Purification and Properties of a Protein Kinase from Bovine Corpus Luteum That Is Stimulated by Cyclic Adenosine 3 ’ , 5 ’-Monophosphate and Luteinizing Hormone *

A protein kinase has been purified from the bovine corpus luteum by acid precipitation, ammonium sulfate fractionation and chromatography on DEAE-cellulose and hydroxylapatite columns. The enzyme has a molecular weight of approximately 159,000. DEAE-cellulose chromatography resolved the protein kinase activity into two peaks, designated as KI and KII. Both peaks possessed adenosine 3’,5’-monophosphate (cyclic AMP)-binding activities and both kinase activities were stimulated in vitro by cyclic AMP. The extent of stimulation of KII by cyclic AMP was greater than that of KI. (K, for cyclic AMP, 2.0 X lop8 M.) KII was further purified by chromatography on hydroxylapatite. In addition to stimulation by cyclic AMP, the enzyme recovered from hydroxylapatite was also stimulated by luteinizing hormone (LH) in vitro. The response to LH was concentration dependent and specific. Other pituitary hormones were ineffective. The effects of LH and cyclic AMP were not additive and the binding of cyclic [3H]AMP to the hydroxylapatite-treated enzyme was not inhibited by the presence of LH. It is concluded that LH may have a direct control on the activity of protein kinase in the corpus luteum that is independent of cyclic AMP.


SUMMARY
A protein kinase has been purified from the bovine corpus luteum by acid precipitation, ammonium sulfate fractionation and chromatography on DEAE-cellulose and hydroxylapatite columns.
The enzyme has a molecular weight of approximately 159,000.
DEAE-cellulose chromatography resolved the protein kinase activity into two peaks, designated as KI and KII.
Both peaks possessed adenosine 3',5'-monophosphate (cyclic AMP)-binding activities and both kinase activities were stimulated in vitro by cyclic AMP. The extent of stimulation of KII by cyclic AMP was greater than that of KI.
(K, for cyclic AMP, 2.0 X lop8 M.) KII was further purified by chromatography on hydroxylapatite. In addition to stimulation by cyclic AMP, the enzyme recovered from hydroxylapatite was also stimulated by luteinizing hormone (LH) in vitro. The response to LH was concentration dependent and specific.
Other pituitary hormones were ineffective.
The effects of LH and cyclic AMP were not additive and the binding of cyclic [3H]AMP to the hydroxylapatite-treated enzyme was not inhibited by the presence of LH.
It is concluded that LH may have a direct control on the activity of protein kinase in the corpus luteum that is independent of cyclic AMP.
In the bovine corpus luteum, luteinizing hormone activates adenylate cyclase to convert ATP to cyclic adenosine 3', 5'monophosphate (1). Both LH1 and cyclic AMP are capable of stimulating progesterone synthesis in this tissue from acetate (2). Also, it has been demonstrated that prior to an increase in progesterone synthesis, under the influence of LH, there is an increase in cyclic AMP concentration in the luteal tissue (3). It was therefore inferred that cyclic AMP acts as * This investigation was support,ed, in part, by a program project HD 05318 from NICHD, and a grant from the Phoenix Memorial Project. an intracellular messenger of LH to stimulate the synthesis of progesterone in bovine corpus luteum.
Although cyclic AMP is a mediator of the actions of a variety of hormones, the mechanism of action of the cyclic nucleotide in the target tissues, is not known.
Since the discovery that cyclic AMP is involved as a mediator in the glycogenolytic effect of epinephrine in the liver (4) and that this effect is mediated by the activation of phosphorylase b kinase kinase (5), a large number of kinases have been isolated from different tissues which are responsive to low concentrations of cyclic AMP. Furthermore, it has been postulated that the diverse biological effects of cyclic AMP are mediated by a family of enzymes known as "protein kinases" (6).
The present investigation is an attempt to understand the mechanism of regulation of cellular events in bovine corpus luteum by LH. This communication describes the partial purification and characterization of protein kinase from bovine corpus luteum.
The data suggest that in addition to cyclic AMP, LH itself is capable of directly stimulating protein kinase in vitro in this tissue.

MATERIALS AND METHODS
Collection of Bovine Corpora Lutea-Bovine corpora lutea were obtained from a local slaughterhouse.
The corpora lutea were collected only during the cycle; tissues from pregnant cows were not included.
The tissues were transported to the laboratory in ice and subsequently frozen at -80".
ChemicaZs-[y-32P]ATP was obtained from New England Nuclear Corporation and cyclic [3H]AMP was purchased from Schwarz Mann Chemical Company.
All other chemicals were conventional commercial products. LH (NIH-LH-B,), FSH (NIH-FSH-PI), and prolactin, were generously donated by the Endocrine Study Section, National Institute of Health. ACTH was obtained from Squibb and HCG from Ayerst Laboratories.
&say of Protein Kinase-Protein kinase was assayed by the procedure described by Kuo et al. (7). The assays were performed in a final volume of 200 ~1 containing the following addition: 1 nmole [y-32P]ATP (1 X lo6 cpm), 2 pmoles of NaF, 2 pmoles of magnesium acetate, 0.4 pmole of theophylline, 0.4 mg of calf t,hymus histone, 10 pmoles of a-glycerophosphate buffer, pH 6.0, and a suitable amount of the enzyme preparation. 495 min), and t.he react,ions were stopped by the addition of 4 ml of 7.5% trichloroacetic acid. To this 0.2 ml of 0.63% bovine serum albumin was added. The supernatant fluid was separated from the precipitate after centrifugation and the precipitate was washed three times with 5'r0 trichloroacetic acid, 2 ml each. The washed precipitate was dissolved in 0.1 ml of 1 N NaOH and the radioacti+-in the aqueous residue was determined by liquid scintillat'ion counting after the addition of 10 ml of Herberg Solution (8) and 0.05 ml of formic acid.

Assay of Cyclic AVP Binding
Activity-Cyclic AMP binding activity was assayed by the procedure described by Gilman (9) with minor modificat~ions. The reaction was carried out in a final volume of 200 ~1 by the following procedure.
The incubation mixture contained 1 ~BI cyclic [3H]AMP (10,000 cpm), 50 mM ammonium acetate, pH 4.0, and a suitable volume of the column efiluent (usually 100 ~1). The incubations were carried out at 0" for 2 hours and were terminated by the addition of 1 ml of 20 ml~ pot,assium phosphat~e buffer, pH 6.0. After 5 min, the solut,ious were filtered through cellulose nitrate membrane filters (0.45 p). The filters were then washed with 10 ml of potassium phosphate buffer, pH 6.0 and dried under a lamp. The dried filters were then transferred to scintillation vials and dissolved in 1 ml of cellosolrc.
Protein Detern,inat.ion-l'roteia was determined by the method of Lowry et d. (IO). Protein profiles in column effluents were assessed by measuring absorbance at 280 nm.
10% Excllnnge C%ro,,latography-DEAE-cellulose (Whatman DE-23) was washed with acid and alkali as described by the manufacturer (What.tnan Technical Bulletin, IE2) and was packed into a column in the presence of 1 M potassium chloride buffered wit,h Buffer A (10 mM Tris-HCl, pH 7.5, 6 mm mercaptoethanol, and 10cC glycerol). After the packed column had been washed with this solution, it was washed with water and then with the appropriate starting buffer until the pH and the conductivity of the effluent and the buffer entering the column were identical.
The column was then washed with 2 column volumes of Buffer B until the pH and the conductivity of the solution leaving the column were identical to the buffer entering the column.
Purificatio~z of Protein Kkase-Unless stated otherwise, all operations were carried out at 4". Centrifugations were carried out in a Sorvall RC 2B refrigerated centrifuge using either a GSA rotor or a SS 34 rotor.
Ultraccntrifugation was carried out in a Beckman L2 65B centrifuge equipped with a 60 Ti rotor.
The distribution of the enzyme amougst subcellular fractions is shown in Table I and the procedure for purification for the soluble fractiou is summarized in Table II. Step I. Preparation oj Soluble Enzyme-Frozen corpora lutea (124 g) were thawed and major blood vessels were removed, and the t'issue was then homogenized in 3 volumes of Buffer A. The homogenization, in a jacketed Waring Blendor, was carried out for 1 min at 70 volt,s followed by 5 min at 40 volts. The homogenate (490 ml) was centrifuged at 6200 rpm (GSA rotor) a Corpus luteum (9 g) was homogenized in the medium described under "Materials and Methods" to yield the whole homogenate. This was centrifuged at 2200 rpm for 10 min (SS 34 rotor) to sediment the nuclear fraction which was suspended in homogenizing medium.
The nuclear supernatant was centrifuged at 7200 rpm for 25 min (SS 34) to yield mitochondrial pellet which was resuspended in t,he homogenizing medium.
The resultant supernatant was centrifuged at 40,000 rpm in a Beckman model L2 ultracentrifuge for 90 min.
b One enzyme unit is nanomoles of SzP from [Y-~~P]ATP incorporated into histone per min at 30". a Frozen corpora lutea (124 g) were treated as shown in the text.
b One activity unit is defined as that amount of enzyme necessary to catalyze the transfer of 1 pmole of 3ZP from [-+zP]ATP into histone per minute at 30". 0 Specific activity is the picomoles of 32P from [yJzP]ATP incorporated into histone per min per mg of protein at 30".
for 25 min. The supernatant liquid was decanted and ultracentrifuged at 38,000 rpm (60 Ti rotor) for 90 min. The supernatant (350 ml) contains the soluble enzyme (Step I enzyme).
Step II. Acid Precipitation-To Step I enzyme was added 1.0 N acetic acid until a pH of 5 was reached.
The solution was allowed to remain at this pH for 30 min after which the solution was centrifuged at 15,000 rpm (SS 34 rotor) for 10 min. The supernatant liquid was readjusted to pH 7.4 by slow addition of 1.0 N NHdOH.
This solution (380 ml) is the Step II enzyme fraction.
Step III. Ammonium Xuljate Fractionation-Ammonium sulfate (148.2 g) was slowly added, with stirring, to the Step II enzyme. After 1 hour, the mixture was centrifuged at 20,000 rpm (SS 34) for 10 min. The residue containing the enzyme was dissolved in Buffer A (40 ml). The solution was dialyzed against two changes of 2 liters of the same buffer. The resulting solution was centrifuged at 20,000 rpm (SS 34 rotor) for 10 min to remove a precipitate formed during dialysis. The supernat,ant liquid is the Step III enzyme.
Step IV. Chromatography on DEAE-cellulose-The Step III enzyme was diluted to a protein concentration of 14 mg per ml with Buffer A (final volume 87 ml), and applied to a column (43 x 2.5 cm diameter) of DEL4E-cellulose which had been previously equilibrated with the same buffer just prior to loading. Subsequently, the column was washed with buffer until the effluent had an absorbance of 0.02 at 280 nm. The proteins were eluted using a linearly increasing salt gradient with Buffer A (1 liter) in the mixing vessel and Buffer A containing 0.4 M potassium chloride (1 liter), in the reservoir.
Fractions (20 ml) were collected at 20-min intervals (Fig. 1). The enzyme was eluted in two peaks (Peak I and Peak II).
Peak II had a higher specific activity than Peak I and responded to cyclic AMP more than Peak I. Fractions (50 to 80) corresponding to Peak II were pooled together (660 ml) and the enzyme precipitated with ammonium sulfate (80% saturation).
The precipitate was allowed to settle overnight and was then collected by centrifugation at 20,000 rpm (SS 34 rotor) for 10 min. It was dissolved in 0.05 M sodium phosphate, pH 7.4 (13 ml) and dialyzed against two changes of the same buffer (2 liters).
The dialyzed enzyme was centrifuged at 20,000 rpm (SS 34 rotor) for 10 min to yield Step IV enzyme.
Step V. Chromatography on Ilydroxylapatite-Step IV enzyme was applied to a column (17 x 2 cm diameter) of hydroxylapatite (Bio-Rad) packed in 0.005 nr potassium phosphate, pH 7.0. After loading, the column was washed with t,he same buffer (100 ml), fractions of 2.5 ml were collected at lo-min intervals.
The column was then eluted with a linearly increasing concentration of salt with 0.005 M potassium phosphate (500 ml) in the mixing chamber and 0.4 nl potassium phosphate (500 ml) in the reservoir (Fig. 2). Fractions containing the enzyme were pooled and the protein precipitate was collected by centrifugation at 20,000 rpm (SS 34 rotor) for 10 min and was dissolved in 0.005 M potassium phosphate, pH 7.0, to yield the Step V enzyme.
Chromatography on Sephadex G-dOO-Step T' enzyme (0.5 ml) was applied to a column (39 x 2.5 cm diameter) of Sephadex G-200, packed in 0.05 M potassium phosphate, pH 7.0. The column was eluted with the same buffer, fractions of 2 ml were collected at IO-min intervals.

RESULTS
The distribution of the protein kinase activity is shown in Table  I. Since the soluble fraction contained significant FIG. 1. Chromatography of protein kinase from bovine corpus lutea on DEAE-cellulose.
Step III enzyme (1218 mg) was diluted to a protein concentration of 14 mg per ml (final volume 57 ml) with Buffer A and chromatographed on DEAE-cellulose column (43 X 2.5 cm diameter) as described in text. The elution of protein was measured by absorbance at 280 nm. The eluate (200 ~1) were used for the assay of protein kinase and cyclic ANP binding activities. amounts of the enzyme activity, purification of the enzyme was carried out using t,his fraction. Table  II shows the summary of the purification procedure.
DEAE-cellulose chromatography ( Step IV enzyme) resolved the kinase activity into two peaks, kinase I and kinase II. Since the kinase II was responsive to cyclic AMP to a greater extent than the kinase I (data presented under "Properties of Kinase"), further purification was performed only with a pool of the fractions having kinase II activity.
Therefore, no attempt is made to calculate the recovery of Step V enzyme.

Effect of Incubation Conditions on Step V Enzyme Activity
EJect of Enzyme Concentration-Incorporation of 32P from [y32P]ATP into histone with increasing enzyme concentration is shown in Fig. 3A. The incorporation was linear up to 6.0 pg concentrat.ion of Step V enzyme.
In subsequent experiments 2.5 pg of enzyme, were used. Effect of Incubation Time-A linear rate of incorporation of 3*P was observed up to 30 min at 30" (Fig. 3B).
E$ect of pH-Using histone as substrate, two pH optima were found; pH 6.0 and pH 7.5. In subsequent experiments, pH 6.0 was chosen as the pH of the incubation medium.

Properties of
Step V Enzyme &ability-The kinase was stable for 30 min exposures to temperatures up to 40" (Fig. 4) The enzyme was eluted in three peaks, which probably is an indication of the dissociation of subunits. Approximately 80% of the total enzyme appeared in the major peak; all three peaks were catalytically active (Fig. 5A). The position of elution of the second peak (87.75 ml) from a column of Sephadex G-200 (39 x 2.5 cm diameter) relative to aldolase (88.0 ml), ovalbumin (110 ml), chymotrypsinogen A (127 ml) and ribonuclease A (187 ml) implies a molecular weight of approximately 160,000 (Fig. 5B). 498

Effect of Nucleotides on Step V Enzyme Activity
Since the kinase activities from other sources (11) are sensitive to small concentrations of cyclic AMP, the responsiveness of Step V enzyme to nucleotides was studied.
A summary of the observations is presented in Table III. Both cyclic AMP and cyclic GMP stimulated protein kinase activity from bovine corpus luteum.
%Iasimal stimulation of kinase activity was found at 1O-6 M and lo+ q respectively.
Since the Step V enzyme possesses both the kinase and cyclic AMP binding properties, the K, for cyclic -4MP was determined. A Scatchard plot (12) of cyclic [31-I]AMP binding to Step V enzyme is presented in Fig. 6. Two binding constants were calculated, a high affinity binding site with K,, 2.0 x lo* liters per Step V enzyme (2.5 rg) was incubated with varying concentrations of cyclic AMP (all tubes contained 10,000 cpm cyclic [aHlAMP) in a final volume of 150 ~1 containing 50 rnM ammonium acetate, pH 4.0. Incubations were performed at 0" for 15 min and the reactions terminated by the addition of 1 ml of 20 mM potassium phosphate, pH 6.0. After 5 min the solutions were filtered through cellulose nit,rate membrane filters (0.45 p) and washed with 10 ml of 20 m&t pot.assium phosphate buffer, pH 6.0. The fiIters were then dried and radioactivity determined as described in the text. mole and a low affinity binding site, with K,, 0.91 x lo4 liters per mole. The latter may represent nonspecific interaction of macromolecules with small ligands and as such, may not, contribute to physiologica activity.
In Vitro E$ect of Luteinizing Hornwne on Kinase ktivity Incubation of Step V enzyme wit,h varying concentrations of LH is presented in Table IV. At 0.1 PL$ concent'ration of LH, there was about 5OL;I, increase of the kinase activity.
The response to LH in vitro was concentration dependent as evidenced by an increase of almost 4-fold at a concentration of 20.0 pg of LH.
Bovine LH inactivated by treatment with H202 did not stimulate the kinase activity.
Both a and j3 subunit,s of bovine LH were also without effect. According to the SIH specifications, the mean relative potencies of bovine and r;heep LH preparations are 1.16 and 1.09 units per mg, respcct.irely.
Other pituit,ary hormones and HCG failed to cause an increase in the kinase activity (Table  V) Fig. 7, the presence of unlabeled cyclic AhlP caused a reduction in the radioactivity bound to Step V enzyme, but     were performed at 0" for 2 hours and the reactions were stopped by the addition of 1 ml of 20 mM potassium phosphate buffer, pH 6.0. The assays were completed as described in the text.

499
lation of the protein kinase did not contain endogenous cyclic AMP.

Test for Phosphorylation of LH by
Step V Enzyme-The possibility that the stimulatory effect of LH on t.he kinase was a result of the phosphorylation of LH itself was tested and the results are shown in Table VI. When LH was substituted for histone in a standard incubation mixture, no significant amount of radioactivity was found in the trichloroacetic acid precipitate. However, when LH (5.0 kg) was added to an incubation mixture containing histone, the extent of phosphorylation was almost 100% greater than the phosphorylation of histone in the absence of LH.
This experiment suggests that the observed stimulation of the histone phosphorylation caused by LH is not a result of phosphorylation of LH itself. The stimulatory effects of LH and cyclic AMP in vitro on the kinase activity were not additive (Table VII).
When 5.0 pg of LH were added to a standard incubation mixture containing lo-* M cyclic AMP, the extent of stimulation reached only equal to that produced by cyclic AMP alone. Even at subopti-ma1 concentrations, LH and cyclic AMP did not produce additive effects.
Test for Protein Kinase Activity in LH Preparations-The possibility that the LH preparations used in the investigations may be contaminated by protein kinase was assessed by incubating varying concentrations of LH under the standard assay conditions, in the absence of the enzyme preparation itself. As can be seen in Table VIII, LH preparations in the absence of added protein kinase did not cause phosphorylation of histone. When the enzyme was added, significant extent of phosphoryla-

This communication
describes the isolation of a protein kinase from bovine corpus luteum, which has, in general, properties similar to corresponding enzymes from other tissues (11). The purificat,ion of the enzyme was conducted by established procedures.
The molecular weight of the enzyme, as determined from it,s position of elution from Sephadex G-200, is approximately 160,000. This large size is comparable to that reported by Reimann et al. (13) for skeletal muscle protein kinase (6.8 S): Gill and Garren (14) for the adrenal cortex protein kinase (7 S), Tao et al. (15) for the rabbit reticulocyte protein kinase, Rubin et al. (16) for enzyme from bovine heart muscle, and Miyamoto et al. (17) for the bovine brain protein kiiase.
Electrophoresis of Step V enzyme on polyacrylamide gels (18) showed the presence of three protein components which may represent &her dissociation of subunit structure or the presence of ot,her contaminating proteins. One of the unique characteristics of the protein kinases is their in vitro activation by low concentrations of cyclic AMP. It has been hypothesized that the diverse cellular effects of cyclic BMP are mediated through activation of tissue specific protein kinases as has been illustrated by the glycogenolytic effect of cyclic AMP (5). The results reported here show that protein kinase from corpus luteum is also extremely sensitive to activation by 10~ concentrations of cyclic AMP.
The present, dat,a also suggests that in addition to control by cyclic SMP, t,he prot,ein kinase present in bovine luteal tissue may be stimulat.ed by LH itself, in vitro. The direct stimulatory effect of LH on t'he kinase was rather an unexpected observation and it has been ascertained esperimentally that the stimulatory effect of LH is not as a result of (a) contaminating cyclic AMP associated with LH preparations; (b) adenylate cyclase activity associn.ted with protein kinase and LH preparations; (c) phosphorylation of LH itself by protein kinase; and (cZ) protein kinase associated with the LH preparations.
The first possibility was examined by the nature of inhibition of the binding of cyclic [3H]A11P to protein kinase. As shown in Fig. 7 No cyclic BMP was detected in the heat denatured LH preparation, when assayed by the competitive protein binding assay of Gilman (9). The second possibility was tested by assaying adenylate cyclase activities in the Step V enzyme and also in the LH preparations.
Adenylate cyclase was assayed by previously published procedures (19,20). Even at concentrations of Step V enzyme 20 times greater than that used in the incubations of the standard kinase reaction, there was no detectable adenylate cyclase activity associated with Step V enzyme in the presence or absence of LH.
Thirdly, LH itself was not phosphorylated by Step V enzyme and therefore this cannot account for the observed stimulation of histone phosphorylation by LH.
The last possibility was ruled out by the absence of protein kinase activity associated with the LH preparation, as shown in Table VIII.
The effects of LH and cyclic AMP were not additive, suggesting that both LH and cyclic AMP may have separate receptors and yet act on the same catalytic subunit. Such a possibility has been earlier proposed by Sutherland and co-workers (21) for the adenylate cyclase system in rat adipocytes in which glucagon and epinephrine stimulate the enzyme, but the adenylate cyclase in the presence of two hormones only reached the activity produced by the more effective hormone of the two.
The stimulation of kinase activity in target tissue by pituitary hormones without concurrent increase in cyclic AMP has previously been reported.
In a recent study, Majumdar and Turkington (22) have shown that prolactin and insulin caused an increase in the activity of a cyclic AMP-dependent protein kinase in the mouse mammary gland grown in organ cultures without an increase in the adenyl cyclase activity.
These results were interpreted to suggest that prolactin caused an increase in the availability of the binding protein for cyclic AMP. Thus, the increased activity of protein kinase in mammary gland by treatment with prolactin was thought to be due to increased availability of cyclic AMP-binding protein associated with protein kinase. Activation of protein kinases by histone (23) and protamine (24) has also been reported recently.
Analysis of the enzymes treated with histone and protamine by sucrose density gradient centrifugation revealed that the protein kinases were dissociated into subunits.
It is not known whether the mechanism of stimulation of the protein kinase from corpus luteum by LH might also occur in a similar manner.
The activation of the protein kinase by LH in the corpus luteum is particularly interesting, because the role of LII in maintaining the integrity of the corpus luteum during early pregnancy and the stimulatory role of LH on progesterone synthesis in the corpus luteum are well established.
Partially purified protein kinase from human term placenta is not stimulated by LH although this enzyme is stimulated by cyclic AMP suggesting some degree of specificity. One obvious difficulty in attaching physiological significance to the observed effect of protein kinase activation directly by a large molecule, such as LH, is the question of permeability of t,he hormone into the luteal cell. So far, there 501 is no direct evidence demonstrating the entry of LH into the target cell. Also, the concentrations of LH used in these experiments were greater than the physiological concentrations of LH. However, the observed activation of the kinase by LH directly in the luteal tissue might offer an alternate mechanism of regulation of intracellular functions in the luteal tissue by LH, independent of cyclic AMP.