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THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol.
1, Issueof January Phfed
10,PP. 1622, 1972
AN ASSAY METHOD FOR THE OUS BIOLOGICAL MATERIALS AND BRAIN*
Protein MEASUREMENT AND A STUDY
JYH-FA Kuof, TEE-PING LEE$, JR.~, AND PAUL GREENGARD
OF GUANOSINE 3’,5’-MONOPHOSPHATE OF AGENTS REGULATING ITS LEVELS
(Received for publication,
August 23, 1971)
From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510
in cyclic GMP levels accompanied by a great elevation in cyclic AMP levels. These data support the concept that the levels of cyclic GMP and cyclic AMP are under separate regulatory control.
An assay method has been developed for the measurement of tissue levels of guanosine 3’,5’-monophosphate (cyclic GMP) based upon the ability of the cyclic nucleotide to activate cyclic GMP-dependent protein kiiase prepared from The either lobster tail muscle or silkmoth pupal fat body. method has been used to measure cyclic GMP levels in various biological tissues and human urine, and in slices of heart and brain incubated under various conditions. The results obtained with this new method agree well with published values of tissue levels of cyclic GMP. The limit of the sensitivity of the assay method is about 0.5 pmole of cyclic GMP in an incubation volume of 0.2 ml (2.5 x 10eg M). Acetylcholine caused a rapid (within 0.5 min) increase of 5- to lo-fold in the cyclic GMP level in rat ventricular slices. The cyclic GMP level returned to the basal value within 5 min. Acetylcholine also effectively lowered the increase in myocardial adenosine 3’,5’-monophosphate (cyclic AMP) caused by isoproterenol or glucagon. Isoproterenol or glucagon, in reverse fashion, effectively lowered the increase in myocardial cyclic GMP level caused by acetylcholine. The basal level of myocardial cyclic AMP was not significantly influenced by acetylcholine, nor the basal cyclic GMP level by isoproterenol or glucagon alone. Acetylcholine increased cyclic GMP levels and decreased cyclic AMP levels in rabbit cerebellar slices. In cerebral cortex slices, acetylcholine increased cyclic GMP levels without affecting cyclic AMP levels. Norepinephrine, on the other hand, lowered the cyclic GMP levels while greatly increasing the cyclic AMP levels of cerebellum. Histamine was without effect on cyclic GMP levels in cerebellum but increased cyclic AMP levels. In cerebral cortex, histamine caused a slight increase
Guanosine 3’) 5’-monophosphate has been found in many biological materials (e.g. l-4). Several lines of evidence, although somewhat fragmentary, suggest that cyclic GMP’ may be involved in a second messenger system distinct from that involving cyclic AMP, i.e. under separate hormonal or metabolic control and with separate regulatory functions (2-13). Some recent, data suggest that cyclic GMP may be involved in cholinergic neurotransmission in heart (7) and brain (8). It is becoming increasingly important to have reliable methods for the measurement of cyclic Gh/IP in biological materials in order to elucidate the role of this nucleotide in cellular metabolism and function. Currently, there are several assay methods available for the measurement of cyclic GMP, including enzymatic coupling methods (2-4, ll), a radioimmunoassay method (la), a radioisotopic displacement method with phosphodiesterase (13), and a method (14) based upon the binding of cyclic GMP to a protein kinase. In the course of our studies on cyclic nucleotide receptor proteins, we have found a group of cyclic GMP-dependent protein kinases, first in lobster tail muscle (9) and subsequently in tissues from several other species and phyla (10, 15). We were able to separate these enzymes from cyclic AMP-dependent enzymes present in the same sources (9, 10, 15). These various cyclic GMP-dependent protein kinases have K, values for cyclic GMP in the range of 2 x lo-* to 3 X 10m7 &f (9, 10). Since we had earlier developed a met,hod for assaying cyclic AhIP based upon its ability to activate cyclic AMP-dependent protein kinases (16), the discovery of cyclic GMP-dependent protein kinases suggested the possibility of developing an analogous assay method for cyclic GMP. In this paper we report a sensitive, specific, and accurate assay method for cyclic GMP employing cyclic
* This work was supported by Grants HE-13305, NS-08440, and MH-17387 from the United States Public Health Service, by Grant G-70-31 from the Life Insurance Medical Research Fund, and by Grant GB-27510 from the National Science Foundation. Papers VIII
and IX 2 Recbient
in this series of Research
GR;f-5016%) from the United 0 Recipient
are References 16 and Career Development
10, respectively. Award (1 K4
States Public Health Service.
Public Health Service. 7 Recipient of Predoctoral States Public Health Service.
from the United
used are: cyclic GMP, AMP,
Issue of January 10, 1972 GMP-dependent protein kinase tail muscle or silkmoth pupal fat volume of 0.2 ml, the sensitivity was found to be about 0.5 pmole EXPERIMENTAL
Kuo, Lee, Reyes, Walton, Donnelly, and Greengaul prepared from either lobster In the usual incubation body. limit of this new assay method of cyclic GRIP. PROCEDURE
39ateriaZs-Cyclic GblP-dependent protein kinase from lobster tail muscle (9) and cyclic AMP-dependent protein kinase from bovine heart (17, 18) were purified to the step of DEAE-cellulose chromatography according to the procedure described earlier. Cyclic GMP-dependent protein kinase from the fat body of Cecropin silkmoth pupae was purified to the step of calcium phosphate gel as reported elsewhere (10). A protein factor which inhibits cyclic AMP-dependent protein kinase (19, 20) was isolated from lobster tail muscle by a procedure similar to that reported by Appleman et al. (19) who used rat skeletal muscle as the starting material. Cyclic 3’) 5’-nucleotide phosphodiesterase was purified from bovine heart according to the method of Butcher and Sutherland (21). Acetylcholine, glucagon, norepinephrine, and isoproterenol were purchased from Sigma; cyclic [GJH]GMP (1.0 mCi/0.081 me;) and cyclic [G-3H]AMP (1.0 mCi/0.0144 mg), from New England Nuclear; histone mixture (calf thymus), from Schwarz-Mann; Cecropia silkmoth pupal fat bodies were kindly provided by Dr. G. R. Wyatt of the Department of Biology. Live lobsters were purchased from a local fish market. Other materials used in the present study were the same as in previous papers (9, 17, 18). Preparation of Tissue Extracts-The procedure used to prepare tissue extracts for cyclic nucleotide analysis was essentially the same as reported earlier (16). Male Sprague-Dawley rats, weighing about 250 g each, were killed by decapitation. Each of the rat tissues reported in Tables I and II was removed, sliced into small pieces, and homogenized in 2 or 3 volumes of cold 5% trichloroacetic acid as rapidly as possible. Cecropia silkmoth pupal fat body, lobst.er tail muscle, and human urine were processed in the same manner as the rat tissues. Cyclic [G-3H]GMP (about 5.0 x 1Oj cpm) and cyclic [G-3H]AMP (1.7 x lo5 cpm) were added to separate aliquots of the homogenates for the purpose of determining the recovery of tissue cyclic GMP and cyclic AMP during purification of the cyclic nucleotides. After removal of the precipitate by centrifugation, the supernatant solution was adjusted to pH 7.0 with 1 M Tris. Preparation and Incubation of Heart and Brain Slices-Six Sprague-Dawlcy rats, each weighing 200 to 250 g, were anesthetized with ether and the hearts were quickly excised after decapitation. The ventricles were sliced (0.5 mm thick) with a Stadie-Riggs tissue slicer (Arthur H. Thomas Co.), and the slices thus obtained were further cut into pieces of 0.5 mm, with a JlcIlwain tissue chopper (Brinkmann Instruments). The ventricular slices were suspended in 30 ml of ice-cold KrebsRinger bicarbonate buffer, pH 7.4, containing 0.4 InM calcium, i.e. one-half the usual amount of calcium, and were collected by centrifugation. This washing procedure was repeated once again. The washed slices were finally suspended in the bicarbonate buffer containing 3 mrvr theophylline. One-milliliter aliquots of the tissue suspension, containing about 15 to 25 mg of protein, were incubated at 37” for varying times under various conditions as indicated in the individual tables. At the end of the incubation period, the medium was removed from the slices by centrifugation in a table model centrifuge followed by aspiration, the reaction stopped by the addition of 1 ml of cold 5%
trichloroacetic acid, and the tissue homogenized. The centrifugation, aspiration, and addition of trichloroacetic acid took less than 15 sec. The procedure for the preparation and incubation of rabbit brain slices was essentially the same as described by Kakiuchi and Rail (22). The balanced salt solution of Sattin and Rall (23) was used for the incubation of the slices. -4t the end of the experiment the incubation medium was removed and the slicrs were homogenized with 1.5 volumes of cold 5y0 trichloroacctic acid. Puri$cation of Cyclic GMP and Cyclic AMP from ExfractsFor purification of cyclic GMP, aliquots (0.5 to 1.5 ml) of the neutralized trichloroacet.ic acid extracts were charged onto columns, 0.5 x 2.0 cm, of AC l-X8 (formate form, 200 to 400 mesh, Rio-Rad). The columns were then washed with 6 ml of 0.5 N formic acid and the eluate, which contained cyclic AMP, was discarded. Cyclic GMP was then eluted from the columns with 3 ml of 4 N formic acid and the fractions were lyophilized. The dried materials were taken up in 0.2 ml of water and quantitatively spotted on thin layer chromatographic plates (SilicAT TLC7GF, Mallinckrodt). The plates were developed with the solvent system described by Goldberg et al. (3) consisting of isopropyl alcohol-H029% NH40H (7 :2 : 1, v/v). Cyclic GMP was eluted with two l-ml aliquots of absolute ethanol from the area on the plates corresponding to the spot of authentic compound. (Goldberg et al. (3) used 50yo et.hanol for eluting cyclic GMP from the gel. We found that a considerable amount of the gel material was soluble in 50% ethanol and that this material interfered with the assay.) Over-all recovery of tissue cyclic GMP, determined from measurement of cyclic [G-3H]GMP, was between 71 and 80%. The data reported for each experiment have been corrected for recovery. Aliquots (usually 0.02 to 1.0 ml) of the alcoholic eluates of cyclic GMP were dried at 65”, in vacua, in the small test tubes (1.3 x 10.0 cm) in which the cyclic nueleotide was to be assayed. Purification of cyclic AMP from aliquots (usually 0.2 ml) of the neutralized extracts was carried out by the procedure described earlier (16). Assay for Cyyclic GllfP and Cyclic AlUP-Tissue levels of cyclic GA11 were assayed with cyclic GMP-dependent protein kinare purified from either lobster tail muscle or the fat body of Cecropia silkmoth pupae. Cyclic AMP levels were assayed with cyrlic AMP-dependent protein kinase from bovine heart. The incubation conditions and reagents used for the assay of cyclic GMP and cyclic AMP were the same as previously described for cyclic AMP analysis (16). The standard incubation mixture used for the assay of cyclic GMP and cyclic AMP coiltained, in a final volume of 0.2 ml, sodium acetate buffer, 1’11 6.0, 10 pmoles; mixed histone, 40 pg; [+P]ATP, 1.0 nmole, con taining about 1.8 x lo6 cpm; magnesium acetate, 2 pmoles; cyclic GMP or cyclic ALIP, ranging from 0.5 to 50 pmoles; and either cyclic GMP-dependent protein kinase from lobster muscle (40 pg) or silkmoth fat body (35 pg), or cyclic AMP-dependent protein kinase (3 pg) from bovine heart. Incubations were carried out at 30” for 5 min in a shaking water bath. The react.ion was terminated by addition of 4 ml of 5% trichloroacetic acid containing 0.25% sodium tungstate, pH 2.0; 0.2 ml of 0.63% bovine serum albumin was added as a carrier protein. After standing at 0” for 5 min, the mixture was centrifuged, and the supernatant solut,ion was removed by aspiration. The precipitate was dissolved in 0.1 ml of 1 N NaOH, and 2 ml of 50/, tri-
chloroacetic acid-0.257, sodium tungstate solution, pH 2.0, were added. The protein was reprecipitated from the solution by acidifying it with 0.1 ml of 1.2 x HzS04. The procedure of centrifugation, removing the supernatant, dissolving the protein in alkali, and then reprecipitating the protein was repeat.ed once more. The protein was finally collected by centrifugation and dissolved in 0.1 ml of 1 N NaOH, and the radioactivity was The amount of cyclic measured in a liquid scintillation counter. GMP or of cyclic AMP present in the samples was determined from standard curves with known quantities of pure cyclic GMP or cyclic AMP. Protein was determined by the method of Lowry et al. (24).
Vol. 247, p\‘o. 1
linear up to 20 pmoles of cyclic GMP. As low as 0.5 pmole of cyclic GMP could be measured accurately in the usual incubation volume of 0.2 ml. The linearity of the lobster muscle protein kinase activity extended to about 40 pmoles of cyclic GMP when the heat-stable protein factor was included in the incubation mixture. However, the sensitivity of the cyclic GMP assay was slightly lower in the presence than in the absence of the inhibitory factor. When a cyclic GMP-dependent protein kinase purified from the fat body of Cecropia silkmoth pupae was used as the enzyme, the results were similar to those obtained with the lobster enzyme. I
GMP in presence and absence internal cyclic GMP standard and phosphodiesterase Preparation of tissue and urine extracts, and isolation of cyclic
Standard Curves for Xeasurement GJIP-dependent Protein Kinase-Curves cyclic GMP-dependent protein kinase the amount of cyclic GJ’IP are shown of the heat-stable protein factor, the
of Cyclic GMP with Cyclic relating the activity of from lobster tail muscle to in Fig. 1. In the absence aetivit.y of the enzyme was
Procedure.” Aliquots from the ethanol eluate of the thin layer chromatographic plates were dried in a vacuum oven, with or without the prior addition of 2 pmoles of authentic cyclic GMP as internal standard. The lyophilized samples were treated, where indicated, with 96 rg of bovine heart phosphodiesterase for 30 min at 30” in a final acetate buffer (pH
of 0.1 ml
6.0), 10 mM magnesium acetate, and 30 mM The incubation mixture was then placed in a boiling
The amount of cyclic GMP was assayed by water bath for 3 min. the standard method, with 40 fig of cyclic GMP-dependent protein kinase from lobster tail muscle. One milliliter of eluate from the chromatographic plates was equivalent to 634.8 mg of protein of rat brain, 33.8 mg of protein of rat heart, 24.1 mg of protein of rat lung, 27.3 mg of protein of silkmoth fat body, 41.0 mg of protein of lobster tail muscle, and 0.10 ml of human urine.
Fro. 1. Standard curve for the measurement of cyclic GMP with a partially purified preparation of cyclic GMP-dependent protein kinase from lobster tail muscle. Assay conditions were as described under “Experimental Procedure.” The enzyme (40 pg) used was from the DEAE-cellulose step of purification (9). Activity is shown in the absence (0-O) and in the presence (O-O) of 40 pg of the heat-stable protein factor from lobster tail muscle. One picomole of a2P incorporated represented about -.-1WJ to WOO cpm.
i 8:::: D Boiled for 3 min.
0.01 0.01 0.05 0.05 0.25 0.25 0.50 0.50 0.02 0.02 0.05 0.05 0.20 0.20 0.50 0.50
1.5 3.3 7.3
2.5 4.4 4.8 6.8 3.0 5.2 7.4
2.1 4.2 5.3 7.6 3.1
11.1 4.7 6.5 13.8 15.2