Assay of protein kinases with peptide substrates

A method for assaying protein kinases that phosphorylate peptides such as Kemptide, such as cAMP-dependent protein kinase, or a glycogen synthase peptide, which is an excellent substrate for protein kinase C. Upon sequentially processing of reaction mixtures through tandem columns of cation and anion exchange resins improved separation of ATP from phosphorylated peptides is achieved such that radioactivity in background samples is nearly nil and the yield of phosphorylated peptides is high. This method is generally applicable to any protein kinase so long as the substrate peptide is appropriately structured such that the peptide retains a net positive charge when fully phosphorylated so that the peptide will adhere to the cation exchange resin and pass through the anion exchange resin. This method reduces labor, radioactivity, enzyme requirements, and costs of assaying protein kinases.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention is directed to a method for separating 
phosphopeptides from ATP which is useful for detecting protein kinase 
activities and specifically, to a chromatographic column method in which a 
stopped reaction mixture containing phosphopeptides and ATP is passed 
through a column containing a cation exchange resin and then through 
another column containing an anion exchange resin, and the eluate which 
contains phosphopeptides free from ATP is then recovered. 
2. Description of Related Art 
Protein kinases are a large class of biologically important molecules. 
Protein kinase activities are generally assayed by measuring the transfer 
of phosphate from [.gamma.-.sup.32 P]ATP to a substrate. The sensitivity 
of the assay relies on effective separation of the radiolabeled product 
from ATP. With a protein as the substrate, the phosphoprotein may be 
precipitated with acid, redissolved in base to remove trapped ATP, (D. A. 
Walsh et al, (1971) J. Biol. Chem. 246, 1977-1985), followed by 
reprecipitation with acid and trapping on paper filter disks, (E. M. 
Reimann et al, (1971) J. Biol. Chem. 246, 1986-1995), glass fiber filters, 
(J. Erlichman et al, (1971) Proc. Natl. Acad. Sci. USA 68, 731-735), or 
cellulose acetate filters (J. L. Goldstein et al (1973) J. Biol. Chem. 
248, 6300-6307). Synthetic peptides have also been employed as protein 
kinase substrates. With the use of an anion exchange resin, one may 
achieve quantitative recovery of a phosphopeptide and effective separation 
of the phosphopeptide from the radioactive ATP (G. Tessmer et al (1973) 
Biochem. Biophys. Res. Commun. 50, 1-7; and B. E. Kemp et al, (1976) Proc. 
Natl. Acad. Sci. USA 73, 1038-1042). Another method involves trapping of 
phosphoproteins (J. J. Witt et al,. (1975) Anal. Biochem. 66, 253- 258) 
and phosphopeptides (D. B. Glass et al, (1978) Anal. Biochem. 87, 566-575) 
on phosphocellulose paper under acidic conditions. ATP is removed more 
effectively with this phosphocellulose method in the presence of 
phosphoric acid (R. Roskoski (1983) in Methods in Enzymology (J. D. Corbin 
et al, Eds.), Vol. 99, pp. 3-6, Academic Press, New York). 
Although the above procedures are well established for measuring protein 
kinase activities, all have certain drawbacks. First, the typical 
background in any of the published procedures is 0.04 to 0.1% of the 
initial radioactivity. Thus, under typical protein kinase assay 
conditions, with 1,000,000 cpm of [.gamma.-.sup.32 P]ATP in the reaction 
mixture, the background radioactivity in assay blanks is 400 to 1000 cpm 
of .sup.32 P. It is this background that determines sensitivity and 
dictates the amount of radioactive substrate and enzyme required for the 
detection of protein kinase activity. Second, all of the methods are 
somewhat tedious. It is therefore desirable to reduce both assay 
background and labor involved in assaying protein kinases. 
SUMMARY OF THE INVENTION 
Therefore, it is an object of the present invention to provide a protein 
kinase assay method in which substrates are completely separated from the 
radioactive ATP in order to produce a high signal-to-noise ratio. 
It is a further object of the present invention to provide a method which 
reduces expense in assaying protein kinases by requiring less radioactive 
ATP. 
It is yet a further object of the present invention to provide a method in 
which .sup.32 P-phosphorylated synthetic peptides are quantitatively 
recovered with little or no background radioactivity. 
It is still a further object of the present invention to provide a method 
in which the processing of samples requires minimum labor. 
It is another object of the present invention to provide a method for 
measuring protein kinase activities by separating phosphorylated peptides 
from ATP. 
It is yet another object of the present invention to provide a method for 
measuring protein kinase activities which is effective with synthetic 
substrates for both cAMP-dependent protein kinase (A-kinase) and protein 
kinase C (C-kinase). 
It is still another object of the present invention to provide a protein 
kinase assay method in which exposure of the investigator to the 
radioactive ATP is greatly reduced. 
The foregoing objects and others are accomplished in accordance with the 
present invention, generally speaking, by providing a method for 
separating phosphopeptides from ATP in order to measure protein kinase 
activity comprising the steps of (a) adding an amount of washing solution 
to a stopped reaction mixture of a majority of ATP and phosphopeptides so 
as to form a prepared solution; (b) adding said prepared solution into a 
first column containing a cation exchange resin so as to retain said 
phosphopeptides in said first column and remove ATP; (c) adding an 
effective amount of an elution acid to said first column and collecting 
from said first column a first eluate of said phosphopeptides; (d) passing 
said first eluate through a second column containing an anion exchange 
resin so as to trap residual ATP and collecting from said second column a 
second eluate which contains said phosphopeptides which are free from ATP. 
Optionally, a second amount of washing solution may be added to the first 
column after step (b) so as to remove further ATP. 
Further scope of the applicability of the present invention will become 
apparent from the detailed description and drawings provided below. 
However, it should be understood that the detailed description and 
specific examples, while indicating preferred embodiments of the 
invention, are given by way of illustration only, since various changes 
and modifications within the spirit and scope of the invention will become 
apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION 
The general chromatographic scheme for separating ATP from phosphopeptides 
is shown in FIG. 1 which outlines a procedure for the processing of either 
A-kinase or C-kinase assays for example. The eluates from Steps A and B 
are discarded, while the eluates from Step C are collected in liquid 
scintillation (LS) vials. Depending on the purity of the protein kinase 
sample tested, any of a number of elution procedures may be employed in 
this method. The method of the present invention is generally applicable 
to any protein kinase so long as the substrate peptide is appropriately 
structured such that the peptide retains a net positive charge when fully 
phosphorylated so that the peptide will adhere to the cation exchange 
resin and pass through the anion exchange resin. 
In general, a volume of the ATP washing solution is added to a stopped 
reaction mix containing the phosphorylated peptide and ATP, and the 
contents are poured into a first column containing a cation exchange resin 
such as CM-Sephadex (Step A). The stopped reaction mix is boiled if 
extraordinarily high amounts of protein are present, before the washing 
solution is added. The washing solution contains a sufficient amount of 
ATP so as to provide a two-fold purpose for (i) effective displacement of 
radioactive [.gamma.-.sup.32 P]ATP, and (ii) with respect to the pH of the 
solution, to allow for quantitative binding of .sup.32 P-peptide to the 
cation exchange resin (lower pH limit) and for chemical compatibility of 
the cation exchange resin within the pH rang e (upper pH limit) and has a 
concentration of ATP from about 0.5 mM to 5.0 mM and a pH of about 6.0 to 
8.0. For example, an ATP washing solution of about 5 mM ATP with a pH of 
about 6.8 may be used. The cation exchange resin employed in the first 
column may be a cross-linked polymer and may be in the form of beads 
having a bead size in the range of 120 .mu.m, and preferably in the range 
of about 40-120 .mu.m depending upon the packing properties desired of the 
cation exchange resin which will determine the flow rate of liquid through 
the resin. Cation exchange resins useful in the method of the present 
invention include resins which bind to phosphorylated peptides but not to 
ATP, such as CM-Sephadex, and any other resin which contains an ionizable, 
reactive-group which has as its property a pK of 3.5 to 4.0, as does the 
carboxy-group of CM-Sephadex, such that the peptide, which binds to the 
reactive-group, is displaced (exchanged) upon eluting the resin with any 
acid, the pH of which is lower than that of the pK of the reactive group. 
CM-Sephadex is a carboxymethyl derivative of Sephadex which is a 
bead-formed, cross-linked dextran gel which swells in water and aqueous 
salt solution. Such resin must not irreversibly (permanently) bind the 
peptide, and such acid must not be chemically incompatible with the matrix 
or reactive group of such resin. It follows that such acid must not be 
chemically incompatible with the second resin, over which the acid eluate 
must flow (for the purpose of binding ATP). Preferably CM-Sephadex may be 
used. 
The eluate from step A, containing most of the ATP, is discarded, after 
which the first column may optionally be washed again with a volume of the 
ATP washing solution, and the eluate again discarded (Step B). This step 
is optional but reduces background values with kinase samples which 
contain high concentrations of proteins. 
The first column, containing the phosphorylated peptides bound to the ion 
exchange resin, is mounted atop a second column which contains an anion 
exchange resin, such as Dowex AGl-X8, and a volume of an elution acid, 
such as acetic acid, is then added to the first column (Step C). The 
eluate from the first column passes directly through the second column and 
into a recovery means, such as a scintillation vial. The anion exchange 
resin employed in the second column is a resin suitable for trapping 
residual ATP and allowing phosphorylated peptides to pass thereover, such 
as Dowex AGl-X8, and any other resins which contain an ionizable group 
that will in the presence of the acid, permit quantitative binding of the 
negatively-charged phosphates of ATP, and permit the net 
positively-charged peptide (which contains a net positive charge when 
fully phosphorylated and when in the presence of any acid employed in 
elution from the cation exchange resin, above) to quantitatively elute 
from the anion exchange resin. Dowex AGl-X8 is a strongly basic anion 
exchanger and more specifically an 8% cross-linked styrene-divinylbenzene 
matrix for separating inorganic and organic anions. Such resin must not be 
incompatible with the acid employed in the elution from the first column, 
and such resin must not irreversibly (permanently) bind the peptide. Also, 
with respect to any acid employed in elution from either of the resins 
(cation- or anion-exchange resins) such acid must not be incompatible with 
the chemical scintillant vehicle, which is used in the process of liquid 
scintillation which follows the assay and permits measurement of 
radioactivity. The resin may have a size in the range of 200 mesh and 
preferably in the range of about 100-200 mesh depending upon the packing 
properties desired of the anion exchange resin, which will determine the 
flow rate through the resin . The elution acid used in Step C is an acid 
suitable for extracting the phosphorylated peptides from the cation 
exchange resin and allowing for residual ATP to be trapped in the anion 
exchange resin. Suitable elution acids include for example acetic acid, 
which is employed because it is (1) compatible with both the cation and 
anion exchange resins, (2) allows for quantitative elution of peptide from 
the cation exchange resin, (3) allows for quantitative binding of ATP to 
the anion exchange resin, (4) allows for quantitative elution of peptide 
through the anion exchange resin, and (5) does not interfere with the 
scintillation chemicals employed in the liquid scintillation measurement 
of radioactivity. Any acid employed other than acetic acid must adhere to 
these properties and need not be an organic acid. 
EXAMPLES 
The procedures presented below are especially useful for relatively pure 
protein kinases and relatively crude fat cell extracts. 
Materials. ATP, cAMP, CM-Sephadex cation exchange resin, bead size 40-120 
.mu.m (C-25-120), dithiothreitol (DTT), A-kinase inhibitor (PKI), Kemptide 
(synthetic heptapeptide substrate for A-kinase, H.sub.2 
N-Leu-Arg-Arg-Ala-Ser-Leu-Gly-COOH), Mops (3-[N-Morpholino]propanesulfonic 
acid (free acid)), sodium dodecyl sulfate (SDS), histone H1, and Tris were 
obtained from Sigma Chemical Co. The ATP used in bulk eluting solutions 
was the least expensive grade (Sigma, No. 3377), prepared by 
phosphorylation of adenosine. Bovine brain phosphatidylserine and 
1,2-diolein were from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Dowex 
AGl-X8 anion exchange resin (100-200 mesh) was from Bio-Rad Laboratories 
(Richmond, Calif.). [.gamma.-.sup.32 P]ATP (10-25 Ci/mmol) was from either 
International Chemical and Nuclear Corp. (Irvine, Calif.) or New England 
Nuclear (Boston, Mass.). The C-kinase dodecapeptide substrate (C. House et 
al, (1987) J. Biol. Chem. 262, 772-777), GS-peptide, i.e. H.sub.2 
N-Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys-COOH, was a gift from 
Dr. Bruce E. Kemp (Department of Medicine, University of Melbourne, 
Repatriation General Hospital, Heidelberg, Australia). 
Preparation, regeneration, and storage of chromatographic columns. 
CM-Sephadex cation exchange resin was hydrated in H.sub.2 O, the fines 
were decanted, and 2 ml of a 1:1 suspension was introduced into glass 
columns fitted with glass wool plugs. The following procedure permits 
rapid and accurate distribution of resins to columns. Two-milliliter 
aliquots of a vigorously stirring 50% resin suspension are rapidly 
transferred to the columns with a 2-ml plastic pipet linked with flexible 
tubing to a Becton-Dickinson Cornwall continuous pipettor. The tip of the 
plastic pipet is removed to increase the size of the opening. Columns were 
charged with 8 ml of 30% acetic acid followed by 8 ml of H.sub.2 O. For 
storage between experiments, the columns were washed with 10 ml of 0.02% 
NaN.sub.3 in H.sub.2 O to prevent microbial growth. Regeneration of 
columns prior to each processing cycle was performed with 8 ml of 30% 
acetic acid followed by 8 ml of H.sub.2 O. After those experiments in 
which the protein kinase samples contained extraordinarily large amounts 
of protein, the columns were washed once with 5 ml of 1 M NaCl before 
regeneration for the next assay. 
Occasionally, porosity developed in the CM-Sephadex resin bed, resulting in 
reduced column flow rates. This problem was eliminated by adding H.sub.2 O 
and stirring the resin bed with a wood applicator. Columns which had not 
been used for a prolonged period of time and manner. One day before they 
were to be used, the CM-Sephadex columns were washed with 8 ml of 30% 
acetic acid, followed 1-2 h later by 8 ml of H.sub.2 O. The resin bed was 
stirred with a wooden applicator and the columns were allowed to drain. 
The columns were again washed with 8 ml of H.sub.2 O. The day they were to 
be used, the columns were regenerated as above with acetic acid followed 
by H.sub.2 O, and, after the columns had drained, the column resin was 
packed by tamping the column rack on a bench top four or five times. 
Proper rehydration of the CM Sephadex resin was essential for optimal 
peptide recovery. However, as column performance was not altered by 
repeated drying and rehydrating over the course of many months, it was not 
necessary to store the resin under hydrated conditions. 
The Dowex AGl-X8 anion exchange resin (100-200 mesh) was washed three times 
with H.sub.2 O followed by 5 bed vol each of 1 N NaOH, glacial acetic 
acid, and 30% acetic acid. Two milliliters of a 50% suspension of resin in 
30% acetic acid were introduced into glass columns fitted with glass wool 
plugs. At the completion of each experiment, the resin was washed with 8 
ml of 30% acetic acid. Columns treated in this manner were ready for the 
next experiment. For experiments in which the protein kinase samples 
contained a large amount of protein, the resin was washed with 5 ml of 3 N 
NaOH prior to washing with acetic acid. Following prolonged storage, the 
columns were prepared for reuse by washing with 30% acetic acid (1-2 ml) 
and stirring with a wooden applicator to remove porosity in the resin for 
reasons described above. As with the CM-Sephadex columns, the Dowex AGl-X8 
columns were unaffected by repeated drying and rehydrating between 
experiments. 
Glass columns, identical to those used in the tandem-column adenylate 
cyclase assay method (Y. Salomon et al (1974) Anal. Biochem. 58, 541-548), 
were used for both resins. The columns were 21 cm in length and the 
internal diameter of the stem portion was 0.7 cm. The columns were 
arranged in identical racks, accommodating 50 columns each, which may be 
stacked one atop the other, and matching trays holding 50 scintillation 
vials were used to collect the final eluates. 
Assay of protein kinase activities. All protein kinase assays were 
conducted in 13.times.100-mm glass test tubes. The assay of A-kinase was 
performed with 100 um Kemptide, 20 mM Mops, pH 7.0, 16 mM magnesium 
acetate, 100 .mu.M ATP, 4 mM DTT, 0.5 .mu.Ci[.gamma.-.sup.32 P]ATP, and, 
where indicated, 16 .mu.M cAMP. With the reaction tubes in a 4.degree. C. 
ice bath, the assay ingredients were added to give a total volume of 60 
.mu.1, and the reaction was initiated by transferring the rack of reaction 
tubes to a 30.degree. C. bath. At the times indicated in the description 
of FIG. 2 and in the Tables, the reaction was stopped by transferring the 
rack of tubes to the 4.degree. C. ice bath and 20 .mu.l of a stopping 
solution was added quickly with a Hamilton repeating syringe. The stopping 
solution contained 100 mM ATP, pH 7.0, and where removal of free magnesium 
was desired, 100 mM EDTA was included. Also, for reactions containing 
large amounts of bovine serum albumin (BSA), such as adipocyte extracts, 
the 20-.mu.l stopping solution also contained 250 mM DTT and 5% SDS, pH 
7.0. Samples were kept in the ice bath until the completion of the 
experiment. For experiments in which the enzyme samples contained 
extraordinarily high protein concentrations, the rack containing the 
reaction tubes was immersed in a boiling H.sub.2 O bath for 2 min. 
A-Kinase sources were the cytosolic extracts of rat adipocyte homogenates, 
prepared as described by Honnor et al (R. C. Honnor et al (1985) J. Biol. 
Chem. 260, 15122-15129), the commercial catalytic subunit, and partially 
purified holoenzyme prepared according to Beavo et al (J. A. Beavo et al 
(1974) in Methods in Enzymology (K. Moldave and L. Grossman Eds.), Vol. 
30(C), pp. 299-308, Academic Press, New York). Under the above assay 
conditions, greater than 95% of Kemptide phosphorylation by these enzyme 
samples was attributable to A-kinase as assessed by either cAMP activation 
or inhibition by the specific inhibitor of this protein kinase (C. D. 
Ashby et al (1972), J. Biol. Chem. 247, 6637-6642), or by both criteria 
with adipocyte preparations. 
The C-kinase reaction mixture contained 20 uM GS-peptide, 10 mM Tris-HCl, 
pH 7.5, 5 mM magnesium acetate, 20 .mu.M ATP, 0.5 mM CaCl.sub.2, and 0.5 
.mu.Ci[.gamma.-.sup.32 P]ATP, in a total volume of 100 .mu.l. Where 
indicated, lipids were added to give final concentrations of 100 .mu.g/ml 
of phosphatidylserine and 10 .mu.g/ml of diolein. The lipid mixture was 
prepared by sonication in 20 mM Tris-HCl, pH 7.5, for 1 min at 30.degree. 
C. The reaction was initiated by the addition of approximately 10 mU of 
C-kinase and stopped as described above for the A-kinase reaction. Highly 
purified C-kinase from rat brain (K. P. Huang et al (1986), J. Biol. Chem. 
261, 12134-12140) was a gift from Dr. Kuo-Ping Huang, National Institutes 
of Health. 
Preparation of phosphopeptides. In order to follow the elution pattern of 
phosphopeptides over the chromatographic columns, .sup.32 P-labeled 
peptides were prepared and purified. Under the assay conditions described 
above, 20 U of the commercial catalytic subunit of A-kinase were reacted 
with 10 uM Kemptide and 5 .mu.M [.gamma..sup.32 P]ATP for 60 min. [.sup.32 
p]Kemptide was separated from ATP by two passages through a Dowex AGl-X8 
column (B. E. Kemp et al (1976), Proc. Natl. Acad. Sci. USA 73, 
1038-1042), and the purified material bound quantitatively to P-81 
phosphocellulose paper. Phosphorylated GS-peptide was prepared by reacting 
10 mU of purified C-kinase with 20 .mu.M [.gamma..sup.32 P]ATP and 20 
.mu.M peptide for 30 min. The phosphorylated peptide, which was purified 
by the method used to purify the phosphorylated Kemptide, also bound 
quantitatively to phosphocellulose paper. 
FIG. 2 shows the elution profiles of test compounds, such as 
[.gamma.-.sup.32 P]ATP and [.sup.32 P]-Kemptide, through the tandem column 
procedure of the present invention. Elution of compounds from the 
CM-Sephadex column are shown to the left of the vertical line in the 
figure, while elutions from both the CM-Sephadex and the AGl-X8 columns 
are shown to the right of the vertical line. FIG. 2A to the right of the 
vertical line shows materials emerging from the CM-Sephadex column and 
destined to pass through the AGl-X8 column, while FIG. 2B shows the 
materials that emerge from the AGl-X8 column. Note the log scale of the 
ordinate in FIG. 2. For illustrative purposes, 1 ml of the ATP washing 
solution was added to the stopped reaction mix, and the contents of the 
reaction tube were applied directly onto the resin bed with a Pasteur 
pipet. (Typically, as described below, the reaction tube contents, usually 
5 ml, are merely poured into the column, which broadens the radioactivity 
peaks but does not increase background values.) The elution of radioactive 
materials with successive 1-ml aliquots of the ATP washing solution is 
shown. Upon emergence of the fourth milliliter of washing solution, 
approximately 99% of the radioactive ATP was eluted, while all of the 
[.sup.32 P]Kemptide remained bound. Again, for illustrative purposes, FIG. 
2 depicts the elution of [.sup.32 P]ATP and [.sup.32 P]Kemptide from the 
CM-Sephadex column with successive 1-ml aliquots of 30% acetic acid before 
(FIG. 2A) and after (FIG. 2B) passage through the Dowex AGl-X8 column. The 
acetic acid continued to remove radiolabeled ATP and quantitatively eluted 
the [.sup.32 P]Kemptide from the CM-Sephadex column. Upon passage of the 
acetic acid eluate through the AGl-X8 column, all residual ATP was trapped 
in the resin, while the [.sup.32 P]Kemptide passed through the AGl-X8 
resin. In assays lacking a large amount of extraneous protein, the amount 
of [.sup.32 P]ATP that passed through the Dowex resin was barely 
discernible above the background counting rate of the scintillation 
counter. In all cases, the recovery of [.sup.32 P]Kemptide was at least 
95% of that which was applied initially to the CM-Sephadex column. FIG. 2B 
also shows the elution profile of [.sup.32 P]GS-peptide which was applied 
and eluted under conditions identical to those described above for the 
[.sup.32 P]Kemptide. Again, recovery of the radiolabeled GS-peptide was 
greater than 95% of that which was applied initially to the CM-Sephadex 
column. 
The general elution method according to the present invention 
advantageously reduces labor in assaying protein kinases and may be 
employed generally as follows. A volume of the ATP washing solution, 
usually 5 ml, is added to the stopped protein kinase reaction mixture with 
a repeating syringe; this addition is performed with sufficient vigor to 
mix the stopped reaction mixture into the ATP washing solution. The 
contents are poured into the CM-Sephadex column and the tubes are 
permitted to drain for a few minutes while resting inverted in the bowl 
portion of the columns. After the liquid has drained from the CM-Sephadex 
resin, these columns are mounted over the Dowex AGl-X8 columns. Eight 
milliliters of 30% acetic acid is applied to the CM-Sephadex columns and 
the total eluate from the AGl-X8 columns is collected in scintillation 
vials. In such an elution scheme, somewhat more radioactive ATP is carried 
through to the Dowex AGl-X8 resin than in the elution scheme depicted in 
FIG. 2 but, again, little ATP passes into the scintillation vial. 
Typically (Table 1), in this rapid elution method, fewer than 10 cpm of 
.sup.32 P from the nucleotide elutes from the Dowex AGl-X8 column, an 
assay background value which is barely discernible above the machine 
background counting value, 14 cpm, of the scintillation counter used in 
these experiments. However, nearly all (&gt;94%) of the [.sup.32 P]Kemptide 
or [.sup.32 P]GS-peptide is recovered (Table 2). Also, although 5 mM ATP 
was used in the bulk eluting solutions for the experiments presented in 
this paper, we have found that lowering the ATP to 0.5 mM does not change 
the column performance. 
TABLE 1 
______________________________________ 
MEASUREMENT OF PROTEIN KINASE ACTIVITIES IN 
RAT ADIPOCYTE EXTRACTS WITH KEMPTIDE 
AND GS-PEPTIDE AS SUBSTRATES 
Assay [.sup.32 P]Peptide formed (cpm) 
condition 
Control Stimulated Blank 
______________________________________ 
A-Kinase 811 .+-. 36 22,025 .+-. 289 
22 .+-. 0.6 
C-Kinase 2825 .+-. 124 
12,050 .+-. 318 
20 .+-. 0.4 
______________________________________ 
With regard to Table 1, it is noted that "Assay condition" indicates the 
assay mixture employed as described above, for measuring A-kinase activity 
with Kemptide or C-kinase activity with the GS-peptide. The stimulated 
condition with A-kinase was achieved with cAMP, and with C-kinase by the 
addition of calcium plus lipids, as described above. The enzyme source for 
A-kinase was the crude cytosolic extract of unstimulated adipocytes 
prepared according to Honnor et al (R. C. Honnor et al (1985) J. Biol. 
Chem. 260, 15122-15129). Since C-kinase activity in the crude cytosolic 
extract exhibited little stimulation by calcium plus lipid, the enzyme was 
partially purified as follows. After acidification to pH 5.2 with acetic 
acid and centrifugation to remove insoluble materials, the supernate was 
neutralized and diluted into a solution containing 25 mM Tris-HCl, pH 7.5, 
1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM DTT, 
and 30% glycerol. This solution was applied to a column of DEAE-cellulose, 
and the C-kinase used for the above experiment was eluted by 70-100 mM 
NaCl. Assays were conducted for 15 min with approximately 1.times.10.sup.6 
cpm of [.gamma.-.sup.32 P]ATP per tube, and the reactions were terminated 
with SDS and DTT, as described above. Samples were processed by the rapid 
elution method as described in the text. The final concentration of SDS in 
samples applied to the CM-Sephadex columns for assay of A-kinase 
activities was 0.1% while that for C-kinase was 0.02% (see discussion 
under Table 2). "Blank" represents cpm of .sup.32 P carried through the 
elution scheme when all assay ingredients were combined but no incubation 
was performed. That is, the reaction was terminated on ice immediately 
after addition of the enzyme sample. The "Blank" values are total cpm 
without subtraction of machine counting background, which was 
approximately 14 cpm. Values shown represent triplicate determinations 
(.mu..+-.SE). For the A-kinase experiment, the amount of enzyme used was 
of the order of 1/600 of the total in the adipocytes from the epididymal 
fat pads from a single rat, whereas the amount of C-kinase was 
approximately 1/10 of the total from an equivalent number of adipocytes. 
That is, in rat adipocytes, the capacity to phosphorylate Kemptide is far 
greater than the capacity to phosphorylate the GS-peptide. 
With a wide variety of enzyme preparations, both crude and pure, the same 
set of CM-Sephadex and Dowex AGl-X8 columns were reused for over 100 
different assays with no change in assay background or any change in 
recovery of phosphorylated peptides. The upper limit for the number of 
assay cycles that could be performed with a single set of columns was not 
found. The columns were repacked only to test the performance of different 
lots of ion exchange resins; no differences between lots were found. 
Assay of protein kinase activities in crude cellular extracts. The assay of 
protein kinase activities, e.g. A-kinase, in extracts of adipocyte 
homogenates was conducted using the method of the present invention. Since 
fat cells are incubated in solutions containing relatively high BSA 
concentrations, typically 1-5%, large amounts of this exogenous protein 
may be introduced into the kinase reaction mixture. Assay background 
values increased from the usual negligible levels to approximately 100 cpm 
per 10.sup.6 cpm of [.sup.32 P]ATP upon addition of more than 600 .mu.g of 
BSA to each assay tube (data not shown). Nevertheless, the columns 
performed satisfactorily and reproducibly from day to day if, during 
column regeneration, the NaCl and NaOH washes (as described above) were 
performed with the CM-Sephadex and Dowex AGl-X8 columns, respectively. 
Without these washes, background radioactivity levels continued to rise 
upon successive use of a given set of columns. After continued use through 
numerous cycles with samples containing large amounts of BSA, background 
radioactivity levels returned to negligible values upon subsequent assay 
with low amounts of exogenous protein. Finally, for routine use with all 
enzyme samples tested other than the BSA-laden fat cell extracts, it was 
not necessary to perform the NaCl and NaOH washes during regeneration of 
the columns. 
Inclusion of SDS and a relatively high concentration of DTT nearly 
eliminated this high background radioactivity upon assay of A-kinase and 
C-kinase activities in relatively crude adipocyte samples (Table 1). In 
the diluted sample applied to the CM-Sephadex column, SDS concentrations 
as high as 0.1% did not affect recovery of phosphorylated Kemptide. 
However, as shown in Table 2, SDS interfered with recovery of [.sup.32 
P]GS-peptide when the detergent concentration was above 0.02% in the 
sample applied to the CM-Sephadex column. Thus, when C-kinase assays were 
performed it was necessary to dilute the SDS sufficiently before applying 
samples to the columns. 
TABLE 2 
______________________________________ 
RECOVERY OF PHOSPHORYLATED SYNTHETIC 
PEPTIDES FROM TANDEM CHROMATOGRAPHIC 
COLUMNS: EFFECT OF SDS CONCENTRATION 
Recovered Recovery 
Peptide Condition (cpm).sup.a 
(%) 
______________________________________ 
[.sup.32 P]Kemptide 
0.02% SDS 14,216 .+-. 120 
94 
0.10% SDS 14,301 .+-. 437 
95 
[.sup.32 P]GS-Peptide 
0.02% SDS 9,024 .+-. 116 
96 
0.10% SDS 564 .+-. 29 
6 
______________________________________ 
It is noted that with regard to Table 2 that Kemptide and GS-peptide 
phosphorylated with A-kinase and C-kinase, respectively, were prepared and 
purified as described above. Each peptide was carried through the routine 
elution scheme designed for rapid processing as described in the text. 
Briefly, after addition of 5 ml of washing solution, samples were poured 
into CM-Sephadex columns, and after these columns were mounted atop the 
Dowex AGl-X8 columns, peptides were eluted directly into scintillation 
vials with 8 ml of acetic acid. The amount of [.sup.32 P]Kemptide and 
[.sup.32 P]GS-peptide applied was 15,060.+-.621 and 9400.+-.201, 
respectively. "Condition" refers to the SDS concentration in the sample 
applied to the CM-Sephadex column. This represents the final SDS 
concentration in the stopped reaction mix after addition of the washing 
solution to expand the sample volume for pouring into the column. 
EQU .sup.1 .mu..+-.SE; n=10 
Table 3 presents a comparison between the tandem column method of the 
present invention with Kemptide as the substrate and the filter trap 
method with histone as the substrate, a method used routinely to determine 
A-kinase activity ratios in cellular extracts (R. C. Honnor et al, (1985) 
J. Biol. Chem. 260, 15122-15129). The A-kinase (-/+)cAMP activity ratios 
of both slightly and moderately stimulated cells were comparable in both 
methods, an indication of the suitability of the tandem column method for 
measuring activity in crude extracts. Moreover, the data in Table 3 
provide a clear demonstration of the benefits in reduced background 
radioactivity, i.e., vastly improved signal to noise ratio. It should be 
noted that the background radioactivity in the filter trap method used in 
Table 3, approximately 400 cpm per 10.sup.6 cpm of substrate, is as low as 
the lowest values obtainable with Kemptide as the substrate when 
processing with phosphocellulose strips (R. Roskoski (1983) in Methods in 
Enzymology (J.D. Corbin and J. G. Hardman, Eds.), Vol. 99, pp. 3-6, 
Academic Press, New York). 
TABLE 3 
______________________________________ 
COMISON OF A-KINASE ACTIVITY RATIOS 
BY THE FILTER TRAP METHOD AND BY THE 
TANDEM CHROMATOGRAPHIC COLUMN METHOD 
Filter trap method 
Tandem column method 
.sup.32 P trapped (cpm) 
.sup.32 P eluted (cpm) 
16 nM 1000 nM 16 nM 1000 nM 
ISO ISO ISO ISO 
______________________________________ 
Blank 425 10 
(-) cAMP 1282 2713 1239 4582 
(+) cAMP 5918 4911 9385 8894 
(+) PKI 690 716 N.D. N.D. 
Activity ratio 
0.113 0.476 0.132 0.515 
(-/+) cAMP 
______________________________________ 
It is noted with regard to Table 3 that A-Kinase activities were assayed as 
described above, with Kemptide as the substrate for processing via the 
tandem column method of the present invention and with histone H1 as the 
substrate for processing via the filter trap method (R. C. Honnor et al, 
(1985) J. Biol. Chem. 260, 15122-15129). Each sample was assayed for 15 
min with 0.5 uCi of [.gamma.-.sup.32 P]ATP. The enzyme sources were 
cytosolic extracts of adipocyte homogenates prepared according to Honnor 
et al (R. C. Honnor et al, (1985) J. Biol. Chem. 260, 15122-15129). 
Isolated rat adipocytes were incubated in the presence of 3 nM PIA and the 
indicated concentrations of isoproterenol (ISO) for 5 min prior to 
homogenization. The A-kinase inhibitor, PKI, was present where indicated 
at 0.6 mg/ml. N.D. refers to "Not determined" in this experiment. 
As noted by Corbin (J. D. Corbin (1983) in Methods in Enzymology (J. D. 
Corbin and J. G. Hardman, Eds.), Vol. 99, pp. 227-232, Academic Press, New 
York), the use of the synthetic substrate instead of histone is 
advantageous with tissues containing high levels of cAMP-independent 
protein kinases. Previously, with the use of the specific A-kinase 
inhibitor, it was found that non-A-kinase activities account for 5-15% of 
total histone phosphorylating activity in fat cell extracts (R. C. Honnor 
et al, (1985) J. Biol. Chem. 260, 15122-15129). On the other hand, with 
Kemptide as the substrate, it has been found that non-A-kinase activities 
account for &lt;5% of total Kemptide phosphorylating activity (data not 
shown). Thus, with the synthetic substrate, as employed in the method of 
the present invention, little error is introduced into A-kinase activity 
ratio calculations by not accounting for non-A-kinase activities, as is 
evident in Table 3. 
After using the tandem column method of the present invention extensively 
for assaying both A-kinase and C-kinase in a variety of tissue samples, 
the only difficulty encountered was the increased background resulting 
from the introduction of unusually high protein concentrations in extracts 
of adipocyte homogenates. It should be noted that the tests performed with 
extraordinarily high BSA concentrations were designed to "stress" the 
assay system. It is evident from the data in Table 3 that A-kinase 
activity may be measured easily in 1/10 or 1/20 dilutions of fat cell 
extracts, or in greater dilutions if the radioactive substrate is 
increased. Although repeated use with crude samples containing relatively 
high protein concentrations from a variety of other sources produced no 
difficulties in column performance, it is possible that altered assay 
backgrounds might result from enzyme samples containing extremely high 
concentrations of proteins, either exogenous or endogenous. Since the 
addition of SDS and DTT to samples before boiling and loading onto the 
CM-Sephadex columns virtually eliminated the elevated backgrounds due to 
BSA, this technique is suggested for at tempting to reduce protein-induced 
elevated background activity, should this become a problem. To determine 
if components of the enzyme samples are contributing to increased 
background radioactivity, it may be necessary to test enzyme samples 
occasionally in combination with the radioactive substrate both before and 
after performing the protein kinase incubation reaction. Also, to guard 
against artifacts resulting from phosphorylation of endogenous proteins 
which may migrate through the columns with the peptide substrates, one 
should perform incubations without exogenous substrates but with 
[.gamma..sup.32 P]ATP. 
The method of the present invention offers several advantages over existing 
procedures. For the assay of a large number of samples, the labor-saving 
features may be beneficial. One need not accurately sample an aliquot of 
the terminated kinase reaction mix, nor is handling of individual filter 
strips or disks necessary. The entire sample is simply poured into a 
column, and the only labor involved thereafter is the addition of washing 
and eluting solutions to the columns with repeating syringes. Typically, 
one person may process 100 samples in less than 40 min. of which less than 
10 min. attendance time is required; the remaining time is to allow 
columns to drain. 
If protein kinase activity is limiting, the advantage in the method of the 
present invention lies in the extremely low background. Since the assay 
background radioactivity with the tandem-column method is lower by at 
least a factor of 30 than most existing methods, it follows that 
sensitivity in detecting kinases is increased by this same factor. 
Alternatively, if the kinase is not limiting, one may choose to lower the 
amount of radioactive substrate, [.gamma.-.sup.32 P]ATP. For example, 
previously A-kinase activities were assayed with histone as the substrate, 
and the phosphohistone was collected on glass fiber filters (R.C. Honnor 
et al, J. Biol Chem. 260, (1985) pp. 15122-15129). This required the use 
of approximately 0.5 uCi of radioactive ATP to detect the A-kinase 
activity in the extracts of 1000-2000 adipocytes. With the method of the 
present invention, A-kinase activities in extracts of 1000 cells are 
easily determined with 0.05 uCi of [.gamma..sup.32 P]ATP. Also, this assay 
would be beneficial in detecting relatively sparse protein kinase species, 
such as the insulin-stimulated protein kinases in cellular extracts. Yu et 
al (Yu et al, J. Biol. Chem. 262, (1987) pp. 16677-16685) added 18-75 
.mu.Ci of [.gamma.-.sup.32 P]ATP per assay tube in order to detect an 
insulin-stimulated serine kinase in adipocytes with Kemptide as the 
phosphate acceptor. With the tandem column method of the present 
invention, these investigators would have required 1 uCi or less of 
substrate for each determination, a considerable reduction in exposure to 
radioactivity. Finally, the cost savings resulting from a reduction in 
substrate may be considerable, especially for laboratories assaying large 
numbers of samples with several .mu.Ci of [.gamma.-.sup.32 P]ATP per 
determination, typical for many investigators in this field, or for those 
who require prodigious amounts of substrate. 
The invention being thus described, it will be obvious the same may be 
varied in many ways. Such variations are not to be regarded as a departure 
from the spirit and scope of the invention and all such modifications as 
would be obvious to one skilled in the art are intended to be included 
within the scope of the following claims.