436
RENATURATION OF PROTEIN KINASES
[36]
[36] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h i c S e p a r a t i o n a n d R e n a t u r a t i o n o f P r o t e i n K i n a s e S u b u n i t s : A p p l i c a t i o n to Catalytic Subunit of Phosphorylase Kinase
By SCOTT M. KEE, CmUN-JYE YUAN, and DONALD J. GRAVES Any attempt to relate the catalytic and regulatory properties of an oligomeric enzyme to its structure involves an understanding of the roles of the different subunits and how they interact with one another. Perhaps the most efficient means of arriving at such understanding is the characterization of isolated subunits in their native states. For some enzymes, however, dissociation and isolation of subunits under nondenaturing conditions are extremely difficult because of strong intersubunit bonding. Such is the case with phosphorylase kinase. ~ This enzyme plays an important role in the regulation of glycogenolysis and consists of four different subunits, a,/3, y, and 8, with a stoichiometry of Ot4/34'Y4~4 .2-4 The a and 13 subunits are regulatory subunits. 2,5 The 8 subunit is shown to be identical to the calcium-binding protein, calmodulin (CAM), by its primary structure and ability to activate CaM-dependent enzymes. 4 The catalytic role of y was proved by Skuster et al. 6 based on the treatment of the holoenzyme with LiBr, a denaturant. Dissociation of the enzyme into its subunits occurred and an active fraction containing the y subunit was obtained by gel filtration and chromatography. Subsequently, catalytically active subunit complexes of ay6 and y8 were found to retain the full catalytic activity of the holoenzyme. 7 Amino acid sequencing provided proof that the 3/subunit contains structural elements similar to those found in the active region of other protein kinases. 8 The catalytic properties of the active y subunit have been described. 9 Methods are described here for the separation of phosphorylase kinase I G. M. Carlson, P. J. Bechtel, and D. J. Graves, Adv. Enzymol. 50, 41 (1979). 2 p. Cohen, Eur. J. Biochem. 34, 1 (1973). T. Hayakawa, J. P. Perkins, D. A. Walsh, and E. G. Krebs, Biochemistry 12, 567 (1973). 4 p. Cohen, A. Burchell, J. G. Foulkes, P. T. W. Cohen, T. C. Vanaman, and A. C. Nairn, FEBS Lett. 92, 287 (1978). 5 T. Hayakawa, J. P. Perkins, and E. G. Krebs, Biochemistry 12, 574 (1973). 6 j. R. Skuster, K.-F. J. Chan, and D. J. Graves, J. Biol. Chem. 255, 2203 (1980). 7 K.-F. J. Chan and D. J. Graves, J. Biol. Chem. 257, 5948 (1982). 8 E. M. Reimann, K. Titani, L. H. Ericsson, R. D. Wade, E. H. Fischer, and D. A. Walsh, Biochemistry 23, 4185 (1984). 9 S. M. Kee and D. J. Graves, J. Biol. Chem. 262, 9448 (1987).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright© 1991by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[36]
H P L C OF PROTEIN KINASE SUBUNITS
437
subunits under denaturing conditions and subsequent reactivation of the 3t subunit ~°'~ and for renaturation of the bacterial expressed form of the subunit of phosphorylase kinase) 2'13 Materials
and Methods
Phosphorylase kinase can be prepared from rabbit skeletal muscle as described by Hayakawa et al. 3 Further purification is obtained by using DEAE-cellulose chromatography. 2 Phosphorylase b is prepared as described by Fischer and Krebs. 14 The kinase activity may be assayed as described by Kee and Graves. 9 The standard assay mixture contains 62 mM HEPES, 42 mM Tris, 2.5 mM dithiothreitol, 0.1 mM CaC12, 10 mM MgC12 , 3 mM ATP, and 100 /xM phosphorylase. The final pH of the reaction mixture is 8.2. The reactions are performed at 30° and initiated by adding MgATP. The reaction is terminated by dilution into ice-cold buffer followed by phosphorylase assay. Phosphorylase activity can be determined directly by measuring the amount of inorganic phosphate released from glucose 1-phosphate in the reaction of glycogen synthesis.~5 H i g h - P e r f o r m a n c e Liquid Chromatographic Separation o f Phosphorylase Kinase Subunits
The separation of phosphorylase kinase subunits on reversed-phase high-performance liquid chromatography (HPLC) was investigated by Crabb and Heilm eyer ) 6 They were successful in completely separating the four subunits using programmed gradients that started with 0.1% (v/v) trifluoroacetic acid (TFA) in water and finished with 0.1% TFA in 62% (v/v) acetonitrile. The subunits are eluted according to size, with the 8 subunit eluting first between 43 and 50% acetonitrile and tz subunit eluting last at about 58% acetonitrile. They found that optimal resolution and recovery was dependent on the amount of protein applied. With a 5-/zm Vydac C18 column (25 x 0.46 cm i.d.), for example, phosphorylase kinase l0 S. M. Kee and D. J. Graves, J. Biol. Chem. 261, 4732 (1986). 11C.-J. Yuan and D. J. Graves, Arch. Biochem. Biophys. 274, 317 (1989). t~ L.-R. Chen, C.-J. Yuan, G. Somasekhar, P. Wejksnora, J. E. Peterson, A. M. Myers, L. Graves, P. T. W. Cohen, E. F. da Cruz e Silva, and D. J. Graves,Biochem. Biophys. Res. Commun. 161, 746 (1989). 13L.-R. Chen, C.-J. Yuan, G. Somasekhar,P. Wejksnora, J. E. Peterson,A. M. Myers, and D. J. Graves, in "Informationtransduction and processing systems from cell to whole body" (O. Hatase and J. E. Wang, eds.), p. 19. Elsevier, New York, 1989. 14E. H. Fischer and E. G. Krebs, J. Biol. Chem. 231, 65 (1958). 15B. Illingworthand G. T. Coli, Biochem. Prep. 3, 1 (1953). 16j. W. Crabb and L. M. G. Heilmeyer,Jr., J. Chromatogr. 296, 129 (1984).
438
RENATURATION OF PROTEIN KINASES
[36]
applications of greater than 0.3 mg result in decreased recovery of all subunits and partial contamination of the 13 subunit with the a subunit. When 0.25 mg sample is applied, individual subunit recoveries ranged from 66 to 88%. A much shorter column (3-6 cm) has little effect on resolution and it is found that a 6 × I cm column allowed higher yields with higher loads while still maintaining excellent resolution. Procedure. Separation of phosphorylase kinase for the purpose of recovering an isolated, catalytically active 3' subunit is achieved using a 5-/zm Vydac C18 column (25 x 0.46 cm i.d.) and a gradient program and other conditions as described by Crabb and Heilmeyer.17 Phosphorylase kinase (1-2 mg/ml in 50 m M glycerol 1-phosphate, pH 7.0, 2 mM EDTA, and 1 mM dithiothreitol) is injected directly to the column previously equilibrated in 0.1% TFA. The 3' subunit is eluted at approximately 50% acetonitrile and 0.09% TFA, with a pH of 2.5. Fractions containing the 3' subunit can be stored in plastic tubes on ice for 1 to 2 weeks. The 3' subunit prepared in this way is pure, judged by SDS-PAGE.I°
Renaturation of High-Performance Liquid Chromatography-Isolated 3"Subunit of Phosphorylase Kinase Initially, the HPLC-recovered 3' subunit (about 100/zg/ml in HPLC solvent) has no activity when assayed according to the usual conditions, with no dilution or after a 20-fold dilution in 40 mM HEPES, pH 6.8, and 5 mM dithiothreitol (buffer A) before the assay. Presumably, the 3' subunit is denatured after undergoing exposure to the low pH and high organic solvent concentrations of the HPLC solvents. A small amount of activity (about 2% of the molar specific activity of the holoenzyme) is observed, however, when the 3' subunit is assayed in the presence of Ca 2+ and the 8 subunit of phosphorylase kinase or its equivalent, CaM. The activity is significantly enhanced if 3' is preincubated with calmodulin and Ca 2+. Because CaM interacts strongly with the 3' subunit the presence of native CaM in the renaturation medium is thought to affect the renaturation process by binding to the 3' subunit and influencing its folding or by trapping the 3' in its native state, facilitating further conversion of denatured 3' to native y. Procedure. The HPLC-purified 3' subunit is diluted to 1-5/~g/ml in an ice-cold solution containing one part assay buffer (0.25 M HEPES, 0.25 M Tris, 0.6 m M CaC12, pH 8.6) and four parts buffer A plus 60-100/zg/ml calmodulin and 1.5 mg/ml phosphorylase b or bovine serum albumin. This results in a final pH of 8.2 and final concentrations of no more than 5% 17j. W. Crabb and L. M. G. H¢ilmeyer, Jr., J. Biol. Chem. 259, 6346 (1984).
[36]
H P L C OF PROTEIN KINASE SUBUNITS 400
'
'
'
'
'
20
30
40
60
439
~O
300
__. 200 I.--
lOOI
0
I0
60
R e a c t i v a t i o n Time (rain)
FIG. I. Temperature effects on reactivation of 3' subunit.
acetonitrile and 0.01% TFA. The reactivation mixture is kept on ice for several days to attain optimum catalytic activity. With these conditions, the molar specific activity of the y subunit reaches approximately 70% of the molar specific activity of nonactivated phosphorylase kinase. Figure 1 illustrates how different temperatures affect reactivation of the y subunit. The initial activity drops 100-fold within about 25 min of incubation at 30°, while at 22 ° activity stays constant for at least 1 hr. In contrast, there is a marked increase in activity when incubated on ice. After only 1 hr the phosphorylase kinase activity of the y subunit increases fourfold. Figure 2 shows the effect other factors have on reactivation. As mentioned above, Ca 2÷ and calmodulin were used for reactivation. It is clear that only the calcium-bound conformation of calmodulin facilitates the reactivation in this case because if Ca 2÷ is absent from the reactivation mixture no reactivation occurs. This is true even though another protein, phosphorylase b, is present. Thus, it is clear that reactivation does not occur in a nonspecific manner requiring only the presence of another protein for success. Reactivation also is pH dependent. Reactivation is greatest at pH 8.2 and only 20% as good at pH 6.8 (result not shown). Finally, as shown in Figs. 1 and 2, reactivation is time dependent and under optimal conditions maximum activity requires 1-2 days. When stored on ice, the reactivated 3' subunit loses no activity for at least 1 month. In the presence of a low concentration of CaM, phosphorylase b or bovine serum albumin does enhance reactivation of the y subunit. If high amounts of CaM (0.5 mg/ml) and 1 mM Ca 2÷ are used, however, the
440
RENATURATION OF PROTEIN KINASES
[36]
70
I - 31 I
m
> m
U 2O
U m n
U m a_ IO ull
r -
A '
i
'
8
'
//12
TIME (hrs) FIG. 2. Effect of other factors on reactivation.
HPLC-purified 7 subunit can be reactivated even better (Fig. 3). A second procedure for renaturing the HPLC-purified 7 subunit is to dialyze the enzyme against 8 M urea followed by dilution and assay as described. ]] With this procedure CaM is not required for renaturation of the y subunit. HPLC-purified y is flushed with N 2 to remove acetonitrile and followed by dialysis against 8 M urea, pH 6.8, containing 100 mM HEPES, and 1 mM dithiothreitol, at 4° for 12 hr. The y subunit is reactivated by a dilution of 8 M urea 20-fold with pH 6.8 HEPES buffer or pH 8.2 Tris buffer containing l mM dithiothreitol and 10% (v/v) glycerol on ice for several hours to reach a maximum activity. By this method the renaturation of the 7 subunit at pH 8.2 is also more favorable than the renaturation at pH 6.8. Although a total of 4 hr of incubation on ice is needed for HPLCpurified 7 to reach a maximum reactivation, the y subunit/8 M urea is reactivated as soon as 8 M urea is diluted with buffers.
Isolation of Reactivated y Subunit from CaM Using CaM-Agarose To obtain the isolated, reactivated 7 subunit free of CaM, agarosebound CaM is used in place of free CaM or 8 in the first reactivation protocol above. Specifically, the reactivation mixture is the same as described in the above section, except that it contained 20% CaM-agarose (Sigma, St. Louis, MO or Bio-Rad, Richmond, CA) and no free CaM. During reactivation the CaM-agarose resin is suspended often. Also, the
[36]
HPLC
OF P R O T E I N K I N A S E SUBUNITS
441
140
120 ml CaM 11111
I-- 8o
_> I-
40
20
2
4
6 Time
8 (hrs)
10
12
FIG. 3. Effect of CaM, phosphorylase b, and BSA on reactivation of 3' subunit. final yield can be increased by removing the supernatant from the resin after 1 or 2 days and replacing it with an identical solution containing fresh HPLC-isolated 3'. When reactivation is complete, and the mixture is poured into a small column, the column is washed well with buffer A at pH 8.2. This removes most of the phosphorylase but not the phosphorylase kinase activity. The 3,-8 bond is known to be extremely strong in the holoenzyme and the LiBr-isolated 3'8 complex. Thus, it is not surprising that elution is difficult and is not achieved by a number of reagents, including 10 m M EGTA and 1 mM EGTA plus either 1 M NaCI, 2 M LiBr, 5 mg/ml heparin, or 0.1 M NaCO3 (pH 10). Partial elution occurs with either 1% (v/v) Triton X-100 or an incubation of several hours in the presence of 1 M Tris, pH 7.0, 5 mM dithiothreitol, and 1 mM EGTA. With a combined solution of 1% (v/v) Triton X-100, 1 M Tris, pH 7.0, 1 mM EGTA, and 5 m M dithiothreitol all activity is immediately eluted. This activity decreases significantly (70% in 1 day) if left in the elution buffer. Thus, the Triton X-100 is removed by passing the eluted 2/subunit activity through a Bio-Beads SM-2 column, but this step results in about 60% loss of activity. The active fractions can then be concentrated, dialyzed against 50% glycerol, 0.1 M HEPES, pH 7.0, 5 mM dithiothreitol, and 0. I mM CaC12, and stored at - 20 ° for at least 3 months with little loss of activity.
442
RENATURATION OF PROTEIN KINASES
[36]
The resulting T subunit solution is shown to be free of CaM,l° but contaminated with residual phosphorylase b from the reactivation mixture.
Renaturation of Bacterial Expressed Chloramphenicol Acetyl Transferase-T The fusion chloramphenicol acetyl transferase (CAT)-T subunit in Escherichia coli is prepared as described by Chen eta/. 12'13 A fusion plasmid containing a ptac promoter, the DNA coding for N-terminal 73 amino acids of bacterial CAT protein, and the cDNA coding for the entire T subunit of phosphorylase kinase is first constructed. Escherichia coli D1210 containing lacI q is, then, transformed with this CAT-kinase plasmid. The plasmid-containing cells are grown at 37° to an OD of 0.2 at 600 nm. At this stage, cells are induced with 2 mM isopropyl-/3-D-thiogalactoside (IPTG) and allowed to grow for another 4 hr. The CAT-T fusion protein is synthesized and large inclusion bodies are formed. Essentially, 20% of the cell protein is the fusion protein. Cells with inclusion bodies are then pelleted and fusion protein is isolated as described by Nagai and Thogersen. is The fusion CAT-T subunit, however, is insoluble and is found in the inclusion bodies, It can be dissolved in 6 M guanidine hydrochloride and partially dissolved in 8 M urea solution. The renaturation of the fusion protein (around 0.6 mg/ml in guanidine hydrochloride) can be achieved by the dilution of 6 M guanidine hydrochloride 60-fold or more with either pH 8.2 Tris buffer or pH 6.8 HEPES buffer containing CaM. When incubated with CaM (0.5 mg/ml), 1 mM Ca 2÷ , and 600 mM glycine on ice for 24 hr around 10-15% molecular specific activity of holoenzyme of CAT-T is found (C.-Y. Huang and D. J. Graves, unpublished result, 1990). Two to 3% of the molecular specific activity of holoenzyme of CAT-T can also be observed when the high concentration of denaturant, guanidine or urea, is simply diluted with pH 8.2 or 6.8 buffer. The CAT-T can be further purified by using reversed-phase HPLC or a CaM-Sepharose column as essentially described by Kee and Graves. 1° Conclusion The general applicability of these methods is quite promising. Reversed-phase HPLC is used to separate the subunits of rat liver branched chain ot-ketoacid dehydrogenase 2-oxoisovalerate dehydrogenase, 19 and Is K. Nagai and H. C. Thogersen, in "Methods in Enzymology" (R. Wu and L. Grossman, eds.), Vol. 153, p. 461. Academic Press, San Diego, California, 1987. 19B. Zhang, M. J. Kuntz, G. W. Goodwin, R. A. Harris, and D. W. Crabb, J. Biol. Chem. 262, 15220 (1987).
[36]
H P L C OF PROTEIN KINASE SUBUNITS
443
CaM treatment results in reactivation of calcineurin after immobilization on nitrocellulose.2° Other proteins which strongly interact with other protein subunits might be renatured by dilution of the denaturant in the presence of these subunits. Alternative methods have been designed by Carlson's laboratory (U. of Mississippi Medical Center, Jackson, Mississippi) for renaturation. In one case, a high concentration of denaturant, 8 M urea, is used for the separation of the subunits of phosphorylase kinase21 by gel filtration, and subsequent dilution of the denaturant achieved enzyme activity of the 3' subunit. A mixture of native odfl subunits also can be obtained by this method. Acknowledgment This work was supported by the National Institutesof Health (Grant No. GM-09587).
2o M. J. Hubbard and C. B. Klee, J. Biol. Chem. 262, 15062 (1987). 21 H. K. Paudel and G. M. Carlson, J. Biol. Chem. 262, 11912 (1987).
RENATURATION OF PROTEIN KINASES
[36]
[36] H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h i c S e p a r a t i o n a n d R e n a t u r a t i o n o f P r o t e i n K i n a s e S u b u n i t s : A p p l i c a t i o n to Catalytic Subunit of Phosphorylase Kinase
By SCOTT M. KEE, CmUN-JYE YUAN, and DONALD J. GRAVES Any attempt to relate the catalytic and regulatory properties of an oligomeric enzyme to its structure involves an understanding of the roles of the different subunits and how they interact with one another. Perhaps the most efficient means of arriving at such understanding is the characterization of isolated subunits in their native states. For some enzymes, however, dissociation and isolation of subunits under nondenaturing conditions are extremely difficult because of strong intersubunit bonding. Such is the case with phosphorylase kinase. ~ This enzyme plays an important role in the regulation of glycogenolysis and consists of four different subunits, a,/3, y, and 8, with a stoichiometry of Ot4/34'Y4~4 .2-4 The a and 13 subunits are regulatory subunits. 2,5 The 8 subunit is shown to be identical to the calcium-binding protein, calmodulin (CAM), by its primary structure and ability to activate CaM-dependent enzymes. 4 The catalytic role of y was proved by Skuster et al. 6 based on the treatment of the holoenzyme with LiBr, a denaturant. Dissociation of the enzyme into its subunits occurred and an active fraction containing the y subunit was obtained by gel filtration and chromatography. Subsequently, catalytically active subunit complexes of ay6 and y8 were found to retain the full catalytic activity of the holoenzyme. 7 Amino acid sequencing provided proof that the 3/subunit contains structural elements similar to those found in the active region of other protein kinases. 8 The catalytic properties of the active y subunit have been described. 9 Methods are described here for the separation of phosphorylase kinase I G. M. Carlson, P. J. Bechtel, and D. J. Graves, Adv. Enzymol. 50, 41 (1979). 2 p. Cohen, Eur. J. Biochem. 34, 1 (1973). T. Hayakawa, J. P. Perkins, D. A. Walsh, and E. G. Krebs, Biochemistry 12, 567 (1973). 4 p. Cohen, A. Burchell, J. G. Foulkes, P. T. W. Cohen, T. C. Vanaman, and A. C. Nairn, FEBS Lett. 92, 287 (1978). 5 T. Hayakawa, J. P. Perkins, and E. G. Krebs, Biochemistry 12, 574 (1973). 6 j. R. Skuster, K.-F. J. Chan, and D. J. Graves, J. Biol. Chem. 255, 2203 (1980). 7 K.-F. J. Chan and D. J. Graves, J. Biol. Chem. 257, 5948 (1982). 8 E. M. Reimann, K. Titani, L. H. Ericsson, R. D. Wade, E. H. Fischer, and D. A. Walsh, Biochemistry 23, 4185 (1984). 9 S. M. Kee and D. J. Graves, J. Biol. Chem. 262, 9448 (1987).
METHODS IN ENZYMOLOGY,VOL. 200
Copyright© 1991by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[36]
H P L C OF PROTEIN KINASE SUBUNITS
437
subunits under denaturing conditions and subsequent reactivation of the 3t subunit ~°'~ and for renaturation of the bacterial expressed form of the subunit of phosphorylase kinase) 2'13 Materials
and Methods
Phosphorylase kinase can be prepared from rabbit skeletal muscle as described by Hayakawa et al. 3 Further purification is obtained by using DEAE-cellulose chromatography. 2 Phosphorylase b is prepared as described by Fischer and Krebs. 14 The kinase activity may be assayed as described by Kee and Graves. 9 The standard assay mixture contains 62 mM HEPES, 42 mM Tris, 2.5 mM dithiothreitol, 0.1 mM CaC12, 10 mM MgC12 , 3 mM ATP, and 100 /xM phosphorylase. The final pH of the reaction mixture is 8.2. The reactions are performed at 30° and initiated by adding MgATP. The reaction is terminated by dilution into ice-cold buffer followed by phosphorylase assay. Phosphorylase activity can be determined directly by measuring the amount of inorganic phosphate released from glucose 1-phosphate in the reaction of glycogen synthesis.~5 H i g h - P e r f o r m a n c e Liquid Chromatographic Separation o f Phosphorylase Kinase Subunits
The separation of phosphorylase kinase subunits on reversed-phase high-performance liquid chromatography (HPLC) was investigated by Crabb and Heilm eyer ) 6 They were successful in completely separating the four subunits using programmed gradients that started with 0.1% (v/v) trifluoroacetic acid (TFA) in water and finished with 0.1% TFA in 62% (v/v) acetonitrile. The subunits are eluted according to size, with the 8 subunit eluting first between 43 and 50% acetonitrile and tz subunit eluting last at about 58% acetonitrile. They found that optimal resolution and recovery was dependent on the amount of protein applied. With a 5-/zm Vydac C18 column (25 x 0.46 cm i.d.), for example, phosphorylase kinase l0 S. M. Kee and D. J. Graves, J. Biol. Chem. 261, 4732 (1986). 11C.-J. Yuan and D. J. Graves, Arch. Biochem. Biophys. 274, 317 (1989). t~ L.-R. Chen, C.-J. Yuan, G. Somasekhar, P. Wejksnora, J. E. Peterson, A. M. Myers, L. Graves, P. T. W. Cohen, E. F. da Cruz e Silva, and D. J. Graves,Biochem. Biophys. Res. Commun. 161, 746 (1989). 13L.-R. Chen, C.-J. Yuan, G. Somasekhar,P. Wejksnora, J. E. Peterson,A. M. Myers, and D. J. Graves, in "Informationtransduction and processing systems from cell to whole body" (O. Hatase and J. E. Wang, eds.), p. 19. Elsevier, New York, 1989. 14E. H. Fischer and E. G. Krebs, J. Biol. Chem. 231, 65 (1958). 15B. Illingworthand G. T. Coli, Biochem. Prep. 3, 1 (1953). 16j. W. Crabb and L. M. G. Heilmeyer,Jr., J. Chromatogr. 296, 129 (1984).
438
RENATURATION OF PROTEIN KINASES
[36]
applications of greater than 0.3 mg result in decreased recovery of all subunits and partial contamination of the 13 subunit with the a subunit. When 0.25 mg sample is applied, individual subunit recoveries ranged from 66 to 88%. A much shorter column (3-6 cm) has little effect on resolution and it is found that a 6 × I cm column allowed higher yields with higher loads while still maintaining excellent resolution. Procedure. Separation of phosphorylase kinase for the purpose of recovering an isolated, catalytically active 3' subunit is achieved using a 5-/zm Vydac C18 column (25 x 0.46 cm i.d.) and a gradient program and other conditions as described by Crabb and Heilmeyer.17 Phosphorylase kinase (1-2 mg/ml in 50 m M glycerol 1-phosphate, pH 7.0, 2 mM EDTA, and 1 mM dithiothreitol) is injected directly to the column previously equilibrated in 0.1% TFA. The 3' subunit is eluted at approximately 50% acetonitrile and 0.09% TFA, with a pH of 2.5. Fractions containing the 3' subunit can be stored in plastic tubes on ice for 1 to 2 weeks. The 3' subunit prepared in this way is pure, judged by SDS-PAGE.I°
Renaturation of High-Performance Liquid Chromatography-Isolated 3"Subunit of Phosphorylase Kinase Initially, the HPLC-recovered 3' subunit (about 100/zg/ml in HPLC solvent) has no activity when assayed according to the usual conditions, with no dilution or after a 20-fold dilution in 40 mM HEPES, pH 6.8, and 5 mM dithiothreitol (buffer A) before the assay. Presumably, the 3' subunit is denatured after undergoing exposure to the low pH and high organic solvent concentrations of the HPLC solvents. A small amount of activity (about 2% of the molar specific activity of the holoenzyme) is observed, however, when the 3' subunit is assayed in the presence of Ca 2+ and the 8 subunit of phosphorylase kinase or its equivalent, CaM. The activity is significantly enhanced if 3' is preincubated with calmodulin and Ca 2+. Because CaM interacts strongly with the 3' subunit the presence of native CaM in the renaturation medium is thought to affect the renaturation process by binding to the 3' subunit and influencing its folding or by trapping the 3' in its native state, facilitating further conversion of denatured 3' to native y. Procedure. The HPLC-purified 3' subunit is diluted to 1-5/~g/ml in an ice-cold solution containing one part assay buffer (0.25 M HEPES, 0.25 M Tris, 0.6 m M CaC12, pH 8.6) and four parts buffer A plus 60-100/zg/ml calmodulin and 1.5 mg/ml phosphorylase b or bovine serum albumin. This results in a final pH of 8.2 and final concentrations of no more than 5% 17j. W. Crabb and L. M. G. H¢ilmeyer, Jr., J. Biol. Chem. 259, 6346 (1984).
[36]
H P L C OF PROTEIN KINASE SUBUNITS 400
'
'
'
'
'
20
30
40
60
439
~O
300
__. 200 I.--
lOOI
0
I0
60
R e a c t i v a t i o n Time (rain)
FIG. I. Temperature effects on reactivation of 3' subunit.
acetonitrile and 0.01% TFA. The reactivation mixture is kept on ice for several days to attain optimum catalytic activity. With these conditions, the molar specific activity of the y subunit reaches approximately 70% of the molar specific activity of nonactivated phosphorylase kinase. Figure 1 illustrates how different temperatures affect reactivation of the y subunit. The initial activity drops 100-fold within about 25 min of incubation at 30°, while at 22 ° activity stays constant for at least 1 hr. In contrast, there is a marked increase in activity when incubated on ice. After only 1 hr the phosphorylase kinase activity of the y subunit increases fourfold. Figure 2 shows the effect other factors have on reactivation. As mentioned above, Ca 2÷ and calmodulin were used for reactivation. It is clear that only the calcium-bound conformation of calmodulin facilitates the reactivation in this case because if Ca 2÷ is absent from the reactivation mixture no reactivation occurs. This is true even though another protein, phosphorylase b, is present. Thus, it is clear that reactivation does not occur in a nonspecific manner requiring only the presence of another protein for success. Reactivation also is pH dependent. Reactivation is greatest at pH 8.2 and only 20% as good at pH 6.8 (result not shown). Finally, as shown in Figs. 1 and 2, reactivation is time dependent and under optimal conditions maximum activity requires 1-2 days. When stored on ice, the reactivated 3' subunit loses no activity for at least 1 month. In the presence of a low concentration of CaM, phosphorylase b or bovine serum albumin does enhance reactivation of the y subunit. If high amounts of CaM (0.5 mg/ml) and 1 mM Ca 2÷ are used, however, the
440
RENATURATION OF PROTEIN KINASES
[36]
70
I - 31 I
m
> m
U 2O
U m n
U m a_ IO ull
r -
A '
i
'
8
'
//12
TIME (hrs) FIG. 2. Effect of other factors on reactivation.
HPLC-purified 7 subunit can be reactivated even better (Fig. 3). A second procedure for renaturing the HPLC-purified 7 subunit is to dialyze the enzyme against 8 M urea followed by dilution and assay as described. ]] With this procedure CaM is not required for renaturation of the y subunit. HPLC-purified y is flushed with N 2 to remove acetonitrile and followed by dialysis against 8 M urea, pH 6.8, containing 100 mM HEPES, and 1 mM dithiothreitol, at 4° for 12 hr. The y subunit is reactivated by a dilution of 8 M urea 20-fold with pH 6.8 HEPES buffer or pH 8.2 Tris buffer containing l mM dithiothreitol and 10% (v/v) glycerol on ice for several hours to reach a maximum activity. By this method the renaturation of the 7 subunit at pH 8.2 is also more favorable than the renaturation at pH 6.8. Although a total of 4 hr of incubation on ice is needed for HPLCpurified 7 to reach a maximum reactivation, the y subunit/8 M urea is reactivated as soon as 8 M urea is diluted with buffers.
Isolation of Reactivated y Subunit from CaM Using CaM-Agarose To obtain the isolated, reactivated 7 subunit free of CaM, agarosebound CaM is used in place of free CaM or 8 in the first reactivation protocol above. Specifically, the reactivation mixture is the same as described in the above section, except that it contained 20% CaM-agarose (Sigma, St. Louis, MO or Bio-Rad, Richmond, CA) and no free CaM. During reactivation the CaM-agarose resin is suspended often. Also, the
[36]
HPLC
OF P R O T E I N K I N A S E SUBUNITS
441
140
120 ml CaM 11111
I-- 8o
_> I-
40
20
2
4
6 Time
8 (hrs)
10
12
FIG. 3. Effect of CaM, phosphorylase b, and BSA on reactivation of 3' subunit. final yield can be increased by removing the supernatant from the resin after 1 or 2 days and replacing it with an identical solution containing fresh HPLC-isolated 3'. When reactivation is complete, and the mixture is poured into a small column, the column is washed well with buffer A at pH 8.2. This removes most of the phosphorylase but not the phosphorylase kinase activity. The 3,-8 bond is known to be extremely strong in the holoenzyme and the LiBr-isolated 3'8 complex. Thus, it is not surprising that elution is difficult and is not achieved by a number of reagents, including 10 m M EGTA and 1 mM EGTA plus either 1 M NaCI, 2 M LiBr, 5 mg/ml heparin, or 0.1 M NaCO3 (pH 10). Partial elution occurs with either 1% (v/v) Triton X-100 or an incubation of several hours in the presence of 1 M Tris, pH 7.0, 5 mM dithiothreitol, and 1 mM EGTA. With a combined solution of 1% (v/v) Triton X-100, 1 M Tris, pH 7.0, 1 mM EGTA, and 5 m M dithiothreitol all activity is immediately eluted. This activity decreases significantly (70% in 1 day) if left in the elution buffer. Thus, the Triton X-100 is removed by passing the eluted 2/subunit activity through a Bio-Beads SM-2 column, but this step results in about 60% loss of activity. The active fractions can then be concentrated, dialyzed against 50% glycerol, 0.1 M HEPES, pH 7.0, 5 mM dithiothreitol, and 0. I mM CaC12, and stored at - 20 ° for at least 3 months with little loss of activity.
442
RENATURATION OF PROTEIN KINASES
[36]
The resulting T subunit solution is shown to be free of CaM,l° but contaminated with residual phosphorylase b from the reactivation mixture.
Renaturation of Bacterial Expressed Chloramphenicol Acetyl Transferase-T The fusion chloramphenicol acetyl transferase (CAT)-T subunit in Escherichia coli is prepared as described by Chen eta/. 12'13 A fusion plasmid containing a ptac promoter, the DNA coding for N-terminal 73 amino acids of bacterial CAT protein, and the cDNA coding for the entire T subunit of phosphorylase kinase is first constructed. Escherichia coli D1210 containing lacI q is, then, transformed with this CAT-kinase plasmid. The plasmid-containing cells are grown at 37° to an OD of 0.2 at 600 nm. At this stage, cells are induced with 2 mM isopropyl-/3-D-thiogalactoside (IPTG) and allowed to grow for another 4 hr. The CAT-T fusion protein is synthesized and large inclusion bodies are formed. Essentially, 20% of the cell protein is the fusion protein. Cells with inclusion bodies are then pelleted and fusion protein is isolated as described by Nagai and Thogersen. is The fusion CAT-T subunit, however, is insoluble and is found in the inclusion bodies, It can be dissolved in 6 M guanidine hydrochloride and partially dissolved in 8 M urea solution. The renaturation of the fusion protein (around 0.6 mg/ml in guanidine hydrochloride) can be achieved by the dilution of 6 M guanidine hydrochloride 60-fold or more with either pH 8.2 Tris buffer or pH 6.8 HEPES buffer containing CaM. When incubated with CaM (0.5 mg/ml), 1 mM Ca 2÷ , and 600 mM glycine on ice for 24 hr around 10-15% molecular specific activity of holoenzyme of CAT-T is found (C.-Y. Huang and D. J. Graves, unpublished result, 1990). Two to 3% of the molecular specific activity of holoenzyme of CAT-T can also be observed when the high concentration of denaturant, guanidine or urea, is simply diluted with pH 8.2 or 6.8 buffer. The CAT-T can be further purified by using reversed-phase HPLC or a CaM-Sepharose column as essentially described by Kee and Graves. 1° Conclusion The general applicability of these methods is quite promising. Reversed-phase HPLC is used to separate the subunits of rat liver branched chain ot-ketoacid dehydrogenase 2-oxoisovalerate dehydrogenase, 19 and Is K. Nagai and H. C. Thogersen, in "Methods in Enzymology" (R. Wu and L. Grossman, eds.), Vol. 153, p. 461. Academic Press, San Diego, California, 1987. 19B. Zhang, M. J. Kuntz, G. W. Goodwin, R. A. Harris, and D. W. Crabb, J. Biol. Chem. 262, 15220 (1987).
[36]
H P L C OF PROTEIN KINASE SUBUNITS
443
CaM treatment results in reactivation of calcineurin after immobilization on nitrocellulose.2° Other proteins which strongly interact with other protein subunits might be renatured by dilution of the denaturant in the presence of these subunits. Alternative methods have been designed by Carlson's laboratory (U. of Mississippi Medical Center, Jackson, Mississippi) for renaturation. In one case, a high concentration of denaturant, 8 M urea, is used for the separation of the subunits of phosphorylase kinase21 by gel filtration, and subsequent dilution of the denaturant achieved enzyme activity of the 3' subunit. A mixture of native odfl subunits also can be obtained by this method. Acknowledgment This work was supported by the National Institutesof Health (Grant No. GM-09587).
2o M. J. Hubbard and C. B. Klee, J. Biol. Chem. 262, 15062 (1987). 21 H. K. Paudel and G. M. Carlson, J. Biol. Chem. 262, 11912 (1987).
