- Email: [email protected]
[8] cGMP-dependent protein kinase
62
SPECIFIC
PROTEIN
KINASES
[8]
EDTA containing 40 m M NaCI. Protein is eluted with a 50 ml 40-400 m M NaCI gradient and 25-drop (0.8 ml) fractions are collected. R~ elutes approximately between 12 and 26 ml, whereas RII and the Mr = 42,000 fragment elute between 35 and 60 ml. Regulatory subunits from both isozymes can be isolated from a single tissue source when both types are present in approximately equal amounts, such as in porcine skeletal muscle. 24 Nucleotide-free regulatory subunits are most often prepared by elution from cAMP-affinity columns with 8 M urea followed by dialysis or by treatment of the R • cAMP complex with urea and removal of urea by Sephadex G-25 chromatography. 25 Care should be taken to remove the urea from the R subunits as rapidly as possible, and even rapid removal does not entirely protect against irreversible nucleotide-binding site perturbation. 26 However, both cAMP-eluted and urea-eluted R subunits readily recombine with C subunit and inhibit its activity. A method for the purification of C subunit is presented in this volume. 27 The choice of tissue and method of elution ultimately depends upon the quantity of RI or RII subunits needed and the nature of the studies for which they are to be used. 24 M. J. Zoller, A. R. Kerlavage, and S. S. Taylor, J. Biol. Chem. 254, 2408 (1979). 25 S. E. Builder, J. A. Beavo, and E. G. Krebs, J. Biol. Chem. 255, 3514 (1980). 26 j. D. Corbin and S. R. Rannels, J. Biol. Chem. 256, 11671 (1981). 27 E. M. Reimann and R. A. Beham, this volume [6].
[8] c G M P - D e p e n d e n t
Protein Kinase
by THOMAS M. LINCOLN Since the discovery of cGMP-dependent protein kinase in various arthropod tissues ~ and the mammalian cerebellum, 2 several attempts had been made to purify the enzyme. These procedures, namely ion-exchange chromatography ~ and gel filtration, 3 successfully separated the cGMPdependent enzyme from the cAMP-dependent protein kinase. The small amount of cGMP-dependent protein kinase present in most animal tissues, however, meant that conventional preparative techniques would yield only small amounts of enzyme from large quantities of starting matei j. F. Kuo and P. Greengard, J. Biol. Chem. 245, 2493 (1970). 2 F. Hofmann and G. Sold, Biochem. Biophys. Res. Commun. 49, 1100 (1972). 3 j. F. Kuo, Proc. Natl. Acad. Sci. U.S.A. 71, 4037 (1974).
METHODS IN ENZYMOLOGY, VOL. 99
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181999-X
[8]
cGMP-DEPENDENT PROTEIN KINASE
63
rial. Thus, the application of cyclic nucleotide affinity chromatography seemed to be a plausible alternative to conventional techniques since the cGMP-dependent protein kinase was known to bind its ligand with high affinity. Three immobilized ligands have been successfully used for the purification of mammalian cGMP-dependent protein kinase: 8-2-aminoethylamino-cAMP-Sepharose 4 B , 4 8-6-aminohexylamino-cAMP-Sepharose 4B, 5 and 8-2-aminoethylthio-cGMP-Sepharose 4B.6,7 The preliminary preparative procedures leading up to the affinity chromatographic steps and the methods used for the elution of the cGMP-dependent protein kinase from the three resins in principle are the same. The 8-2-aminoethylaminocAMP-Sepharose 4B resin is commercially available, making the cGMP derivative somewhat disadvantageous unless one undertakes its synthesis. 7'8 The purification procedure for cGMP-dependent protein kinase from bovine lung described below has been performed using all three ligands and has yielded similar results. Experimental Methods
Cyclic GMP-Dependent Protein Kinase Assay. The standard assay for cGMP-dependent protein kinase was conducted in a Volume of 100 /zl containing 20 m M Tris-C1, pH 7.5, 100 mM magnesium acetate, 10 tzM [-/-32p]ATP (200 to 300 cpm/pmol), 10/xg of either mixed histone (type II-A) or F2b histone, and 0.1/zM cGMP when present. The reaction was initiated with the addition of enzyme and incubations were carried out at 30° for 5 rain. The reaction was terminated by pipetting an aliquot of the mixture onto filter paper discs. The discs were immersed in 10% trichloroacetic acid and processed as described previously. 9 One unit of enzyme activity is the amount of enzyme required to transfer 1/~mol of phosphate from ATP to histone in 1 min. Other Procedures. Eight percent polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was performed as described by Laemmli ~°using Coomassie blue G-250 to stain the protein bands. Protein 4 G. N. Gill, K. E. Holdy, G. M. Walton, and C. B. Kanstein, Proc. Natl. Acad. Sci. U.S.A. 73, 3918 (1976). 5 j. D. Corbin, personal communication (1982). 6 T. M. Lincoln, W. L. Dills, and J. D. Corbin, J. Biol. Chem. 252, 4269 (1977). 7 V. Flockerzi, N. Speichermann, and F. Hofmann, J. Biol. Chem. 253, 3395 (1978). 8 W. L. Dills, J. A. Beavo, P. J. Bechtel, K. R. Meyers, L. J Sakai, and E. G. Krebs, Biochemistry 15, 3724 (1976). 9 j. D. Corbin and E. Reimann, this series, Vol. 38, p. 287. l0 U. K. Laemmli, Nature (London) 227, 680 (1970).
64
SPECIFIC PROTEIN KINASES
[8]
was determined by the Lowry method, lj and in some cases by the modified procedures of Ross and Schatz. 128-2-Aminoethylthio-cGMP was synthesized and purified as described previously. 8 Coupling to CNBrSepharose 4B was as described earlier. 6 Materials. Histone was purchased from either Sigma Co. (type II-A) or Worthington Diagnostics (F2b). CNBr-Sepharose 4B was from Pharmacia while 8-2-aminoethylamino-cAMP-Sepharose 4B was obtained from P-L Biochemicals. DE-11 and DE-52 cellulose were from the Whatman Co. Results and Discussion
Purification Procedures: Step 1. Preparation of the Extract. Freshly obtained bovine lungs (2-3 kg) were obtained from a local slaughterhouse. The lungs were transported to the laboratory on ice to keep them as cold as possible. All further procedures were performed at 4°. Dissected lung tissue (1.5 to 2 kg) was removed with a knife leaving behind the trachea, large bronchiolar tubes, and fat tissue. The tissue was ground with a meat grinder into a fine, pulpy mass and suspended in 2 volumes of 20 mM sodium phosphate-2 mM EDTA-25 mM 2-mercaptoethanol, pH 7.0 (PEM buffer) in a 4-liter Waring blender (approximately 3/4 capacity). The tissue was homogenized in 10-sec bursts at each speed (low, medium, and high), and the procedure was repeated twice. The viscous homogenate was centrifuged in a 3-liter capacity rotor at 12,000 g for 20 min. The supernatant was poured through glass wool into a 4-liter flask, and the extract was diluted in an equal volume of PEM buffer and mixed thoroughly. Step 2. DEAE-Celluose Chromatography. The entire extract (approximately 6 liters) was gently mixed for 1 hr with 4 liters of DE-11 (or DE-52) previously equilibrated in the same buffer. The DEAE-extract suspension was then poured into a large column (8-liter capacity) and the extract was passed through the settling DEAE. This procedure, in contrast to batch absorption followed by the removal of the extract before filling the column, yielded a greater recovery of cGMP-dependent protein kinase from the DEAE-cellulose column upon salt elution. After the extract had passed into the DEAE-cellulose bed, the column was washed with 20 liters of PEM containing 50 mM NaC1. This fraction contained a large amount of cAMP-dependent protein kinase type I but less than 1% of the cGMP-dependent protein kinase from the original extract. The cGMPn O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). n E. Ross and G. Schatz, Anal. Biochem. 54, 304 (1973).
[8]
cGMP-DEPENDENT PROTEIN KINASE
65
dependent protein kinase was then eluted with 12 liters of PEM containing 150 mM NaC1, although 80 to 90% of the enzyme could be recovered from the column in the second through the sixth liter of the 150 mM NaCI wash and used as the source for the next purification step. Step 3. Ammonium Sulfate Precipitation. The NaC1 wash was mixed with solid ammonium sulfate (1 kg/2 liters of effluent) for 1 hr with occasional stirring. The suspension was centrifuged at 12,000 g for 20 min and the supernatant was discarded. The pellets were resuspended in a minimum volume of PEM buffer (usually about 300-400 ml for every 2 kg of lung tissue) and placed in dialysis sacks. The suspensions were then dialyzed overnight against 24 liters of PEM. At this point, the cloudy suspension cleared considerably, even though a significant amount of ammonium sulfate was still present in the dialysis sack. This residual amount of salt did not affect the recovery of cGMP-dependent protein kinase at this step nor at the following affinity chromatographic step. Thus, further dialysis was not deemed necessary and only prolonged the purification. In fact, in more recent preparations a 2-hr dialysis was used without noticeable differences in results. 5 The dialysate was collected and centrifuged at 12,000 g for 20 min to remove insoluble material and the supernatant passed through glass wool into a flask. The volume of the suspension had approximately doubled during the dialysis. Step 4. Affinity Chromatography. The entire ammonium sulfate fraction was passed through a 3-ml column of either 8-2-aminoethylaminocAMP, 8-6-aminohexylamino-cAMP, or 8-2-aminoethylthio-cGMP Sepharose 4B (approximately 2-4 t~mol of nucleotide per ml of packed gel) at about 25 ml/hr. Greater than 90% of the cGMP-dependent protein kinase was bound to the column during the loading procedure using either immobilized ligand. Faster flow rates of up to I00 ml/hr did not decrease the binding of the enzyme to the 8-2-aminoethylthio-cGMP-Sepharose 4B presumably due to its extremely high affinity for the cGMP derivative. 6 Elution of the cGMP-dependent protein kinase was performed as follows: The column was first washed with 30 ml of PEM followed by 30 ml of PEM containing 1 M NaCI. These fractions contained only minute amounts of cGMP-dependent protein kinase and were subsequently discarded. The column was then washed with 1 ml of PEM then with 0.5 ml of PEM containing 10 mM cGMP and the flow was halted. The column was removed from the cold and allowed to stand at room temperature for 30 min. This enabled the free cGMP to displace the cGMP-dependent protein kinase from the immobilized ligand. The column was then washed with 3 ml of PEM plus I0 mM cGMP at room temperature and fractions were collected in tubes placed on ice. This procedure was repeated four times. A typical profile for elution of cGMP-dependent protein kinase
66
SPECIFIC PROTEIN KINASES
IS]
10
2.5 10mM cGMP .I
>I.-
I
8-
m I.-o ,,~
2.0
6-
-1.5
w
z
Z
4-
- 1.0
_z iii I--
O n" 0_
k.
o_
2
-0.5
7 0
,
0
4
8
~
12
16
0
20
MILLILITERS
FIG. 1. Elution of cGMP-dependent protein kinase from 8-2-aminoethylthio-cGMPSepharose 4B.
from 8-2-aminoethylthio cGMP-Sepharose 4B is shown in Fig. 1. Greater than 60% of the protein was eluted in the first 6 ml. Continued washing eluted residually bound protein and kinase activity even up to 16 ml, attesting to the high affinity of the enzyme for the immobilized cGMP ligand. An alternate procedure for the elution of residual cGMP-dependent protein kinase was adopted as follows after the first two elutions: the immobilized ligand was removed from the column and incubated overnight in 3 ml of PEM plus 10 mM cGMP in a centrifuge tube. The beads were then removed by centrifugation and the supernatant was saved for cGMP-dependent protein kinase determination. An extra 3 mg of protein could usually be recovered by this procedure. A summary of the purification procedure from 1 kg of bovine lung using the cGMP-Sepharose 4B affinity column is shown in the table. Greater than a 4000-fold purification was obtained through the affinity chromatography step. The enzyme had a specific activity of approximately 0.3 units/mg protein using mixed histone as the substrate or approximately 1 unit/mg protein using the preferred phosphate acceptor, F2b histone. Step 5. Sepharose 6B Chromatography (Optional). The protein eluted by the affinity chromatography step was 80-90% pure as judged by S D S PAGE (Fig. 2). Most of the protein migrated as a single band with an Mr = 80,000. One major contaminant migrating at 50,000 to 55,000 Mr was usually seen and could be removed by gel filtration. The eluted cGMP-
[8]
c G M P - D E P E N D E N T PROTEIN KINASE
67
PURIFICATION OF cGMP-DEPENDENT PROTEIN KINASE FROM BOVINE LUNG BY 8-2-AMINOETHYLTHIO-cGMP AFFINITY CHROMATOGRAPHYa
Step 12,000 g supernatant DEAE-cellulose Ammonium sulfate cGMP-Sepharose
Volume (ml) 3,000 7,000 150 12
Protein (mg) 69,000 11,700 4,950 3.75
Proteinkinaseb (units)
Recovery (%)
5.64 2.44 1.64 1.40
-43 29 25
Two kilograms of bovine lung was used as the starting material, and the preparation was divided into two 1-kg parts: part one (Fig. 1) was chromatographed on 8-2-aminoethylthio-cGMP-Sepharose 4B and part two (not shown) was chromatographed on 8-2-aminoethylamino-cAMP-Sepharose 4B. b Histone (type II-A) was used as the substrate.
a
dependent protein kinase was concentrated by applying the sample to a 0.5-ml DEAE-cellulose column (conveniently poured into a pasteur pipet) equilibrated in PEM. The enzyme was eluted with 1 ml of PEM containing 500 mM NaCI and applied to a Sepharose 6B column (0.9 x 60 cm) equilibrated with PEM buffer. The cGMP-dependent protein kinase eluted as a single peak of protein corresponding to a Mr = 160,000; while the minor contaminant(s) eluted as a lower molecular weight species. The kinase was pooled, concentrated on a 0.5 ml DEAE-cellulose column as described above, and dialyzed exhaustively against PEM. Since the holoenzyme Mr was estimated at 160,000, the enzyme probably consists of a dimer of identical subunits. 6 This hypothesis has been upheld in experiments from several laboratories and now appears incontrovertibleY
C o m m e n t s on the Purification
The purification of the cGMP-dependent protein kinase is simple and rapid using either affinity ligand. Typical purifications can be accomplished in 5 to 7 days following the above procedures. In many cases, the time for purification can be abbreviated even more. For example, the use of a porous fritted disc funnel attached to a vacuum flask rather than a gravity-operated column can decrease dramatically the time required for DEAE-cellulose chromatography. As mentioned earlier, smaller elution volumes from the DEAE-cellulose column can also be used with only minor losses in yield. This not only reduces the elution time required but also reduces considerably the amount of material to be handled at the
_FIG.2. Eight percent SDS-PAGE of cGMP-dependent protein kinase purified by affinity chromatography. Lane a, 20.0/xg protein from 12,000 g supernatant; Lane b, 8.0/zg protein from DEAE-cellulose elution; Lane c, 8.0/zg protein from 60% ammonium sulfate precipitate; Lane d, 4 t~g cGMP-dependent protein kinase from 8-2-aminoethylthio-cGMPSepharose 4B; Lane e, 20 /~g cGMP-dependent protein kinase from 8-2-aminoethylthiocGMP-Sepharose 4B. Molecular weight standards are myosin (200,000), /3-galactosidase (130,000), phosphorylase (94,000), bovine serum albumin (68,000), ovalbumin (43,000), and glyceraldehyde-3-phosphate dehydrogenase (35,000).
[8]
cGMP-DEPENDENT PROTEIN KINASE
69
ammonium sulfate step. Finally, the Sepharose 6B chromatography step may be omitted depending on the purity of enzyme desired. As will be mentioned below, one of the contaminants usually seen is the regulatory subunit of the cAMP-dependent protein kinase and this can be successfully removed by eluting the affinity column with 0.1 mM cAMP prior to the cGMP elution. It has also been observed that there is less contaminating regulatory subunit in the earlier fractions eluted from the immobilized cAMP affinity column. It can be observed from Table I that there is a significant loss of total activity of cGMP-dependent protein kinase between the crude extract and the DEAE-cellulose step. On the other hand, there is very little loss of cGMP binding between these two steps (not shown). The discrepancy is due to the presence in the crude extract of a cGMP-independent protein kinase activity present which is assayed together with cGMP-dependent protein kinase activity using 100 mM Mg 2+ and 10/zM ATP. Thus, the overall recovery of protein kinase activity is artificially low due to the fact that it was not possible to assay specifically the cGMP-dependent protein kinase activity in the crude extract using histone as the substrate.
Potential Problems and Pitfalls Perhaps the most common problem encountered during the purification of the cGMP-dependent protein kinase is loss of the enzyme through proteolysis. It has been observed that limited tryptic proteolysis of cGMP-dependent protein kinase produces three major protein bands detected in 7.5% SDS-polyacrylamide gels at Mr = 75,000, 50,000, and 30,000. 6 Originally, considerable variability in yield was encountered in the purification when the starting material was obtained from local slaughterhouses. It was subsequently learned that some abattoirs treat living animals with proteolytic enzymes to give the meat a tender texture. This could have had an influence on the yield of cGMP-dependent protein kinase, although this cannot be proven. It has been noticed, however, that previously frozen tissue did not yield appreciable amounts of enzyme so that the fresher the source of tissue the better the yield will be. This is not surprising since lung and the large amounts of blood contained in the tissue are rich in proteolytic enzymes. Aging or freeze-thawing the tissue probably releases and activates numerous proteases which could affect enzyme yields. Likewise, longer purification times (particularly at the ammonium sulfate step) encourage proteolytic breakdown. EDTA undoubtedly inhibits much cation-dependent proteolysis, and the inclusion of this compound is recommended throughout the purification. Another problem associated with the purification is contamination by cAMP-dependent protein kinase regulatory subunits and fragments of
70
SPECIFIC PROTEIN KINASES
[8]
regulatory subunits. This is not surprising since both affinity ligands bind cAMP and cGMP receptors. The contamination of cGMP-dependent protein kinase with regulatory subunits can be minimized in two ways: first, the elution of the DEAE column with 50 mM NaC1 removes practically all of the type I cAMP-dependent protein kinase (but unfortunately not the free type I regulatory subunit), which makes up approximately half of the cAMP-dependent protein kinase activity in bovine lung. Furthermore, elution of the cGMP-dependent protein kinase with 150 mM NaCI does not elute a large amount of the type II cAMP-dependent enzyme although some contamination undoubtedly occurs. Second, elution of the immobilized cyclic nucleotide affinity column with small amounts of cAMP (i.e., 0.1 mM) prior to cGMP elution effectively removes the type II R subunit. 7 Of course, if either of these methods fails to yield a pure product, gel filtration or sucrose gradients may be the final answer. The storage of the cGMP-dependent protein kinase does not usually pose a serious problem. Pure enzyme has been stored up to 6 months or more at 4° in PEM buffer or at - 2 0 ° in the same buffer containing 50% glycerol with no significant loss of activity or proteolytic breakdown. The enzyme has been frozen in small aliquots by adding glycerol (10%) followed by quick-freezing in dry ice-acetone. It should be stated that the glycerol is absolutely necessary and repetitive freezing and thawing must be avoided. The storage of the immobilized cyclic nucleotide-Sepharose 4B likewise does not present a problem. After elution of the enzyme, the column can be "cleaned" by washing with several volumes of 8 M urea plus 1% Triton X-100 followed by reequilibration in PEM or any other buffer. The Sepharose 4B beads can be stored in 50% glycerol at - 2 0 ° or at 4 ° in 0.2% sodium azide. The former storage procedure is recommended. The beads have been reused for several years with no significant loss in binding properties. A final problem encountered with the purification of the cGMP-dependent protein kinase is the removal of bound cGMP from the enzyme. Because cGMP is bound with such high affinity (KD < 1 × 10-8 M), ordinary dialysis is insufficient to remove the nucleotide. One approach to this problem is to treat the enzyme with charcoal to bind free cGMP. 6 Typically, pure cGMP-dependent protein kinase is diluted 1 : 100 in PEM containing 2 mg/ml bovine serum albumin. The latter stabilizes the dilute enzyme. Activated charcoal (such as Norite A) is added to a final concentration of 2 to 5 mg/ml charcoal. Higher amounts of charcoal inhibit total enzyme activity presumably by binding to the enzyme itself. The enzyme-charcoal mixture is then incubated for 20 min at room temperature with occasional mixing and the suspension is centrifuged to remove the charcoal. The enzyme treated in this manner can be stimulated some 5- to
[9]
INSECT (cAMP/cGMP)-DEPENDENT PROTEIN KINASE
71
10-fold by cGMP. it is important to dilute the cGMP-dependent protein kinase prior to charcoal treatment in order to lower the probability that dissociated cGMP will rebind to the enzyme. In many instances, however, it is desirable to have a nondiluted enzyme free of cGMP. In this instance, the concentrated enzyme is incubated for 1 hr at room temperature with 1 mM cAMP followed by another hour at 4°. Charcoal is then added as described above and the suspension is incubated for 20 min at room temperature followed by centrifugation. The enzyme can then be dialyzed against PEM buffer to remove traces of cAMP. A 5-fold stimulation of the cGMP-dependent protein kinase by cGMP is observed for enzyme treated in this fashion. An alternate procedure was described by Flockerzi et al. 7 for the removal of cGMP. In this case, cGMP-dependent protein kinase is adsorbed to DEAE-cellulose and washed at room temperature with PEM buffer containing 0.1 mM cAMP. The cAMP is then removed from the enzyme by washing the column with at least 10 volumes of PEM buffer at 4 °. The cGMP-free cGMP-dependent protein kinase can then be eluted from the column with NaC1 and dialyzed. In both instances, little loss of total cGMP-dependent protein kinase activity is observed.
[9] I n s e c t ( c A M P - c G M P ) - D e p e n d e n t
Protein Kinase
By ALEXANDER VARDANIS
This enzyme is one of a number of cyclic nucleotide-dependent protein kinases found in insects. It has the unusual property of similar affinity for either cyclic AMP or cyclic GMp.1 It is present in relatively high titer only in insects that have just molted, and is associated with insect epidermis. 2 It is purified from the thoracic integument and legs of newly emerged (0-24 hr) adults of the grasshopper Melanoplus sanquinipes. Insect Culture A colony of a nondiapausing strain of the grasshopper is grown in a controlled environment room programmed to reproduce a typical summer day. A continuous supply of eggs is obtained from an adult population of approximately 400 females and 200 males. The time from egg to adult is 1 A. Vardanis, J. Biol. Chem. 255, 7238 (1980). 2 A. Vardanis, lnsect Biochem. 12, 399 (1982).
METHODS IN ENZYMOLOGY, VOL. 99
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181999-X
SPECIFIC
PROTEIN
KINASES
[8]
EDTA containing 40 m M NaCI. Protein is eluted with a 50 ml 40-400 m M NaCI gradient and 25-drop (0.8 ml) fractions are collected. R~ elutes approximately between 12 and 26 ml, whereas RII and the Mr = 42,000 fragment elute between 35 and 60 ml. Regulatory subunits from both isozymes can be isolated from a single tissue source when both types are present in approximately equal amounts, such as in porcine skeletal muscle. 24 Nucleotide-free regulatory subunits are most often prepared by elution from cAMP-affinity columns with 8 M urea followed by dialysis or by treatment of the R • cAMP complex with urea and removal of urea by Sephadex G-25 chromatography. 25 Care should be taken to remove the urea from the R subunits as rapidly as possible, and even rapid removal does not entirely protect against irreversible nucleotide-binding site perturbation. 26 However, both cAMP-eluted and urea-eluted R subunits readily recombine with C subunit and inhibit its activity. A method for the purification of C subunit is presented in this volume. 27 The choice of tissue and method of elution ultimately depends upon the quantity of RI or RII subunits needed and the nature of the studies for which they are to be used. 24 M. J. Zoller, A. R. Kerlavage, and S. S. Taylor, J. Biol. Chem. 254, 2408 (1979). 25 S. E. Builder, J. A. Beavo, and E. G. Krebs, J. Biol. Chem. 255, 3514 (1980). 26 j. D. Corbin and S. R. Rannels, J. Biol. Chem. 256, 11671 (1981). 27 E. M. Reimann and R. A. Beham, this volume [6].
[8] c G M P - D e p e n d e n t
Protein Kinase
by THOMAS M. LINCOLN Since the discovery of cGMP-dependent protein kinase in various arthropod tissues ~ and the mammalian cerebellum, 2 several attempts had been made to purify the enzyme. These procedures, namely ion-exchange chromatography ~ and gel filtration, 3 successfully separated the cGMPdependent enzyme from the cAMP-dependent protein kinase. The small amount of cGMP-dependent protein kinase present in most animal tissues, however, meant that conventional preparative techniques would yield only small amounts of enzyme from large quantities of starting matei j. F. Kuo and P. Greengard, J. Biol. Chem. 245, 2493 (1970). 2 F. Hofmann and G. Sold, Biochem. Biophys. Res. Commun. 49, 1100 (1972). 3 j. F. Kuo, Proc. Natl. Acad. Sci. U.S.A. 71, 4037 (1974).
METHODS IN ENZYMOLOGY, VOL. 99
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181999-X
[8]
cGMP-DEPENDENT PROTEIN KINASE
63
rial. Thus, the application of cyclic nucleotide affinity chromatography seemed to be a plausible alternative to conventional techniques since the cGMP-dependent protein kinase was known to bind its ligand with high affinity. Three immobilized ligands have been successfully used for the purification of mammalian cGMP-dependent protein kinase: 8-2-aminoethylamino-cAMP-Sepharose 4 B , 4 8-6-aminohexylamino-cAMP-Sepharose 4B, 5 and 8-2-aminoethylthio-cGMP-Sepharose 4B.6,7 The preliminary preparative procedures leading up to the affinity chromatographic steps and the methods used for the elution of the cGMP-dependent protein kinase from the three resins in principle are the same. The 8-2-aminoethylaminocAMP-Sepharose 4B resin is commercially available, making the cGMP derivative somewhat disadvantageous unless one undertakes its synthesis. 7'8 The purification procedure for cGMP-dependent protein kinase from bovine lung described below has been performed using all three ligands and has yielded similar results. Experimental Methods
Cyclic GMP-Dependent Protein Kinase Assay. The standard assay for cGMP-dependent protein kinase was conducted in a Volume of 100 /zl containing 20 m M Tris-C1, pH 7.5, 100 mM magnesium acetate, 10 tzM [-/-32p]ATP (200 to 300 cpm/pmol), 10/xg of either mixed histone (type II-A) or F2b histone, and 0.1/zM cGMP when present. The reaction was initiated with the addition of enzyme and incubations were carried out at 30° for 5 rain. The reaction was terminated by pipetting an aliquot of the mixture onto filter paper discs. The discs were immersed in 10% trichloroacetic acid and processed as described previously. 9 One unit of enzyme activity is the amount of enzyme required to transfer 1/~mol of phosphate from ATP to histone in 1 min. Other Procedures. Eight percent polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was performed as described by Laemmli ~°using Coomassie blue G-250 to stain the protein bands. Protein 4 G. N. Gill, K. E. Holdy, G. M. Walton, and C. B. Kanstein, Proc. Natl. Acad. Sci. U.S.A. 73, 3918 (1976). 5 j. D. Corbin, personal communication (1982). 6 T. M. Lincoln, W. L. Dills, and J. D. Corbin, J. Biol. Chem. 252, 4269 (1977). 7 V. Flockerzi, N. Speichermann, and F. Hofmann, J. Biol. Chem. 253, 3395 (1978). 8 W. L. Dills, J. A. Beavo, P. J. Bechtel, K. R. Meyers, L. J Sakai, and E. G. Krebs, Biochemistry 15, 3724 (1976). 9 j. D. Corbin and E. Reimann, this series, Vol. 38, p. 287. l0 U. K. Laemmli, Nature (London) 227, 680 (1970).
64
SPECIFIC PROTEIN KINASES
[8]
was determined by the Lowry method, lj and in some cases by the modified procedures of Ross and Schatz. 128-2-Aminoethylthio-cGMP was synthesized and purified as described previously. 8 Coupling to CNBrSepharose 4B was as described earlier. 6 Materials. Histone was purchased from either Sigma Co. (type II-A) or Worthington Diagnostics (F2b). CNBr-Sepharose 4B was from Pharmacia while 8-2-aminoethylamino-cAMP-Sepharose 4B was obtained from P-L Biochemicals. DE-11 and DE-52 cellulose were from the Whatman Co. Results and Discussion
Purification Procedures: Step 1. Preparation of the Extract. Freshly obtained bovine lungs (2-3 kg) were obtained from a local slaughterhouse. The lungs were transported to the laboratory on ice to keep them as cold as possible. All further procedures were performed at 4°. Dissected lung tissue (1.5 to 2 kg) was removed with a knife leaving behind the trachea, large bronchiolar tubes, and fat tissue. The tissue was ground with a meat grinder into a fine, pulpy mass and suspended in 2 volumes of 20 mM sodium phosphate-2 mM EDTA-25 mM 2-mercaptoethanol, pH 7.0 (PEM buffer) in a 4-liter Waring blender (approximately 3/4 capacity). The tissue was homogenized in 10-sec bursts at each speed (low, medium, and high), and the procedure was repeated twice. The viscous homogenate was centrifuged in a 3-liter capacity rotor at 12,000 g for 20 min. The supernatant was poured through glass wool into a 4-liter flask, and the extract was diluted in an equal volume of PEM buffer and mixed thoroughly. Step 2. DEAE-Celluose Chromatography. The entire extract (approximately 6 liters) was gently mixed for 1 hr with 4 liters of DE-11 (or DE-52) previously equilibrated in the same buffer. The DEAE-extract suspension was then poured into a large column (8-liter capacity) and the extract was passed through the settling DEAE. This procedure, in contrast to batch absorption followed by the removal of the extract before filling the column, yielded a greater recovery of cGMP-dependent protein kinase from the DEAE-cellulose column upon salt elution. After the extract had passed into the DEAE-cellulose bed, the column was washed with 20 liters of PEM containing 50 mM NaC1. This fraction contained a large amount of cAMP-dependent protein kinase type I but less than 1% of the cGMP-dependent protein kinase from the original extract. The cGMPn O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). n E. Ross and G. Schatz, Anal. Biochem. 54, 304 (1973).
[8]
cGMP-DEPENDENT PROTEIN KINASE
65
dependent protein kinase was then eluted with 12 liters of PEM containing 150 mM NaC1, although 80 to 90% of the enzyme could be recovered from the column in the second through the sixth liter of the 150 mM NaCI wash and used as the source for the next purification step. Step 3. Ammonium Sulfate Precipitation. The NaC1 wash was mixed with solid ammonium sulfate (1 kg/2 liters of effluent) for 1 hr with occasional stirring. The suspension was centrifuged at 12,000 g for 20 min and the supernatant was discarded. The pellets were resuspended in a minimum volume of PEM buffer (usually about 300-400 ml for every 2 kg of lung tissue) and placed in dialysis sacks. The suspensions were then dialyzed overnight against 24 liters of PEM. At this point, the cloudy suspension cleared considerably, even though a significant amount of ammonium sulfate was still present in the dialysis sack. This residual amount of salt did not affect the recovery of cGMP-dependent protein kinase at this step nor at the following affinity chromatographic step. Thus, further dialysis was not deemed necessary and only prolonged the purification. In fact, in more recent preparations a 2-hr dialysis was used without noticeable differences in results. 5 The dialysate was collected and centrifuged at 12,000 g for 20 min to remove insoluble material and the supernatant passed through glass wool into a flask. The volume of the suspension had approximately doubled during the dialysis. Step 4. Affinity Chromatography. The entire ammonium sulfate fraction was passed through a 3-ml column of either 8-2-aminoethylaminocAMP, 8-6-aminohexylamino-cAMP, or 8-2-aminoethylthio-cGMP Sepharose 4B (approximately 2-4 t~mol of nucleotide per ml of packed gel) at about 25 ml/hr. Greater than 90% of the cGMP-dependent protein kinase was bound to the column during the loading procedure using either immobilized ligand. Faster flow rates of up to I00 ml/hr did not decrease the binding of the enzyme to the 8-2-aminoethylthio-cGMP-Sepharose 4B presumably due to its extremely high affinity for the cGMP derivative. 6 Elution of the cGMP-dependent protein kinase was performed as follows: The column was first washed with 30 ml of PEM followed by 30 ml of PEM containing 1 M NaCI. These fractions contained only minute amounts of cGMP-dependent protein kinase and were subsequently discarded. The column was then washed with 1 ml of PEM then with 0.5 ml of PEM containing 10 mM cGMP and the flow was halted. The column was removed from the cold and allowed to stand at room temperature for 30 min. This enabled the free cGMP to displace the cGMP-dependent protein kinase from the immobilized ligand. The column was then washed with 3 ml of PEM plus I0 mM cGMP at room temperature and fractions were collected in tubes placed on ice. This procedure was repeated four times. A typical profile for elution of cGMP-dependent protein kinase
66
SPECIFIC PROTEIN KINASES
IS]
10
2.5 10mM cGMP .I
>I.-
I
8-
m I.-o ,,~
2.0
6-
-1.5
w
z
Z
4-
- 1.0
_z iii I--
O n" 0_
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o_
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-0.5
7 0
,
0
4
8
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12
16
0
20
MILLILITERS
FIG. 1. Elution of cGMP-dependent protein kinase from 8-2-aminoethylthio-cGMPSepharose 4B.
from 8-2-aminoethylthio cGMP-Sepharose 4B is shown in Fig. 1. Greater than 60% of the protein was eluted in the first 6 ml. Continued washing eluted residually bound protein and kinase activity even up to 16 ml, attesting to the high affinity of the enzyme for the immobilized cGMP ligand. An alternate procedure for the elution of residual cGMP-dependent protein kinase was adopted as follows after the first two elutions: the immobilized ligand was removed from the column and incubated overnight in 3 ml of PEM plus 10 mM cGMP in a centrifuge tube. The beads were then removed by centrifugation and the supernatant was saved for cGMP-dependent protein kinase determination. An extra 3 mg of protein could usually be recovered by this procedure. A summary of the purification procedure from 1 kg of bovine lung using the cGMP-Sepharose 4B affinity column is shown in the table. Greater than a 4000-fold purification was obtained through the affinity chromatography step. The enzyme had a specific activity of approximately 0.3 units/mg protein using mixed histone as the substrate or approximately 1 unit/mg protein using the preferred phosphate acceptor, F2b histone. Step 5. Sepharose 6B Chromatography (Optional). The protein eluted by the affinity chromatography step was 80-90% pure as judged by S D S PAGE (Fig. 2). Most of the protein migrated as a single band with an Mr = 80,000. One major contaminant migrating at 50,000 to 55,000 Mr was usually seen and could be removed by gel filtration. The eluted cGMP-
[8]
c G M P - D E P E N D E N T PROTEIN KINASE
67
PURIFICATION OF cGMP-DEPENDENT PROTEIN KINASE FROM BOVINE LUNG BY 8-2-AMINOETHYLTHIO-cGMP AFFINITY CHROMATOGRAPHYa
Step 12,000 g supernatant DEAE-cellulose Ammonium sulfate cGMP-Sepharose
Volume (ml) 3,000 7,000 150 12
Protein (mg) 69,000 11,700 4,950 3.75
Proteinkinaseb (units)
Recovery (%)
5.64 2.44 1.64 1.40
-43 29 25
Two kilograms of bovine lung was used as the starting material, and the preparation was divided into two 1-kg parts: part one (Fig. 1) was chromatographed on 8-2-aminoethylthio-cGMP-Sepharose 4B and part two (not shown) was chromatographed on 8-2-aminoethylamino-cAMP-Sepharose 4B. b Histone (type II-A) was used as the substrate.
a
dependent protein kinase was concentrated by applying the sample to a 0.5-ml DEAE-cellulose column (conveniently poured into a pasteur pipet) equilibrated in PEM. The enzyme was eluted with 1 ml of PEM containing 500 mM NaCI and applied to a Sepharose 6B column (0.9 x 60 cm) equilibrated with PEM buffer. The cGMP-dependent protein kinase eluted as a single peak of protein corresponding to a Mr = 160,000; while the minor contaminant(s) eluted as a lower molecular weight species. The kinase was pooled, concentrated on a 0.5 ml DEAE-cellulose column as described above, and dialyzed exhaustively against PEM. Since the holoenzyme Mr was estimated at 160,000, the enzyme probably consists of a dimer of identical subunits. 6 This hypothesis has been upheld in experiments from several laboratories and now appears incontrovertibleY
C o m m e n t s on the Purification
The purification of the cGMP-dependent protein kinase is simple and rapid using either affinity ligand. Typical purifications can be accomplished in 5 to 7 days following the above procedures. In many cases, the time for purification can be abbreviated even more. For example, the use of a porous fritted disc funnel attached to a vacuum flask rather than a gravity-operated column can decrease dramatically the time required for DEAE-cellulose chromatography. As mentioned earlier, smaller elution volumes from the DEAE-cellulose column can also be used with only minor losses in yield. This not only reduces the elution time required but also reduces considerably the amount of material to be handled at the
_FIG.2. Eight percent SDS-PAGE of cGMP-dependent protein kinase purified by affinity chromatography. Lane a, 20.0/xg protein from 12,000 g supernatant; Lane b, 8.0/zg protein from DEAE-cellulose elution; Lane c, 8.0/zg protein from 60% ammonium sulfate precipitate; Lane d, 4 t~g cGMP-dependent protein kinase from 8-2-aminoethylthio-cGMPSepharose 4B; Lane e, 20 /~g cGMP-dependent protein kinase from 8-2-aminoethylthiocGMP-Sepharose 4B. Molecular weight standards are myosin (200,000), /3-galactosidase (130,000), phosphorylase (94,000), bovine serum albumin (68,000), ovalbumin (43,000), and glyceraldehyde-3-phosphate dehydrogenase (35,000).
[8]
cGMP-DEPENDENT PROTEIN KINASE
69
ammonium sulfate step. Finally, the Sepharose 6B chromatography step may be omitted depending on the purity of enzyme desired. As will be mentioned below, one of the contaminants usually seen is the regulatory subunit of the cAMP-dependent protein kinase and this can be successfully removed by eluting the affinity column with 0.1 mM cAMP prior to the cGMP elution. It has also been observed that there is less contaminating regulatory subunit in the earlier fractions eluted from the immobilized cAMP affinity column. It can be observed from Table I that there is a significant loss of total activity of cGMP-dependent protein kinase between the crude extract and the DEAE-cellulose step. On the other hand, there is very little loss of cGMP binding between these two steps (not shown). The discrepancy is due to the presence in the crude extract of a cGMP-independent protein kinase activity present which is assayed together with cGMP-dependent protein kinase activity using 100 mM Mg 2+ and 10/zM ATP. Thus, the overall recovery of protein kinase activity is artificially low due to the fact that it was not possible to assay specifically the cGMP-dependent protein kinase activity in the crude extract using histone as the substrate.
Potential Problems and Pitfalls Perhaps the most common problem encountered during the purification of the cGMP-dependent protein kinase is loss of the enzyme through proteolysis. It has been observed that limited tryptic proteolysis of cGMP-dependent protein kinase produces three major protein bands detected in 7.5% SDS-polyacrylamide gels at Mr = 75,000, 50,000, and 30,000. 6 Originally, considerable variability in yield was encountered in the purification when the starting material was obtained from local slaughterhouses. It was subsequently learned that some abattoirs treat living animals with proteolytic enzymes to give the meat a tender texture. This could have had an influence on the yield of cGMP-dependent protein kinase, although this cannot be proven. It has been noticed, however, that previously frozen tissue did not yield appreciable amounts of enzyme so that the fresher the source of tissue the better the yield will be. This is not surprising since lung and the large amounts of blood contained in the tissue are rich in proteolytic enzymes. Aging or freeze-thawing the tissue probably releases and activates numerous proteases which could affect enzyme yields. Likewise, longer purification times (particularly at the ammonium sulfate step) encourage proteolytic breakdown. EDTA undoubtedly inhibits much cation-dependent proteolysis, and the inclusion of this compound is recommended throughout the purification. Another problem associated with the purification is contamination by cAMP-dependent protein kinase regulatory subunits and fragments of
70
SPECIFIC PROTEIN KINASES
[8]
regulatory subunits. This is not surprising since both affinity ligands bind cAMP and cGMP receptors. The contamination of cGMP-dependent protein kinase with regulatory subunits can be minimized in two ways: first, the elution of the DEAE column with 50 mM NaC1 removes practically all of the type I cAMP-dependent protein kinase (but unfortunately not the free type I regulatory subunit), which makes up approximately half of the cAMP-dependent protein kinase activity in bovine lung. Furthermore, elution of the cGMP-dependent protein kinase with 150 mM NaCI does not elute a large amount of the type II cAMP-dependent enzyme although some contamination undoubtedly occurs. Second, elution of the immobilized cyclic nucleotide affinity column with small amounts of cAMP (i.e., 0.1 mM) prior to cGMP elution effectively removes the type II R subunit. 7 Of course, if either of these methods fails to yield a pure product, gel filtration or sucrose gradients may be the final answer. The storage of the cGMP-dependent protein kinase does not usually pose a serious problem. Pure enzyme has been stored up to 6 months or more at 4° in PEM buffer or at - 2 0 ° in the same buffer containing 50% glycerol with no significant loss of activity or proteolytic breakdown. The enzyme has been frozen in small aliquots by adding glycerol (10%) followed by quick-freezing in dry ice-acetone. It should be stated that the glycerol is absolutely necessary and repetitive freezing and thawing must be avoided. The storage of the immobilized cyclic nucleotide-Sepharose 4B likewise does not present a problem. After elution of the enzyme, the column can be "cleaned" by washing with several volumes of 8 M urea plus 1% Triton X-100 followed by reequilibration in PEM or any other buffer. The Sepharose 4B beads can be stored in 50% glycerol at - 2 0 ° or at 4 ° in 0.2% sodium azide. The former storage procedure is recommended. The beads have been reused for several years with no significant loss in binding properties. A final problem encountered with the purification of the cGMP-dependent protein kinase is the removal of bound cGMP from the enzyme. Because cGMP is bound with such high affinity (KD < 1 × 10-8 M), ordinary dialysis is insufficient to remove the nucleotide. One approach to this problem is to treat the enzyme with charcoal to bind free cGMP. 6 Typically, pure cGMP-dependent protein kinase is diluted 1 : 100 in PEM containing 2 mg/ml bovine serum albumin. The latter stabilizes the dilute enzyme. Activated charcoal (such as Norite A) is added to a final concentration of 2 to 5 mg/ml charcoal. Higher amounts of charcoal inhibit total enzyme activity presumably by binding to the enzyme itself. The enzyme-charcoal mixture is then incubated for 20 min at room temperature with occasional mixing and the suspension is centrifuged to remove the charcoal. The enzyme treated in this manner can be stimulated some 5- to
[9]
INSECT (cAMP/cGMP)-DEPENDENT PROTEIN KINASE
71
10-fold by cGMP. it is important to dilute the cGMP-dependent protein kinase prior to charcoal treatment in order to lower the probability that dissociated cGMP will rebind to the enzyme. In many instances, however, it is desirable to have a nondiluted enzyme free of cGMP. In this instance, the concentrated enzyme is incubated for 1 hr at room temperature with 1 mM cAMP followed by another hour at 4°. Charcoal is then added as described above and the suspension is incubated for 20 min at room temperature followed by centrifugation. The enzyme can then be dialyzed against PEM buffer to remove traces of cAMP. A 5-fold stimulation of the cGMP-dependent protein kinase by cGMP is observed for enzyme treated in this fashion. An alternate procedure was described by Flockerzi et al. 7 for the removal of cGMP. In this case, cGMP-dependent protein kinase is adsorbed to DEAE-cellulose and washed at room temperature with PEM buffer containing 0.1 mM cAMP. The cAMP is then removed from the enzyme by washing the column with at least 10 volumes of PEM buffer at 4 °. The cGMP-free cGMP-dependent protein kinase can then be eluted from the column with NaC1 and dialyzed. In both instances, little loss of total cGMP-dependent protein kinase activity is observed.
[9] I n s e c t ( c A M P - c G M P ) - D e p e n d e n t
Protein Kinase
By ALEXANDER VARDANIS
This enzyme is one of a number of cyclic nucleotide-dependent protein kinases found in insects. It has the unusual property of similar affinity for either cyclic AMP or cyclic GMp.1 It is present in relatively high titer only in insects that have just molted, and is associated with insect epidermis. 2 It is purified from the thoracic integument and legs of newly emerged (0-24 hr) adults of the grasshopper Melanoplus sanquinipes. Insect Culture A colony of a nondiapausing strain of the grasshopper is grown in a controlled environment room programmed to reproduce a typical summer day. A continuous supply of eggs is obtained from an adult population of approximately 400 females and 200 males. The time from egg to adult is 1 A. Vardanis, J. Biol. Chem. 255, 7238 (1980). 2 A. Vardanis, lnsect Biochem. 12, 399 (1982).
METHODS IN ENZYMOLOGY, VOL. 99
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181999-X