- Email: [email protected]
An enzymatic method for the measurement of GTP and GDP in biological samples
ANALYTICAL
BIOCHEMISTRY
95,512-519
An Enzymatic
(1979)
Method for the Measurement in Biological Samples’
of GTP and GDP
F. A. M. DE AZEREDO,* G. K. FEUSSNER,W. D. LUST, AND J. V. PASSONNEAU Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda.
and Communicative Maryland 20014
Received December 11, 1978 A method is described for the analysis of GTP/GDP, or GDP alone, in a coupled enzymatic assay. Samples are pretreated with creatine kinase in the presence of phosphocreatine to remove the ADP present. Succinyl CoA synthetase is used to convert GTP to GDP in the presence of succinate and coenzyme A. Endogenous GDP and that formed from GTP are measured in a second step with pyruvate kinase and lactate dehydrogenase in the presence of excess NADH. The sensitivity of the assay is in the nanomole range. A further modification is described, where the final product NAD+ is cycled in yet another enzyme system, extending the sensitivity to the picomole range. Sample analyses are given for a variety of tissues using 7 mg of sample and for 1-pg freeze-dried sections from the cerebellum of mouse brain
sensitivities have been used for the measurement of these compounds; combined paper chromatography and enzymatic assay, 10eg mol (4), high-pressure liquid chromatography, lo-lo mol(5), and enzymic procedures, lo-* mol(6-8). Another method has been reported for the determination of lo-lo mol of the sum of GTP and GDP, using an enzymic cycling procedure (9,lO). A modification of that procedure was devised by Goldberg et al. (11) which increased the sensitivity to the detection of lo-l2 mol. In the present investigation following removal of ADP from samples with creatine kinase and phosphocreatine, GTP is converted to GDP in the presence of succinyl CoA synthetase. The only other nucleotide which is acted upon by the enzyme ’ The U. S. Government’s right to retain a non- is ITP, which is present in much lower exclusive royalty-free license in and to the copyright concentrations than GTP. Using high-prescovering this paper, for governmental purposes, is sure liquid chromatography in a system acknowledged. where 0.2 nmoYmg protein was the lower * Recipient of a postdoctoral fellowship from Conlimit of sensitivity, neither ITP nor IDP selho National de Desenvolvemento Cientifico e Technologico, Rio de Janeiro, Brazil. was detectable in brain extracts (Dr. L.
Guanine nucleotides are involved in a number of important biological pathways; for example GTP plays a key role in protein synthesis, is the substrate for cyclic GMP formation, and is a potential energy source. Special interest has been focused on guanine nucleotides in nervous tissue because of the high concentrations of cyclic GMP in the cerebellum, 5 pmol/mg protein (l), and retina outer segments, SO-150 pmoYmg dry wt (2,3). The measurement of guanine nucleotides by enzymic methods has been made difficult because they are present in one-third the amount of adenine nucleotides, and enzymes which are guanine specific have not been readily available. Several different methods of varying
0003-2697/79/080512-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
512
ENZYMATIC
ANALYSIS
GTP
GDP
7.84 t 0.78
(12)
2.20 k 0.26 (11)
+ 0.37 (11)
2.20 + 0.25 (11)
Tissue Preparation
2 0.52
1.37 + 0.17 (13)
1
CONCENTRATIONS OF GTP AND GDP IN TISSUE SAMPLESO Concentration (nmohmg protein)
cortex
Muscle
6.18
Heart
6.59
(12) Liver
6.63 ? 0.44
2.51 2 0.18
(12) Retina
7.60 2 0.27 (10)
(12) 1.56 2 0.24 (10)
0 The tissues were prepared and analyzed as described in the text. All tissues were from mice, except for the retinas from 3-day chicks. Values are for the mean + SEM for the number of animals given in parentheses.
Rodichok, personal communication). Since the levels of GTP reported here are nearly 40 times greater (Table 1) the contribution of ITP would not exceed 2.5% and could be even less. The GDP is measured by the disappearance of NADH in a coupled pyruvate kinase-lactate dehydrogenase system. In the microprocedure described, the NAD+ formed in that reaction is used in an enzymatic cycle to generate a 3000fold accumulation of product. A sample procedure is included for lo-l2 mol of GDP or GTP, with analyses of 0.3- to 1.5-pg samples of cerebellar layers. MATERIALS
513
GTP/GDP
EC 2.7.3.2), phosphocreatine, and coenzyme A were obtained from BoehringerMannheim Biochemicals (Indianapolis, Ind.). Beef heart lactate dehydrogenase (Llactate:NAD+ oxidoreductase EC 1.1.1.27) was purchased from Worthington Biochemicals (Freehold, N. J.). All other coenzymes and nucleotides were purchased from Sigma Chemical Company (St. Louis, MO.).
TABLE
Cerebral
OF
AND METHODS
Succinyl coenzyme A synthetase from pig heart (succinate:CoA ligase EC 6.2.1.4), pyruvate kinase (ATPpyruvate 2-O-phosphotransferase EC 2.7.1.40), creatine kinase (ATP:creatine N-phosphotransferase
General purpose NIH mice fed ad fibitum , and weighing 24-29 g, were frozen in liquid nitrogen. Samples of cerebral cortex, skeletal muscle, heart, and liver were dissected in a cryostat at -30°C. The tissues were extracted by a minor modification of the methanol-HCl method described by Lowry and Passonneau (12). To approximately 50 mg of tissue was added 100 ~1 of 0.1 N HCl in methanol and the mixture was homogenized at -20 to -30°C. After penetration of the solvent was complete, 1 ml of 0.3 N HC104 containing 1 mM EGTA” was added. Cold-adapted or hibernating hamsters were frozen and extracted in an identical manner (13). Retinas from 3-day-old chicks (Truslow Farms, Chestertown, Md.) were prepared and incubated as described previously (14). The medium containing the retinas was aerated with a 95% O,-5% CO, mixture for 30 min, after which the retinas (approximately 30 mg of tissue) were homogenized in 1 ml of 0.3 N HClO, containing 1 mM EGTA. In both cases, the homogenates were centrifuged and the supernatant acid was removed and neutralized with 100 ~1 of 3 N KHC03 (final pH approximately 7). The pellet was dissolved in 1 N NaOH at 60°C for 10 min and analyzed forprotein content (15). ’ Abbreviation used: EGTA, ethylene aminoethyl ether) N. N’-tetraacetic acid.
glycol
bis(P-
514
DE AZEREDO
Analytic
ET AL.
Principles
Step 1.
succinyl + GTP + CoA p CoA synthetase
Succinate
succinyl COA + Pi + GDP
Step 2.
GDP + phosphoenol
Pyruvate
+ NADH
pyruvate
+ H+
pyruvate ,GTP kinase
lactate dehydrogenase
The reaction is carried out in two steps in order to achieve stoichiometry of the oxidation of NADH with the GDP present. If the two steps are combined, the GTP produced in the pyruvate kinase step reacts again in the succinyl CoA synthetase step, and a cycle is produced which amplifies the formation of product (9- 11). In the present analytic scheme, the NAD+ formed is instead added to a cycling system which can amplify the product over a range of 3- to 30,000-fold in an hour. Kinetic
+ pyruvate
Considerations
Removal of ADP from samples. Since the pyruvate kinase reacts with other dinucleotides present in tissues, a method was devised to remove the ADP, which is present in tissues in substantially higher amounts than GDP. In addition, succinyl CoA synthetase was found to be contaminated with adenylate kinase which reacted with the ADP in the tissues. Consequently, when succinyl CoA synthetase was present, ADP was converted to ATP and AMP. The amount of ADP in the measurement of GTP plus dinucleotides was lower than if total dinucleotides alone were measured. Since these two numbers are subtracted, the net effect is that the values for GTP were erroneously low. To eliminate these two problems, creatine kinase was used with phosphocreatine to convert the ADP present to ATP. The K,
>
NAD+
+ lactate
for ADP of creatine kinase, under the analytical conditions described below, was found to be approximately 0.15 mM, with a V max of 40 pmol/mg/min. The K, for GDP was about 4 mM and the V,,,,, 10 lmol/mgl min. The apparent first-order rate constants then are 0.27 min-’ and 0.0025 min-’ for ADP and GDP, respectively, an approximate lOO-fold difference. Therefore, it was possible to choose an amount of creatine kinase that would convert the ADP essentially 100% and not remove the GDP. The half-time for conversion of the ADP with 1 CLg/ml creatine kinase was calculated to be approximately 3 min, while that for GDP would be 300 min. After a 20-min incubation the reaction with ADP is virtually complete (six half-times, 98.5% of completion) while less than 5% of the GDP would react. In practice, standard ADP added to the incubation mixture was undetectable, and there was no measurable loss of GDP. Succinyl coenzyme A synthetase
reaction.
The succinyl CoA synthetase enzyme from pig heart reacts with guanine and inosine nucleotides. However, since the amount of inosine nucleotides in tissue is no more than 2.5% of the guanine nucleotides the assay measures primarily guanine nucleotides. Because the succinyl CoA synthetase reaction is reversible, conditions were chosen to insure that the conversion of GTP to GDP was essentially complete. The
ENZYMATIC
equilibrium for the reaction termined to be (16): K =
ANALYSIS
has been de-
(succinate)(CoA)(GTP) (succinyl CoA)(GDP)(PJ
cinate, would be calculated as follows (12): V max =
3.7(GTP)i3 (succinate)( CoA) ’
where (GTP), is the final concentration of GTP. For the reaction to be 99% complete, (GTP)r = 0.01 (GTP),. Then the equation becomes (GTP): 370 = (succinate) (CoA). With GTP concentrations of 10, 25, or 100 PM in the reaction, and for the reaction to proceed to 99%, the product of succinate and CoA must be equal or greater than 0.04, 0.23, and 3.7 mM*, respectively. In practice, 10 mM succinate was chosen since there seemed to be no disadvantage in using this high concentration and it allowed coenzyme A levels to be low. For example, with 10 mM succinate and 0.15 mM coenzyme A the product would be 1.5 mM* and one could measure up to 65 PM GTP. The coenzyme levels chosen were 2- to 5-fold greater than the GTP concentration, which was sufficient to complete the reaction and did not contribute greatly to the fluorescent blank. For example, with 20 PM GTP as substrate and 5 Z.&ml succinyl CoA synthetase, the GDP formed in 20 min was the same with 40, 100,400, or 800 PM CoA. The contribution to the fluorescent blank was negligible except at the 800 PM CoA level. The amount of succinyl CoA synthetase needed for a half-time of 3 min for 50 PM GTP, based on a K, for GTP of 10 PM (17), protein, and in a Vmax of 10 pmoYmin/mg the presence of saturating levels of suc-
4 x K, + 50 to.98
= 3.7.
For the reaction to proceed to completion, the final concentrations of succinyl CoA, GDP, and Pi will approximate those of the initial concentrations of (GTP) = (GTP)i. Making these substitutions the equation becomes (GTP)r =
515
OF GTP/GDP
= 4 x 10 + 50 20 min = 4.5 pM/min. In practice, because of the complex back reaction, it was necessary to use a concentration of 5 Z&ml of the enzyme, equivalent to 50 Z..&min. The K, of pyruvate kinase for GDP was determined to be 280 pM in the reagent chose, with a V,,, of 28 pmoYmin/mg protein. The amount of pyruvate kinase needed to complete the reaction in 5 min was calculated to be on the order of 10 Z.&ml; as a margin of safety, 20 Z.&ml was used routinely . Procedure
for Tissue Extracts
Reagent I. The reagent is double strength since in this procedure it was diluted by an equal volume of tissue extract: imidazole, 200 mM, pH 7.0; MgC12, 4 mM; succinate, 20 mM; CoA, 0.30 mM; dithiothreitol, 0.5 mM; phosphocreatine, 1 mM; hydrazine, 10 mM; BSA, 0.04%; succinyl CoA synthetase (when added), 10 Z.&ml; creatine kinase (0.025 U, based on supplier’s specifications, or 0.04 U based on analyses under our conditions), 1 &ml. Reagent ZZ. Imidazole-HCl, 50 mM, pH 7.0; MgC12, 2 mrvr; KCl, 75 mM; NADH. 3 PM; phosphoenol pyruvate, 40 PM; hydrazine, 10 mM; lactate dehydrogenase (beef heart), 2 @g/ml; pyruvate kinase, 20 Z&ml. The hydrazine is added to trap tissue pyruvate as well as any contaminant in the reagents. In the direct analyses, readings can be taken after the lactate dehydrogenase step, eliminating the need for a trapping agent. In the method for lo-‘* mol, separate readings cannot be made, and the
516
DE AZEREDO
use of hydrazine eliminates a troublesome blank. In either case hydrazine is recommended for greater sensitivity. The approximate time for hydrazone formation is 20 min; sufficient lactate dehydrogenase is used to measure the newly formed pyruvate before the hydrazone can form (18). Step I. In 10 x 75mm tubes, to 25 ~1 of Reagent I with and without succinyl CoA synthetase were added 25 ~1 of blank, 15-30 PM GDP, 100 PM ADP, or samples. The tubes were incubated at room temperature for 20 min and heated for 3 min at 100°C. Step 2. To the cooled tubes was added 1 ml of Reagent II, a reading was taken so that NADH read nearly full scale on the fluorometer, the pyruvate kinase was added, and a second reading was taken after 20 min. The tubes without succinyl CoA synthetase are a measure of GDP as well as of dinucleotides other than ADP. While the exact concentrations are not known, a value of 1.27 nmol/mg protein for GDP using chromatography has been reported in rat brain (19). Goldberg et al. (20) reported dinucleotides other than ADP to be 1.93 nmoYmg protein; the contribution of other dinucleotides may then be on the order of 0.7 nmol/mg protein. The tubes with succinyl CoA synthetase measure both the triand dinucleotides of guanosine and inosine. The difference between the two sets is, of course, a measure of the trinucleotides. Calculations were based on GDP standardized spectrophotometrically according to the ADP method of Lowry and Passonneau (12), except that the pyruvate kinase was increased to 20 pg/ml. GTP was then standardized with the above procedure, and calculations were based on the GDP. The GTP reaction was found to be linear over at least the range of lo-50 PM under the conditions described. Because the GTP concentrations determined by this method were higher than
ET AL.
many reported in the literature, other methods of standardization were also tried. Since GTP was known to contain GDP by enzymatic analysis, the material was purified by column chromatography for the determination by absorption spectrum (21). Good separation was achieved on the column, but after lyophilization, the resulting material was found by enzymatic analysis to contain GDP and GMP, apparently formed during the drying process. An analysis of the trinucleotide was then made by using phosphoglycerate kinase and glyceraldehyde-P dehydrogenase (12). GTP standards analyzed in this manner agreed within 3% with the succinyl CoA synthetase reaction. While phosphoglycerate kinase will react with ATP and ITP, the amount present must be minor to achieve the agreement between the two determinations. Consequently, the values for tissue levels based on the GTP standardized in this fashion must be within a few percent of actual values. Measurement GDPIGTP
of 1-3
X IO-l2 mol of
The GDPlGTP assay was combined with an enzymic cycling procedure (22) to form a product 3000-fold greater than the initial material. Reagent 1. The conversion of GTP to GDP. Imidazole-HCl buffer, 100 mM, pH
7.0; MgC12, 2 mM; succinate, 10 mM; coenzyme A, 0.04 mM; hydrazine, 10 mM; bovine serum albumin, 0.02%; P-creatine, 1.5 mM; creatine kinase, 0.5 &ml; succinyl CoA synthetase, 5 pg/ml when present. Reagent
2. The measurement
of GDP.
Imidazole-HCl buffer, 100 mM, pH 7.0; MgC12, 2 mM; KCl, 75 mM; P-enol pyruvate, 0.01 mM; hydrazine, 100 mM; ascorbate, 2 mM; NADH, 2 PM; beef heart lactate dehydrogenase, 2 lug/ml; pyruvate kinase, 20 &ml. Reagent 3. The amp@cation in reagent 2. Tris-HCl ~formed _
of the NADi
buffer. , 1DH
ENZYMATIC
ANALYSIS
Step 4. Destruction of unreacted NADH. To all samples was added 5 ~1 0.2 N HCI. Step 5. Cycling step. A ~-PI portion was taken from samples and added to 100 ~1 of reagent 3, in 10 x 75-mm test tubes. To additional tubes containing reagent 3 were added appropriate concentrations of NAD+ standards (5 ~1 of 5 x lo-’ M). The tubes were incubated 60 min at 38”C, then heated 3 min at 100°C to destroy the enzymes. Step 6. Indicator step. To all tubes was added 1 ml of reagent 4. To additional tubes of reagent 4 were added malate standards at the appropriate concentrations (3 PM). The tubes were incubated at room temperature for 10 min and read at a fluorometer setting that provided full-scale deflection for standard and samples.
8.0; ethanol, 300 mM; bovine serum albumin, 0.02%; mercaptoethanol, 2.5 mM. Just before use the following are added: oxalacetate, 2 mM; yeast alcohol dehydrogenase, 45 pg/ml; malate dehydrogenase, 2.5 pg/ml. Reagent 4. Measurement of malate formed in reagent 3. 2-Amino-Zmethyllpropanol-HCI buffer, 50 mM pH 9.9; NAD+, 0.2 mM; glutamate, 10 mM; glutamate-oxalacetate transaminase, 5 kg/ml; malate dehydrogenase, 2.0 puglml. The following reactions were carried out under oil (12). Step 1. Inactivation of tissue enzymes. 0.1 N NaOH, 0.05 ~1, for samples and blanks, or 0.1 N NaOH containing 20-50 PM GDP and/or GTP. The samples were heated 20 min at 80°C. Step 2. GTP reaction with succinyl CoA synthetase. To all samples was added 2 pi of reagent 1 without succinyl CoA synthetase (rack I). From these samples l-p.1 portions were removed to a second rack (rack II), and 1 ~1 of reagent 1 containing succinyl CoA synthetase was added. The samples were incubated 20 min at room temperature, followed by 20 min at 80°C to denature the enzymes. Step 3. Measurement of native GDP and reacted GTP. To all samples 4 ~1 of reagent 2 was added and the samples were incubated 10 min at room temperature.
RESULTS Tissues were prepared from mice or chicks as described under Materials and Methods and analyzed. The results are given in Table I. In the course of investigating the metabolism in the brain of hibernating hamsters, it was considered to be of interest to measure the guanine nucleotides in these animals. We have evidence that the brain of the hibernators, which is approximately 4°C freezes faster than that of the coldadapted animals, which have a body tem-
TABLE GTP
AND
GDP
CONCENTRATIONS
IN THREE
517
OF GTP/GDP
REGIONS
2 OF HAMSTER
CENTRAL
NERVOUS
SYSTEM”
Concentration (nmoVmg protein) Cortex
Cold adapted Hibernator
Cerebellum
Spinal cord
GTP
GDP
GTP
GDP
GTP
GDP
6.46 2 0.19 5.90 k 0.32
1.33 ? 0.09 1.03 2 0.14
4.49 2 0.43 5.12 t 0.38
1.65 2 0.22 1.57 + 0.08
6.51 k 0.54 5.71 2 0.53
1.05 2 0.17 0.70 2 0.19
‘I The tissues were prepared from hamsters which were either cold adapted or hibernating and analyzed as described in the text. Values are means + SEM for six animals, except for hibernator cortex, where there were five animals.
518
DE AZEREDO TABLE
3
GTP AND GDP CONCENTRATION IN LAYERS OF THE CEREBELLUM FROM MOUSE BRAIN” Concentration (nmol/mg dry wt) Layer
GTP
GDP
Molecular (4) Granular (3) White (5)
2.06 2 0.10 1.72 ? 0.40 1.21 2 0.15
1.48 2 0.20 1.53 f 0.36 0.91 2 0.14
a The tissues were prepared and analyzed as described in the text. Values are the means r SEM for the number of samples given in parentheses.
perature of 34°C (13). The concentrations reported in Table 2 show no significant differences between the two sets of hamsters. Samples of the molecular, granular, and white layers of cerebellum were dissected, weighed, and analyzed by oil-well techniques (12) using the above procedures for l-3 x lo-l2 mol. The data are given in Table 3. DISCUSSION A method has been described utilizing succinyl CoA synthetase for the measurement of GTP in biological material. The reaction is coupled to pyruvate kinase to measure the GDP formed in the first reaction. The prior treatment of the tissues with creatine kinase and phosphocreatine removes the ADP which would otherwise react in the pyruvate kinase system, as well as that formed by the myokinase contamination of succinyl CoA synthetase. Samples incubated with and without succinyl CoA synthetase provide a measure of GTP/GDP or GDP alone (plus whatever CDP and UDP are present). In general, the concentrations reported here are substantially higher than those reported using different enzymic determination (67) and chromatographic techniques (19). These
ET AL.
results may be in part due to the sensitivity of the fluorometric analysis. It is possible to measure accurately as little as 0.1 nmol of substance which can be used to oxidize or reduce pyridine nucleotides. In the direct fluorometric analysis, 7.5 mg of tissue was used, and the amount of GTP or GDP measured was on the order of 1 to 5 nmol. A comment may be in order on the higher proportions of GDP (Table 3) found in cerebellar layers than in the mouse cerebral cortex (Table 1) or hamster cerebellum (Table 2). There may be regional differences (cerebral cortex vs cerebellum) and species differences (mouse vs hamster), and finally the concentration in the cortical layers of the cerebellum may not reflect the average concentration of the whole region. The coupling of the assay to a cycling system further extends the sensitivity of the system. In the present demonstration (Table 3), l-2 pg of tissue was analyzed, so that 1 pmol of GTP/GDP was measured. REFERENCES 1. Lust, W. D., Kupferberg, H. J., Yonekawa, W. D., Penry, J. K., Passonneau, J. V., and Wheaton, A. B. (1978) Mol. Pharmacol. 14, 347-356. 2. Orr, H. J., Lowry, 0. H., Cohen, A. J., and Ferrendelli, J. A. (1976) Proc. Nat. Acad. Sci. USA 73, 4442-4445. 3. de Azeredo, F. A. M., Lust, W. D., and Passonneau, J. V. (1978) Biochem. Biophys. Res. Commun. 85, 293-300. 4. Keppler, D. 0. R., Pausch, J., and Decker, K. (1974) J. Biot. Chem. 249, 21 t-216. 5. Jackson, R. C., Boritlki, T. J., Morris, H. P., and Weber, G. (1976) Life Sci. 19, 1531-1536. 6. Keppler, D. 0. R., and Kaiser, W. (1978) Anal. Biochem. 86, 147-153. 7. Gruber, W., Mollering, H., and Bergmeyer, H. V. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 4, p. 2078, Academic Press, New York. 8. Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 4, p. 2162, Academic Press, New York. 9. Cha, S., and Cha, C. M. (1965) Mol. Pharmacof. 1, 178-189.
ENZYMATIC
ANALYSIS
10. Cha, S.. and Cha, C. M. (1970) Anol. Biochem. 33, 174-192. 11. Goldberg, N. D., Dietz, S. B., and O’Toole, A. G. (1969) J. Biol. Chem. 244, 4458-4466. 12. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York. 13. Lust, W. D., Goldman, S., Wheaton, A., Feussner, G., and Passonneau, J. (1978) Trans. Amer. Sot. Neurochem. 9, Abstract 140. 14. de Azeredo, F. A. M., and Martins-Ferreira. H. (1979) Neurochem. Res. 4, 83-91. 15. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275.
OF GTPiGDP
519
16. Kaufman, S., and Alivisatos, S. Cl. A. ( 1955) J. Biol. Chem. 216, 141-152. 17. Cha, S., Parks, R. E., Jr. (1964) J. Bid. Chem. 239, 1%1- 1967. 18. Lowry, 0. H., Passonneau, J. V., Hasselberg, F. X., and Schulz, D. W. ( 1964) J. Bid. Chem. 239, 18-30. 19. Kleihues, P., Kobayashi, K., and Hossman, K.-A. (1974) J. Neurochem. 23, 417-425. 20. Goldberg, N. D.. Passonneau, J. V., and Lowry, 0. H. (1966) J. Bid. Chem. 241, 3997-4003. 21. Krishan, N., and Krishna, G. (1976) Anal. Biothem. 70, 18-31. 22. Kato. T., Berger, S. J., Carter, J. A., and Lowry, 0. H. (1973) And. Biochem.. 53, 86-97.
BIOCHEMISTRY
95,512-519
An Enzymatic
(1979)
Method for the Measurement in Biological Samples’
of GTP and GDP
F. A. M. DE AZEREDO,* G. K. FEUSSNER,W. D. LUST, AND J. V. PASSONNEAU Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda.
and Communicative Maryland 20014
Received December 11, 1978 A method is described for the analysis of GTP/GDP, or GDP alone, in a coupled enzymatic assay. Samples are pretreated with creatine kinase in the presence of phosphocreatine to remove the ADP present. Succinyl CoA synthetase is used to convert GTP to GDP in the presence of succinate and coenzyme A. Endogenous GDP and that formed from GTP are measured in a second step with pyruvate kinase and lactate dehydrogenase in the presence of excess NADH. The sensitivity of the assay is in the nanomole range. A further modification is described, where the final product NAD+ is cycled in yet another enzyme system, extending the sensitivity to the picomole range. Sample analyses are given for a variety of tissues using 7 mg of sample and for 1-pg freeze-dried sections from the cerebellum of mouse brain
sensitivities have been used for the measurement of these compounds; combined paper chromatography and enzymatic assay, 10eg mol (4), high-pressure liquid chromatography, lo-lo mol(5), and enzymic procedures, lo-* mol(6-8). Another method has been reported for the determination of lo-lo mol of the sum of GTP and GDP, using an enzymic cycling procedure (9,lO). A modification of that procedure was devised by Goldberg et al. (11) which increased the sensitivity to the detection of lo-l2 mol. In the present investigation following removal of ADP from samples with creatine kinase and phosphocreatine, GTP is converted to GDP in the presence of succinyl CoA synthetase. The only other nucleotide which is acted upon by the enzyme ’ The U. S. Government’s right to retain a non- is ITP, which is present in much lower exclusive royalty-free license in and to the copyright concentrations than GTP. Using high-prescovering this paper, for governmental purposes, is sure liquid chromatography in a system acknowledged. where 0.2 nmoYmg protein was the lower * Recipient of a postdoctoral fellowship from Conlimit of sensitivity, neither ITP nor IDP selho National de Desenvolvemento Cientifico e Technologico, Rio de Janeiro, Brazil. was detectable in brain extracts (Dr. L.
Guanine nucleotides are involved in a number of important biological pathways; for example GTP plays a key role in protein synthesis, is the substrate for cyclic GMP formation, and is a potential energy source. Special interest has been focused on guanine nucleotides in nervous tissue because of the high concentrations of cyclic GMP in the cerebellum, 5 pmol/mg protein (l), and retina outer segments, SO-150 pmoYmg dry wt (2,3). The measurement of guanine nucleotides by enzymic methods has been made difficult because they are present in one-third the amount of adenine nucleotides, and enzymes which are guanine specific have not been readily available. Several different methods of varying
0003-2697/79/080512-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
512
ENZYMATIC
ANALYSIS
GTP
GDP
7.84 t 0.78
(12)
2.20 k 0.26 (11)
+ 0.37 (11)
2.20 + 0.25 (11)
Tissue Preparation
2 0.52
1.37 + 0.17 (13)
1
CONCENTRATIONS OF GTP AND GDP IN TISSUE SAMPLESO Concentration (nmohmg protein)
cortex
Muscle
6.18
Heart
6.59
(12) Liver
6.63 ? 0.44
2.51 2 0.18
(12) Retina
7.60 2 0.27 (10)
(12) 1.56 2 0.24 (10)
0 The tissues were prepared and analyzed as described in the text. All tissues were from mice, except for the retinas from 3-day chicks. Values are for the mean + SEM for the number of animals given in parentheses.
Rodichok, personal communication). Since the levels of GTP reported here are nearly 40 times greater (Table 1) the contribution of ITP would not exceed 2.5% and could be even less. The GDP is measured by the disappearance of NADH in a coupled pyruvate kinase-lactate dehydrogenase system. In the microprocedure described, the NAD+ formed in that reaction is used in an enzymatic cycle to generate a 3000fold accumulation of product. A sample procedure is included for lo-l2 mol of GDP or GTP, with analyses of 0.3- to 1.5-pg samples of cerebellar layers. MATERIALS
513
GTP/GDP
EC 2.7.3.2), phosphocreatine, and coenzyme A were obtained from BoehringerMannheim Biochemicals (Indianapolis, Ind.). Beef heart lactate dehydrogenase (Llactate:NAD+ oxidoreductase EC 1.1.1.27) was purchased from Worthington Biochemicals (Freehold, N. J.). All other coenzymes and nucleotides were purchased from Sigma Chemical Company (St. Louis, MO.).
TABLE
Cerebral
OF
AND METHODS
Succinyl coenzyme A synthetase from pig heart (succinate:CoA ligase EC 6.2.1.4), pyruvate kinase (ATPpyruvate 2-O-phosphotransferase EC 2.7.1.40), creatine kinase (ATP:creatine N-phosphotransferase
General purpose NIH mice fed ad fibitum , and weighing 24-29 g, were frozen in liquid nitrogen. Samples of cerebral cortex, skeletal muscle, heart, and liver were dissected in a cryostat at -30°C. The tissues were extracted by a minor modification of the methanol-HCl method described by Lowry and Passonneau (12). To approximately 50 mg of tissue was added 100 ~1 of 0.1 N HCl in methanol and the mixture was homogenized at -20 to -30°C. After penetration of the solvent was complete, 1 ml of 0.3 N HC104 containing 1 mM EGTA” was added. Cold-adapted or hibernating hamsters were frozen and extracted in an identical manner (13). Retinas from 3-day-old chicks (Truslow Farms, Chestertown, Md.) were prepared and incubated as described previously (14). The medium containing the retinas was aerated with a 95% O,-5% CO, mixture for 30 min, after which the retinas (approximately 30 mg of tissue) were homogenized in 1 ml of 0.3 N HClO, containing 1 mM EGTA. In both cases, the homogenates were centrifuged and the supernatant acid was removed and neutralized with 100 ~1 of 3 N KHC03 (final pH approximately 7). The pellet was dissolved in 1 N NaOH at 60°C for 10 min and analyzed forprotein content (15). ’ Abbreviation used: EGTA, ethylene aminoethyl ether) N. N’-tetraacetic acid.
glycol
bis(P-
514
DE AZEREDO
Analytic
ET AL.
Principles
Step 1.
succinyl + GTP + CoA p CoA synthetase
Succinate
succinyl COA + Pi + GDP
Step 2.
GDP + phosphoenol
Pyruvate
+ NADH
pyruvate
+ H+
pyruvate ,GTP kinase
lactate dehydrogenase
The reaction is carried out in two steps in order to achieve stoichiometry of the oxidation of NADH with the GDP present. If the two steps are combined, the GTP produced in the pyruvate kinase step reacts again in the succinyl CoA synthetase step, and a cycle is produced which amplifies the formation of product (9- 11). In the present analytic scheme, the NAD+ formed is instead added to a cycling system which can amplify the product over a range of 3- to 30,000-fold in an hour. Kinetic
+ pyruvate
Considerations
Removal of ADP from samples. Since the pyruvate kinase reacts with other dinucleotides present in tissues, a method was devised to remove the ADP, which is present in tissues in substantially higher amounts than GDP. In addition, succinyl CoA synthetase was found to be contaminated with adenylate kinase which reacted with the ADP in the tissues. Consequently, when succinyl CoA synthetase was present, ADP was converted to ATP and AMP. The amount of ADP in the measurement of GTP plus dinucleotides was lower than if total dinucleotides alone were measured. Since these two numbers are subtracted, the net effect is that the values for GTP were erroneously low. To eliminate these two problems, creatine kinase was used with phosphocreatine to convert the ADP present to ATP. The K,
>
NAD+
+ lactate
for ADP of creatine kinase, under the analytical conditions described below, was found to be approximately 0.15 mM, with a V max of 40 pmol/mg/min. The K, for GDP was about 4 mM and the V,,,,, 10 lmol/mgl min. The apparent first-order rate constants then are 0.27 min-’ and 0.0025 min-’ for ADP and GDP, respectively, an approximate lOO-fold difference. Therefore, it was possible to choose an amount of creatine kinase that would convert the ADP essentially 100% and not remove the GDP. The half-time for conversion of the ADP with 1 CLg/ml creatine kinase was calculated to be approximately 3 min, while that for GDP would be 300 min. After a 20-min incubation the reaction with ADP is virtually complete (six half-times, 98.5% of completion) while less than 5% of the GDP would react. In practice, standard ADP added to the incubation mixture was undetectable, and there was no measurable loss of GDP. Succinyl coenzyme A synthetase
reaction.
The succinyl CoA synthetase enzyme from pig heart reacts with guanine and inosine nucleotides. However, since the amount of inosine nucleotides in tissue is no more than 2.5% of the guanine nucleotides the assay measures primarily guanine nucleotides. Because the succinyl CoA synthetase reaction is reversible, conditions were chosen to insure that the conversion of GTP to GDP was essentially complete. The
ENZYMATIC
equilibrium for the reaction termined to be (16): K =
ANALYSIS
has been de-
(succinate)(CoA)(GTP) (succinyl CoA)(GDP)(PJ
cinate, would be calculated as follows (12): V max =
3.7(GTP)i3 (succinate)( CoA) ’
where (GTP), is the final concentration of GTP. For the reaction to be 99% complete, (GTP)r = 0.01 (GTP),. Then the equation becomes (GTP): 370 = (succinate) (CoA). With GTP concentrations of 10, 25, or 100 PM in the reaction, and for the reaction to proceed to 99%, the product of succinate and CoA must be equal or greater than 0.04, 0.23, and 3.7 mM*, respectively. In practice, 10 mM succinate was chosen since there seemed to be no disadvantage in using this high concentration and it allowed coenzyme A levels to be low. For example, with 10 mM succinate and 0.15 mM coenzyme A the product would be 1.5 mM* and one could measure up to 65 PM GTP. The coenzyme levels chosen were 2- to 5-fold greater than the GTP concentration, which was sufficient to complete the reaction and did not contribute greatly to the fluorescent blank. For example, with 20 PM GTP as substrate and 5 Z.&ml succinyl CoA synthetase, the GDP formed in 20 min was the same with 40, 100,400, or 800 PM CoA. The contribution to the fluorescent blank was negligible except at the 800 PM CoA level. The amount of succinyl CoA synthetase needed for a half-time of 3 min for 50 PM GTP, based on a K, for GTP of 10 PM (17), protein, and in a Vmax of 10 pmoYmin/mg the presence of saturating levels of suc-
4 x K, + 50 to.98
= 3.7.
For the reaction to proceed to completion, the final concentrations of succinyl CoA, GDP, and Pi will approximate those of the initial concentrations of (GTP) = (GTP)i. Making these substitutions the equation becomes (GTP)r =
515
OF GTP/GDP
= 4 x 10 + 50 20 min = 4.5 pM/min. In practice, because of the complex back reaction, it was necessary to use a concentration of 5 Z&ml of the enzyme, equivalent to 50 Z..&min. The K, of pyruvate kinase for GDP was determined to be 280 pM in the reagent chose, with a V,,, of 28 pmoYmin/mg protein. The amount of pyruvate kinase needed to complete the reaction in 5 min was calculated to be on the order of 10 Z.&ml; as a margin of safety, 20 Z.&ml was used routinely . Procedure
for Tissue Extracts
Reagent I. The reagent is double strength since in this procedure it was diluted by an equal volume of tissue extract: imidazole, 200 mM, pH 7.0; MgC12, 4 mM; succinate, 20 mM; CoA, 0.30 mM; dithiothreitol, 0.5 mM; phosphocreatine, 1 mM; hydrazine, 10 mM; BSA, 0.04%; succinyl CoA synthetase (when added), 10 Z.&ml; creatine kinase (0.025 U, based on supplier’s specifications, or 0.04 U based on analyses under our conditions), 1 &ml. Reagent ZZ. Imidazole-HCl, 50 mM, pH 7.0; MgC12, 2 mrvr; KCl, 75 mM; NADH. 3 PM; phosphoenol pyruvate, 40 PM; hydrazine, 10 mM; lactate dehydrogenase (beef heart), 2 @g/ml; pyruvate kinase, 20 Z&ml. The hydrazine is added to trap tissue pyruvate as well as any contaminant in the reagents. In the direct analyses, readings can be taken after the lactate dehydrogenase step, eliminating the need for a trapping agent. In the method for lo-‘* mol, separate readings cannot be made, and the
516
DE AZEREDO
use of hydrazine eliminates a troublesome blank. In either case hydrazine is recommended for greater sensitivity. The approximate time for hydrazone formation is 20 min; sufficient lactate dehydrogenase is used to measure the newly formed pyruvate before the hydrazone can form (18). Step I. In 10 x 75mm tubes, to 25 ~1 of Reagent I with and without succinyl CoA synthetase were added 25 ~1 of blank, 15-30 PM GDP, 100 PM ADP, or samples. The tubes were incubated at room temperature for 20 min and heated for 3 min at 100°C. Step 2. To the cooled tubes was added 1 ml of Reagent II, a reading was taken so that NADH read nearly full scale on the fluorometer, the pyruvate kinase was added, and a second reading was taken after 20 min. The tubes without succinyl CoA synthetase are a measure of GDP as well as of dinucleotides other than ADP. While the exact concentrations are not known, a value of 1.27 nmol/mg protein for GDP using chromatography has been reported in rat brain (19). Goldberg et al. (20) reported dinucleotides other than ADP to be 1.93 nmoYmg protein; the contribution of other dinucleotides may then be on the order of 0.7 nmol/mg protein. The tubes with succinyl CoA synthetase measure both the triand dinucleotides of guanosine and inosine. The difference between the two sets is, of course, a measure of the trinucleotides. Calculations were based on GDP standardized spectrophotometrically according to the ADP method of Lowry and Passonneau (12), except that the pyruvate kinase was increased to 20 pg/ml. GTP was then standardized with the above procedure, and calculations were based on the GDP. The GTP reaction was found to be linear over at least the range of lo-50 PM under the conditions described. Because the GTP concentrations determined by this method were higher than
ET AL.
many reported in the literature, other methods of standardization were also tried. Since GTP was known to contain GDP by enzymatic analysis, the material was purified by column chromatography for the determination by absorption spectrum (21). Good separation was achieved on the column, but after lyophilization, the resulting material was found by enzymatic analysis to contain GDP and GMP, apparently formed during the drying process. An analysis of the trinucleotide was then made by using phosphoglycerate kinase and glyceraldehyde-P dehydrogenase (12). GTP standards analyzed in this manner agreed within 3% with the succinyl CoA synthetase reaction. While phosphoglycerate kinase will react with ATP and ITP, the amount present must be minor to achieve the agreement between the two determinations. Consequently, the values for tissue levels based on the GTP standardized in this fashion must be within a few percent of actual values. Measurement GDPIGTP
of 1-3
X IO-l2 mol of
The GDPlGTP assay was combined with an enzymic cycling procedure (22) to form a product 3000-fold greater than the initial material. Reagent 1. The conversion of GTP to GDP. Imidazole-HCl buffer, 100 mM, pH
7.0; MgC12, 2 mM; succinate, 10 mM; coenzyme A, 0.04 mM; hydrazine, 10 mM; bovine serum albumin, 0.02%; P-creatine, 1.5 mM; creatine kinase, 0.5 &ml; succinyl CoA synthetase, 5 pg/ml when present. Reagent
2. The measurement
of GDP.
Imidazole-HCl buffer, 100 mM, pH 7.0; MgC12, 2 mM; KCl, 75 mM; P-enol pyruvate, 0.01 mM; hydrazine, 100 mM; ascorbate, 2 mM; NADH, 2 PM; beef heart lactate dehydrogenase, 2 lug/ml; pyruvate kinase, 20 &ml. Reagent 3. The amp@cation in reagent 2. Tris-HCl ~formed _
of the NADi
buffer. , 1DH
ENZYMATIC
ANALYSIS
Step 4. Destruction of unreacted NADH. To all samples was added 5 ~1 0.2 N HCI. Step 5. Cycling step. A ~-PI portion was taken from samples and added to 100 ~1 of reagent 3, in 10 x 75-mm test tubes. To additional tubes containing reagent 3 were added appropriate concentrations of NAD+ standards (5 ~1 of 5 x lo-’ M). The tubes were incubated 60 min at 38”C, then heated 3 min at 100°C to destroy the enzymes. Step 6. Indicator step. To all tubes was added 1 ml of reagent 4. To additional tubes of reagent 4 were added malate standards at the appropriate concentrations (3 PM). The tubes were incubated at room temperature for 10 min and read at a fluorometer setting that provided full-scale deflection for standard and samples.
8.0; ethanol, 300 mM; bovine serum albumin, 0.02%; mercaptoethanol, 2.5 mM. Just before use the following are added: oxalacetate, 2 mM; yeast alcohol dehydrogenase, 45 pg/ml; malate dehydrogenase, 2.5 pg/ml. Reagent 4. Measurement of malate formed in reagent 3. 2-Amino-Zmethyllpropanol-HCI buffer, 50 mM pH 9.9; NAD+, 0.2 mM; glutamate, 10 mM; glutamate-oxalacetate transaminase, 5 kg/ml; malate dehydrogenase, 2.0 puglml. The following reactions were carried out under oil (12). Step 1. Inactivation of tissue enzymes. 0.1 N NaOH, 0.05 ~1, for samples and blanks, or 0.1 N NaOH containing 20-50 PM GDP and/or GTP. The samples were heated 20 min at 80°C. Step 2. GTP reaction with succinyl CoA synthetase. To all samples was added 2 pi of reagent 1 without succinyl CoA synthetase (rack I). From these samples l-p.1 portions were removed to a second rack (rack II), and 1 ~1 of reagent 1 containing succinyl CoA synthetase was added. The samples were incubated 20 min at room temperature, followed by 20 min at 80°C to denature the enzymes. Step 3. Measurement of native GDP and reacted GTP. To all samples 4 ~1 of reagent 2 was added and the samples were incubated 10 min at room temperature.
RESULTS Tissues were prepared from mice or chicks as described under Materials and Methods and analyzed. The results are given in Table I. In the course of investigating the metabolism in the brain of hibernating hamsters, it was considered to be of interest to measure the guanine nucleotides in these animals. We have evidence that the brain of the hibernators, which is approximately 4°C freezes faster than that of the coldadapted animals, which have a body tem-
TABLE GTP
AND
GDP
CONCENTRATIONS
IN THREE
517
OF GTP/GDP
REGIONS
2 OF HAMSTER
CENTRAL
NERVOUS
SYSTEM”
Concentration (nmoVmg protein) Cortex
Cold adapted Hibernator
Cerebellum
Spinal cord
GTP
GDP
GTP
GDP
GTP
GDP
6.46 2 0.19 5.90 k 0.32
1.33 ? 0.09 1.03 2 0.14
4.49 2 0.43 5.12 t 0.38
1.65 2 0.22 1.57 + 0.08
6.51 k 0.54 5.71 2 0.53
1.05 2 0.17 0.70 2 0.19
‘I The tissues were prepared from hamsters which were either cold adapted or hibernating and analyzed as described in the text. Values are means + SEM for six animals, except for hibernator cortex, where there were five animals.
518
DE AZEREDO TABLE
3
GTP AND GDP CONCENTRATION IN LAYERS OF THE CEREBELLUM FROM MOUSE BRAIN” Concentration (nmol/mg dry wt) Layer
GTP
GDP
Molecular (4) Granular (3) White (5)
2.06 2 0.10 1.72 ? 0.40 1.21 2 0.15
1.48 2 0.20 1.53 f 0.36 0.91 2 0.14
a The tissues were prepared and analyzed as described in the text. Values are the means r SEM for the number of samples given in parentheses.
perature of 34°C (13). The concentrations reported in Table 2 show no significant differences between the two sets of hamsters. Samples of the molecular, granular, and white layers of cerebellum were dissected, weighed, and analyzed by oil-well techniques (12) using the above procedures for l-3 x lo-l2 mol. The data are given in Table 3. DISCUSSION A method has been described utilizing succinyl CoA synthetase for the measurement of GTP in biological material. The reaction is coupled to pyruvate kinase to measure the GDP formed in the first reaction. The prior treatment of the tissues with creatine kinase and phosphocreatine removes the ADP which would otherwise react in the pyruvate kinase system, as well as that formed by the myokinase contamination of succinyl CoA synthetase. Samples incubated with and without succinyl CoA synthetase provide a measure of GTP/GDP or GDP alone (plus whatever CDP and UDP are present). In general, the concentrations reported here are substantially higher than those reported using different enzymic determination (67) and chromatographic techniques (19). These
ET AL.
results may be in part due to the sensitivity of the fluorometric analysis. It is possible to measure accurately as little as 0.1 nmol of substance which can be used to oxidize or reduce pyridine nucleotides. In the direct fluorometric analysis, 7.5 mg of tissue was used, and the amount of GTP or GDP measured was on the order of 1 to 5 nmol. A comment may be in order on the higher proportions of GDP (Table 3) found in cerebellar layers than in the mouse cerebral cortex (Table 1) or hamster cerebellum (Table 2). There may be regional differences (cerebral cortex vs cerebellum) and species differences (mouse vs hamster), and finally the concentration in the cortical layers of the cerebellum may not reflect the average concentration of the whole region. The coupling of the assay to a cycling system further extends the sensitivity of the system. In the present demonstration (Table 3), l-2 pg of tissue was analyzed, so that 1 pmol of GTP/GDP was measured. REFERENCES 1. Lust, W. D., Kupferberg, H. J., Yonekawa, W. D., Penry, J. K., Passonneau, J. V., and Wheaton, A. B. (1978) Mol. Pharmacol. 14, 347-356. 2. Orr, H. J., Lowry, 0. H., Cohen, A. J., and Ferrendelli, J. A. (1976) Proc. Nat. Acad. Sci. USA 73, 4442-4445. 3. de Azeredo, F. A. M., Lust, W. D., and Passonneau, J. V. (1978) Biochem. Biophys. Res. Commun. 85, 293-300. 4. Keppler, D. 0. R., Pausch, J., and Decker, K. (1974) J. Biot. Chem. 249, 21 t-216. 5. Jackson, R. C., Boritlki, T. J., Morris, H. P., and Weber, G. (1976) Life Sci. 19, 1531-1536. 6. Keppler, D. 0. R., and Kaiser, W. (1978) Anal. Biochem. 86, 147-153. 7. Gruber, W., Mollering, H., and Bergmeyer, H. V. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 4, p. 2078, Academic Press, New York. 8. Grassl, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 4, p. 2162, Academic Press, New York. 9. Cha, S., and Cha, C. M. (1965) Mol. Pharmacof. 1, 178-189.
ENZYMATIC
ANALYSIS
10. Cha, S.. and Cha, C. M. (1970) Anol. Biochem. 33, 174-192. 11. Goldberg, N. D., Dietz, S. B., and O’Toole, A. G. (1969) J. Biol. Chem. 244, 4458-4466. 12. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York. 13. Lust, W. D., Goldman, S., Wheaton, A., Feussner, G., and Passonneau, J. (1978) Trans. Amer. Sot. Neurochem. 9, Abstract 140. 14. de Azeredo, F. A. M., and Martins-Ferreira. H. (1979) Neurochem. Res. 4, 83-91. 15. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275.
OF GTPiGDP
519
16. Kaufman, S., and Alivisatos, S. Cl. A. ( 1955) J. Biol. Chem. 216, 141-152. 17. Cha, S., Parks, R. E., Jr. (1964) J. Bid. Chem. 239, 1%1- 1967. 18. Lowry, 0. H., Passonneau, J. V., Hasselberg, F. X., and Schulz, D. W. ( 1964) J. Bid. Chem. 239, 18-30. 19. Kleihues, P., Kobayashi, K., and Hossman, K.-A. (1974) J. Neurochem. 23, 417-425. 20. Goldberg, N. D.. Passonneau, J. V., and Lowry, 0. H. (1966) J. Bid. Chem. 241, 3997-4003. 21. Krishan, N., and Krishna, G. (1976) Anal. Biothem. 70, 18-31. 22. Kato. T., Berger, S. J., Carter, J. A., and Lowry, 0. H. (1973) And. Biochem.. 53, 86-97.