. 2). Prolonged incubation of 32P-labeled histone with subtilisin, a relatively nonspecific protease, completely eliminated the radioactivity remaining at the origin. All the radioactivity applied to the strip was recovered from the solvent front. However, in the early stage of digestion by subtilisin or the partial digestion by trypsin the radioactivity was found to spread over the entire strip after chromatography. Similar results were also obtained when 32P-labeled phosvitin was incubated with these proteases. The recovery of 32P-labeled protein by the method presently described was compared with those reported in the literature (Table 1). Although most of the previous methods are adequate for recovery of [32P]phosvitin, TABLE COMPARISON
OF THE
Method ITLC Reimann ef al. (4) Tao and Hackett (5) Walsh et al. b (2)
RECOVERY
1
OF 3ZP-L~~~~~~
PROTEIN BY DIFFERENT METHOD@
[3*P]histone II-A recovered (%I 99 62 81 69
[3*P]phosvitin recovered (W 100 97 90 76
a The data were obtained by adding 200 pg of “P-labeled protein (2 x lo5 cpm) to the assay mixture without [y-32P]ATP. Duplicate samples were treated according to the various assay methods. b For the precipitation of histone, 24% trichloroacetic acid was used (9). For the precipitation of phosvitin, 10% trichloroacetic was used (8). standard
ASSAY
METHOD
FOR PROTEIN
KINASE
597
none gave good results for the recovery of [3ZP]histone. The poor recovery of [32P]histone by the published methods may be due to the incomplete precipitation of histone by trichloroacetic acid. The present method gave quantitative recoveries of both [32P]histone and [32P]phosvitin. Direct comparison of the previously reported methods and the ITLC method for measuring the incorporation of 32P into histone and phosvitin by protein kinase showed relative activities which paralleled the relative recoveries shown in Table 1. Thus, the higher activity observed with the ITLC method was accounted for by the quantitative recovery of [32P]protein in this procedure. While this work was in progress, Witt and Roskoski (10) reported an assay method for protein kinase using phosphocellulose paper to separate 32P-labeled histone from [y-32P]ATP. As compared with the method of Reimann and co-workers (4), the phosphocellulose-paper method gave a higher protein kinase activity when histone was used as substrate; however, a considerably lower activity was observed when phosvitin was used as substrate. The presently described ITLC method gave higher protein kinase activity with each of these substrates than did the method of Reimann and co-workers (4). This assay method was used in a number of experiments to characterize the protein kinase reaction. The dependence of 32P incorporation on the time of incubation is shown in Fig. 3. The incorporation of 32P into histone by cyclic AMP-dependent protein kinase (Fig. 3A) was linearly related to time for at least 5 min. The activity in the presence of cyclic AMP was increased about 16-fold. When GTP was used as phosphoryl donor,
Time hid
FIG. 3. Dependence of 32P incorporation on the time of incubation. For Fig. A. cyclic AMP-dependent protein kinase was used. The conditions were: histone as substrate and ATP as phosphoryl donor in the presence (- 0 -) and in the absence (- 0 -4 of cyclic AMP; histone as substrate and GTP as phosphoryl donor in the presence of cyclic AMP (- n -). For Fig. B, cyclic AMP-independent phosvitin kinase was used. The conditions were: phosvitin as substrate and ATP as phosphoryl donor in the presence (- 0 -) and in the absence (- 0 -) of cyclic AMP; phosvitin as substrate and GTP as phosphoryl donor in the presence of cyclic AMP (- n -).
598
HUANG
AND
ROBINSON
pg of Protein
FIG. 4. Dependence was carried out under cyclic AMP-dependent independent phosvitin
of protein kinase activity on concentration of protein. The assay standard assay conditions by using histone II-A as substrate for protein kinase (- 0 -) and phosvitin as substrate for cyclic AMPkinase (- 0 -).
cyclic AMP-dependent protein kinase did not detectably phosphorylate histone. The phosphorylation reaction catalyzed by rabbit muscle phosvitin kinase (Fig. 3B) was linear with the time of incubation; the reaction rate was independent of the addition of cyclic AMP; GTP was 50% as efficient as ATP. The reaction rate of the protein kinase-catalyzed reaction was linearly related to the amount of protein used in the assay (Fig. 4). The phosphorylation of various protein substrates by both kinases is shown in Fig. 5. For cyclic AMP-dependent protein kinase (Fig. 5A), both histone and protamine are readily phosphorylated, while casein and
-0
5
10
15
20
0
5
10
15
20
Time (mid
FIG. 5. Comparison of the phosphorylation of various protein substrates by cyclic AMP-dependent protein kinase (Fig. A) and cyclic AMP-independent phosvitin kinase (Fig. B). The reactions were carried out under standard assay conditions. The substrates were: histone III-S (lysine-rich fraction) (- 0 -); histone II-A (histone mixture) (-- 0 -); histone VIII-S (arginine-rich fraction) (- n -); protamine (- 0 -9; casein (-- A -): phosvitin (- a -).
ASSAY
METHOD
FOR PROTEIN TABLE
COMPARISON OF KINETIC AMP-DEPENDENT
599
KINASE
2
CONSTANTS OF PROTEIN SUBSTANCES FOR CYCLIC PROTEIN KINASE AND PHOSVITIN KINA@
Cyclic-AMP dependent protein kinase V mns
Phosvitin kinase V Wl”Z
Protein substrate
(nmol 3ZPlmin/mg enzyme)
(mdml)
Lysine-rich histone Histone mixture Arginine-rich histone Protamine Casein Phosvitin
892.5 250 120 56.3 12.5 -
1.6 0.35 0.45 0.05 0.9 -
K ,,,
(nmol 32P/min/mg enzyme)
K, hdml)
79.3
9.1
83.3
1.6
o The reactions were carried out under standard assay conditions except that the ATP concentration was 0.25 mM and the protein substrate concentration was varied between 0.04 and 4 mg/ml. The kinetic constants of those protein substrates which had insignificant amount of activity over the entire range of concentration are not indicated.
phosvitin are poorly phosphorylated. In contrast, phosvitin and casein are readily phosphorylated by phosvitin kinase; nevertheless, histone and protamine are not (Fig. 5B). The comparison of kinetic constants of protein substrates for both cyclic AMP-dependent protein kinase and phosvitin kinase is shown in Table 2. For cyclic AMP-dependent protein kinase, the order of decrease in I/,,, is lysine-rich histone, histone mixture, arginine-rich histone, protamine, and casein; the order of increase in K, is protamine, histone mixture, arginine-rich histone, casein, and lysine-rich histone. Casein which has relatively low I/,,, and high K, as compared with histone is a poor substrate (I 1). For phosvitin kinase, the V mas for both casein and phosvitin are similar; however, the K, for phosvitin is lower than the K, for casein. REFERENCES 1. Walsh, D. A., Perkins, J. P., and Krebs, E. G. (1968)J. Biol. Chem. 243, 3763-3765. 2. Walsh, D. A., Perkins, J. P., Brostrom. C. 0.. Ho, E. S.. and Krebs, E. G. (1971) J. Biol. Chem. 246, 1968-1976. 3. Kuo, J. F., and Greengard, P. (197O)J. Biol. Chem. 245, 2493-2498. 4. Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971)3. Biol. Chem. 246, 1968- 1995. 5. Tao, M.. and Hackett, P. (1973)J. Biol. Chem. 248, 5324-5332. 6. Li, H-C., and Felmby, D. A. (1973)Anal. Biochem. 52, 300-304. 7. Yamamura, H., Nishiyama, K., Shimonura. R.. andNishizuka. Y. (1973)Biochemistry 12, 856-862.
Goldstein, J. L., and Hasty, M. A. (1973)J. Biol. Chem. 248, 6300-6307. 9. Chen, L-J., and Walsh, D. A. (1971) Biochemistry 10, 3614-3621. 10. Witt, J. J.. and Roskoski, R. Jr. (1973) Anal. Biochem. 66, 253-258. 11. Bylttnd, D. B.. and Krebs. E. G. (1975)J. Biol. Chem. 250, 6355-6361. 8.
BIOCHEMISTRY
72, 593-599 (1976)
A Rapid and Sensitive Assay Method for Protein Kinase KUO-PING
HUANG
AND J. C. ROBINSON
Section on Developmental Enzymology, Laboratory of Biomedical Sciences. National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Mapland 20014 Received August 27. 1975; accepted January 30. 1976 A simplified assay method is described for the determination of protein kinase activity. Enzymatic activity is followed by measuring the incorporation of 32P from the terminal phosphoryl group of nucleoside triphosphates into protein substrate. Separation of the resulting 32P-labeled phosphoprotein from the unreacted 3ZP-labeled nucleoside triphosphate is achieved by chromatography of the reaction mixture on Gelman thin-layer chromatography medium for 5 min. The assay is shown to be more accurate than previous methods. Some properties of cyclic AMP-dependent and cyclic AMP-independent protein kinases from rabbit muscle are compared by using the assay method.
Since the discovery of cyclic AMP-dependent protein kinase (l), considerable effort has been focused on the study of protein kinases from different sources. This enzyme catalyzes the transfer of the terminal phosphoryl group of nucleoside triphosphates to serine and threonine residues in protein substrates. The method routinely used for the determination of protein kinase activity is based on the transfer of the 32Plabeled terminal phosphoryl group of a nucleoside triphosphate to protein substrate, the separation of the 32P-labeled protein from the unreacted 32P-labeled nucleoside triphosphate being achieved by precipitation of the protein with trichloroacetic acid. Removal of the contaminating 32Plabeled nucleoside triphosphate in the precipitated protein is achieved by repeated precipitation and washing (l-3), by prolonged washing of the precipitate on a filter disk (4,5), or by paper chromatography (6). All of these methods are relatively time-consuming. In this paper we describe a simple and accurate assay method for protein kinase. Separation of the labeled protein from unreacted nucleoside triphosphate is achieved by chromatography of the reaction mixture on Gelman chromatography medium (ITLC) for only 5 min. METHODS
Calf thymus histone, type II-A (histone mixture), type III-S (lysinerich fraction), and type VIII-S (arginine-rich fraction); protamine; phosvitin; ATP; GTP; trypsin; and subtilisin were purchased from Sigma Chemical Company. [y-32P]ATP and [-Y-~~P]GTP were obtained from 593 Copyright 0 1976 by Academic Pres\. Inc. All nghta of reproduction anany form rewrved.
594
HUANG
AND
ROBINSON
New England Nuclear Corporation. Vitamin-free casein (Nutritional Biochemicals) was suspended in water according to the method of Riemann et al. (4). Chromatographic sheet (ITLC type SG, 20 x 20 cm) was the product of Gelman Instrument Company. Cyclic AMP-dependent protein kinase from rabbit muscle was purified according to the method of Yamamuraet al. (7); the muscle protein kinase B, fraction was used. Cyclic AMP-independent phosvitin kinase was prepared from rabbit muscle by an isolation procedure which in essential details was identical to the method employed by Goldstein and Hasty (8) for the preparation of phosvitin kinase from rooster liver. Protein kinase activity was measured at 37°C in 0.1 ml of reaction mixture containing 25 mM /3-glycerophosphate, pH 7.0; 1 mM dithiothreitol; 0.5 mM EDTA; 1 mM magnesium acetate; 12.5 PM cyclic AMP; 0.3 mM ethylene glycol bis(P-amino-ethylether)-N,N’-tetraacetate; 0.07 mM [Y-~*P]ATP (400 to 1000 CPM/pmol); 200 pg of histone (for cyclic AMPdependent protein kinase) or 200 pg of phosvitin (for cyclic AMPindependent phosvitin kinase); and protein kinase. At timed intervals IO-p1 samples were spotted on the chromatographic strips (1.5 x 9.8 cm) at the origin (1.5 cm above the bottom edge) at a site which had previously received 15 ~1 of 20% TCA. Ascending chromatography was immediately carried out using a solvent system of 5% trichloroacetic acid containing 0.2 M KCI. An alternate procedure was used to stop the reaction by adding 10 ~1 of the reaction mixture to a test tube containing 20 ~1 of 25% acetic acid. A portion of the terminated reaction mixture was spotted on the chromatographic strip and developed immediately in the same solvent system. After developing for 5 min the strips were removed and dried with warm air. In this solvent system the reaction product (32P-labeled protein) remained at the origin, whereas [32P]ATP moved with the solvent front. A 2.5-cm segment of each strip, from 1.5 cm above to 1.0 cm below the starting line, was cut out and inserted into a scintillation vial. After adding 10 ml of Aquasol the radioactivity was measured in a scintillation counter. 32P-labeled proteins were prepared by incubating either histone or protamine with cyclic AMP-dependent protein kinase or by incubating either phosvitin or casein with cyclic AMP-independent phosvitin kinase in 0.3 ml of reaction mixture for 40 min. The reaction mixture which contained either histone, phosvitin, or casein was then applied to a Sephadex G-50 column (2.0 x 36 cm) previously equilibrated with 0.05 M tris-Cl buffer, pH 7.5, containing 1 mM dithiothreitol. The reaction mixture which contained protamine was applied to a Sephadex G-25 column (2.0 x 38 cm) previously equilibrated with 0.05 M tris-Cl buffer, pH 7.5, containing I mM dithiothreitol and 0.5 M KCl. Fractions of 3 ml were collected. The radioactive protein peak eluted at the void volume was pooled and concentrated in an Amicon ultrafiltration cell fitted with either a UM10 membrane (for histone, phosvitin, and casein) or a UM-05 membrane (for protamine).
ASSAY
METHOD
FOR PROTEIN
595
KlNASE
CPr/l PER SEGMENT 36503
43
111
83
137
272
12
20
27
16
ORIGIN
A
B
C
D
E
57600
1w
56904
177
1395
54
37
32903
9534
7587
F
G
H
FIG. 1. Chromatography of 32P-labeled nucleoside triphosphates and 3ZP-labeled protein on Gelman chromatography medium. After 5 min of chromatography in 5% trichloroacetic acid containing 0.2 M KCI, the strip was cut into one 25cm segment and six I-cm segments. Each segment was counted in scintillation counter. A, [Y-~*PJATP; B, [y-3ZP]GTP; C, [32P]phosvitin; D, [32P]histone II-A; E, [32P]casein; F, [32P]protamine; G, mixture of [32P]phosvitin and [yz2P]ATP; H, mixture of [32P]histone H-A and [Y-~~P]ATP. The radioactivity recovered from each strip accounted for the total amount of radioactivity applied.
RESULTS
AND DISCUSSION
The separation of radioactive ATP, GTP, phosvitin, histone, casein, and protamine on Gelman chromatography medium (ITLC) is shown in Fig. 1. Both ATP and GTP (Fig. 1, A and B) moved away from the origin, whereas phosvitin, histone, casein, and protamine (Fig. 1, C, D, E,
1
60 Time bin)
FIG. 2. Effect of incubation of [32P]histone with trypsin and subtilisin on the retention of radioactivity at the origin. To 120 pg of [3ZP]histone (25,000 cpm), either 2 fig of trypsin (- 0 -) or 30 pg of subtilisin (- 0 -) was added: Ten microliter samples were taken at timed intervals and chromatographed as described under Methods.
596
HUANG
AND ROBINSON
and F) remained at the origin. The recovery of 32P-labeled histone, phosvitin, and casein at the origin of the chromatography strip was quantitative, and the recovery of 32P-protamine was over 95%. Good separation between ATP and phosvitin (Fig. 1, G) and between ATP and histone (Fig. 1, H) was observed when the mixtures were chromatographed in 5% trichloroacetic acid containing 0.2 M KCl. A similar type of separation was observed between ATP and casein and between ATP and protamine. The separation was not affected by the addition of either bovine serum albumin (up to 50 pug) or ATP (up to 300 nmol) to each chromatographic strip. The quantitative retention of 3ZP-labeled protein at the origin on the ITLC strip after chromatography appears to be dependent upon both the precipitation of the protein by the solvent system and the adsorptive properties of the ITLC chromatographic medium. This view is based on the finding that 32P-labeled histone and protamine could not be quantitatively precipitated by 5% trichloroacetic acid containing 0.2 M KCI. Incubation of 32P-labeled histone with either trypsin or subtilisin progressively reduced the amount of radioactivity retained at the origin after chromatography (Fig. 2). Prolonged incubation of 32P-labeled histone with subtilisin, a relatively nonspecific protease, completely eliminated the radioactivity remaining at the origin. All the radioactivity applied to the strip was recovered from the solvent front. However, in the early stage of digestion by subtilisin or the partial digestion by trypsin the radioactivity was found to spread over the entire strip after chromatography. Similar results were also obtained when 32P-labeled phosvitin was incubated with these proteases. The recovery of 32P-labeled protein by the method presently described was compared with those reported in the literature (Table 1). Although most of the previous methods are adequate for recovery of [32P]phosvitin, TABLE COMPARISON
OF THE
Method ITLC Reimann ef al. (4) Tao and Hackett (5) Walsh et al. b (2)
RECOVERY
1
OF 3ZP-L~~~~~~
PROTEIN BY DIFFERENT METHOD@
[3*P]histone II-A recovered (%I 99 62 81 69
[3*P]phosvitin recovered (W 100 97 90 76
a The data were obtained by adding 200 pg of “P-labeled protein (2 x lo5 cpm) to the assay mixture without [y-32P]ATP. Duplicate samples were treated according to the various assay methods. b For the precipitation of histone, 24% trichloroacetic acid was used (9). For the precipitation of phosvitin, 10% trichloroacetic was used (8). standard
ASSAY
METHOD
FOR PROTEIN
KINASE
597
none gave good results for the recovery of [3ZP]histone. The poor recovery of [32P]histone by the published methods may be due to the incomplete precipitation of histone by trichloroacetic acid. The present method gave quantitative recoveries of both [32P]histone and [32P]phosvitin. Direct comparison of the previously reported methods and the ITLC method for measuring the incorporation of 32P into histone and phosvitin by protein kinase showed relative activities which paralleled the relative recoveries shown in Table 1. Thus, the higher activity observed with the ITLC method was accounted for by the quantitative recovery of [32P]protein in this procedure. While this work was in progress, Witt and Roskoski (10) reported an assay method for protein kinase using phosphocellulose paper to separate 32P-labeled histone from [y-32P]ATP. As compared with the method of Reimann and co-workers (4), the phosphocellulose-paper method gave a higher protein kinase activity when histone was used as substrate; however, a considerably lower activity was observed when phosvitin was used as substrate. The presently described ITLC method gave higher protein kinase activity with each of these substrates than did the method of Reimann and co-workers (4). This assay method was used in a number of experiments to characterize the protein kinase reaction. The dependence of 32P incorporation on the time of incubation is shown in Fig. 3. The incorporation of 32P into histone by cyclic AMP-dependent protein kinase (Fig. 3A) was linearly related to time for at least 5 min. The activity in the presence of cyclic AMP was increased about 16-fold. When GTP was used as phosphoryl donor,
Time hid
FIG. 3. Dependence of 32P incorporation on the time of incubation. For Fig. A. cyclic AMP-dependent protein kinase was used. The conditions were: histone as substrate and ATP as phosphoryl donor in the presence (- 0 -) and in the absence (- 0 -4 of cyclic AMP; histone as substrate and GTP as phosphoryl donor in the presence of cyclic AMP (- n -). For Fig. B, cyclic AMP-independent phosvitin kinase was used. The conditions were: phosvitin as substrate and ATP as phosphoryl donor in the presence (- 0 -) and in the absence (- 0 -) of cyclic AMP; phosvitin as substrate and GTP as phosphoryl donor in the presence of cyclic AMP (- n -).
598
HUANG
AND
ROBINSON
pg of Protein
FIG. 4. Dependence was carried out under cyclic AMP-dependent independent phosvitin
of protein kinase activity on concentration of protein. The assay standard assay conditions by using histone II-A as substrate for protein kinase (- 0 -) and phosvitin as substrate for cyclic AMPkinase (- 0 -).
cyclic AMP-dependent protein kinase did not detectably phosphorylate histone. The phosphorylation reaction catalyzed by rabbit muscle phosvitin kinase (Fig. 3B) was linear with the time of incubation; the reaction rate was independent of the addition of cyclic AMP; GTP was 50% as efficient as ATP. The reaction rate of the protein kinase-catalyzed reaction was linearly related to the amount of protein used in the assay (Fig. 4). The phosphorylation of various protein substrates by both kinases is shown in Fig. 5. For cyclic AMP-dependent protein kinase (Fig. 5A), both histone and protamine are readily phosphorylated, while casein and
-0
5
10
15
20
0
5
10
15
20
Time (mid
FIG. 5. Comparison of the phosphorylation of various protein substrates by cyclic AMP-dependent protein kinase (Fig. A) and cyclic AMP-independent phosvitin kinase (Fig. B). The reactions were carried out under standard assay conditions. The substrates were: histone III-S (lysine-rich fraction) (- 0 -); histone II-A (histone mixture) (-- 0 -); histone VIII-S (arginine-rich fraction) (- n -); protamine (- 0 -9; casein (-- A -): phosvitin (- a -).
ASSAY
METHOD
FOR PROTEIN TABLE
COMPARISON OF KINETIC AMP-DEPENDENT
599
KINASE
2
CONSTANTS OF PROTEIN SUBSTANCES FOR CYCLIC PROTEIN KINASE AND PHOSVITIN KINA@
Cyclic-AMP dependent protein kinase V mns
Phosvitin kinase V Wl”Z
Protein substrate
(nmol 3ZPlmin/mg enzyme)
(mdml)
Lysine-rich histone Histone mixture Arginine-rich histone Protamine Casein Phosvitin
892.5 250 120 56.3 12.5 -
1.6 0.35 0.45 0.05 0.9 -
K ,,,
(nmol 32P/min/mg enzyme)
K, hdml)
79.3
9.1
83.3
1.6
o The reactions were carried out under standard assay conditions except that the ATP concentration was 0.25 mM and the protein substrate concentration was varied between 0.04 and 4 mg/ml. The kinetic constants of those protein substrates which had insignificant amount of activity over the entire range of concentration are not indicated.
phosvitin are poorly phosphorylated. In contrast, phosvitin and casein are readily phosphorylated by phosvitin kinase; nevertheless, histone and protamine are not (Fig. 5B). The comparison of kinetic constants of protein substrates for both cyclic AMP-dependent protein kinase and phosvitin kinase is shown in Table 2. For cyclic AMP-dependent protein kinase, the order of decrease in I/,,, is lysine-rich histone, histone mixture, arginine-rich histone, protamine, and casein; the order of increase in K, is protamine, histone mixture, arginine-rich histone, casein, and lysine-rich histone. Casein which has relatively low I/,,, and high K, as compared with histone is a poor substrate (I 1). For phosvitin kinase, the V mas for both casein and phosvitin are similar; however, the K, for phosvitin is lower than the K, for casein. REFERENCES 1. Walsh, D. A., Perkins, J. P., and Krebs, E. G. (1968)J. Biol. Chem. 243, 3763-3765. 2. Walsh, D. A., Perkins, J. P., Brostrom. C. 0.. Ho, E. S.. and Krebs, E. G. (1971) J. Biol. Chem. 246, 1968-1976. 3. Kuo, J. F., and Greengard, P. (197O)J. Biol. Chem. 245, 2493-2498. 4. Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971)3. Biol. Chem. 246, 1968- 1995. 5. Tao, M.. and Hackett, P. (1973)J. Biol. Chem. 248, 5324-5332. 6. Li, H-C., and Felmby, D. A. (1973)Anal. Biochem. 52, 300-304. 7. Yamamura, H., Nishiyama, K., Shimonura. R.. andNishizuka. Y. (1973)Biochemistry 12, 856-862.
Goldstein, J. L., and Hasty, M. A. (1973)J. Biol. Chem. 248, 6300-6307. 9. Chen, L-J., and Walsh, D. A. (1971) Biochemistry 10, 3614-3621. 10. Witt, J. J.. and Roskoski, R. Jr. (1973) Anal. Biochem. 66, 253-258. 11. Bylttnd, D. B.. and Krebs. E. G. (1975)J. Biol. Chem. 250, 6355-6361. 8.
