- Email: [email protected]
A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples
ANALYTICAL BIOCHEMISTRY 170,376-38 1 ( 1988)
A Bioluminescent Assay for the Determination of Phosphoenolpyruvate Carboxykinase Activity in Nanogram-Sized Tissue Samples MONIKA
WIMMER
Department of Anatomy, University of Bask Pestalozzistrasse 20, CH-4056 Basel, Switzerland Received October 26, 1987 A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels. @ 1988 Academic Press,Inc. KBY WORDS: phosphoenolpyruvate carboxykinase; bioluminescence method; picomole range.
Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) catalyzes the irreversible conversion of oxaloacetate to phosphoenolpyruvate. The expression of the enzyme is subjected to hormonal control (l), and it is generally supposed to be one of the control points in gluconeogenesis. In liver and kidney, i.e., the primary sites of gluconeogenesis, however, the enzyme exhibits different activity in various cells depending on their localization (2-5). Extremely sensitive methods of enzyme activity determination are required to measure the distribution of the PEPCK’ activity within these tissues. Up to now, two different methods have been used to determine the activity of the enzyme in nanogram-sized tissue samples: (i) determination of the phosphoenolpyruvate formed in the PEPCK reaction by a coupled fluorometric method (4), and (ii) determination of yp2P incorporation in phosphoenolpyruvate from [T-~*P]GTP, which is used by the enzyme as a phosphate donor (2,3,5). Method (i) is highly sensitive (detection limit
ca. 5 X lo-‘* mol) but complicated and time-consuming. Method (ii) is fairly simple but less sensitive (detection limit lo-‘* mol) (6) than the fluorometric method. This report describes a third method providing a highly sensitive and convenient means for the determination of PEPCK activity in small tissue samples. The assay relies on the conversion of oxaloacetate to phosphoenolpyruvate and CO2 by PEPCK in the presence of ITP. In a second step ATP and pyruvate are formed from ADP and phosphoenolpyruvate in the presence of pyruvate kinase. The resulting concentration of ATP is quantified by a luminometric method which employs the firefly luciferin/luciferase system. MATERIALS
Substrates, streptozotocin, and ATP bioluminescence CLS kit were supplied by Boehringer (Mannheim, FRG). Adenosine diphosphate (A 4386), pyruvate kinase (P 7889), oxaloacetate, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). The other chemicals were supplied by Merck (Darmstadt, FRG).
’ Abbreviations used: PEPCK, phosphoenolpyruvate carboxykinase; PEP, phosphoenolpyruvate; Hepes, 4(2-hydroxyethyl)-I-piperazineethanesulfonic acid. 0003-2697188 $3.00 Copyrigbt Q 1988 by Academic PISS, Inc. All rights of reproduction in any form resewed.
AND METHODS
376
PHOSPHOENOLPYRUVATE
CARBOXYIUNASE
ACTIVITY
371
(50 3-Mercaptopicolinic acid was a generous gift media was added: K2HP04/KH2P04 mM, pH 7.0), ADP (2 mM), KC1 (2.5 mM), from Smith Kline and French, Inc. (Philadelphia, PA). MgC12 (5 mM), pyruvate kinase (0.05 IU), Male Wistar rats were used. Diabetes was and bovine serum albumin (0.05%). The reaction was terminated after 45 min of incuinduced by a single intravenous application bation at 25°C by the addition of 0.69 ~1 of of streptozotocin (7 mg/ 100 g body wt, disNaOH (0.7 N). solved in 50 mM citrate buffer, pH 4.5) (7). Third step: Luminometric determination Control animals received an injection of 0.1 ml/ 100 g body wt of 0.9% NaCl. The onset of ofATP. One microliter of the assay mixture diabetes was controlled by the increase of was transferred into a cuvette using a Hamilblood glucose values from 8.42 (kO.7) mM in ton Microlab 1000 and diluted with 199 ~1 of the control group to 17.16 (k2.5) mM in the redistilled water. Then 50 ~1 of the reagent streptozotocin-treated group. Blood glucose solution from the ATP luminescence CLS kit concentrations were determined by the glu- (Boehringer) was added and the peak signal of emitted light was recorded by an LKBcose oxidase method using the GOD-Perid Wallac 125 1 luminometer. Different lots of kit from Boehringer. Homogenates. For the preparation of ho- the luminescence kit gave variable yields of signal. Therefore, the effimogenates, liver samples frozen in liquid ni- luminescence trogen and kept at -80°C were weighed and ciency of each reagent kit was standardized immediately homogenized in 9 vol of ice- with known concentrations of phosphoenolcold 50 mM Hepes’ buffer, pH 7.5, by using a pyruvate and ATP. Standards. The reaction was standardized glass homogenizer fitted with a Teflon pestel. The PEPCK activity was determined by a with phosphoenolpyruvate as well as with three step luminometric procedure. Steps 1 ATP of known concentrations added to the and 2 were performed under oil following the first step reaction mixture. Blanks and controls. Blank reactions were oil-well technique of Lowry and Passonneau (8). Volumes less than 5 ~1 were pipetted performed in the presence of the reaction with the equipment described by Fink and mixture incubated with and without substrate. Samples incubated without substrate Pette (9). were used as tissue blanks. First step: Formation of phosphoenolpyruThe blank values did not change over a vate in the PEPCK reaction. The assays ( 1.04 ~1) contained potassium phosphate buffer period of 2 h. Tissue blanks were variable (50 mM, pH 7.5) MgS04 (10 mM), MnSO, presumably depending on the remaining ATP content. The typical standard deviation (0.1 mM) EGTA (0.1 mM), mercaptoethanol of a series of tissue blank measurements of (1 mM), oxaloacetate (2.5 mM), and bovine serum albumin (0.05%). the same sample was less than 10% of the The reaction was initiated by the addition mean value. of 0.69 ~1 inosine-5’-triphosphate (5 mM). Specificity test. The specificity of the The samples were incubated at 25°C for 20 PEPCK reaction was controlled by the addimin unless otherwise indicated. The reaction tion of 3-mercaptopicolinic acid (0.5 mrvr) to was terminated by the addition of 0.69 ~1 the assay mixture used for the first step. This Na3P04/K2HP04 (250 mM, pH 12.0) and concentration effectively inhibited the subsequent heating to 70°C for 10 min. PEPCK reaction ( 10). Second step: Formation of A TP from phosphoenolpyruvate and ADP. After terminaRESULTS tion of the first step the samples were brought to neutral pH by the addition of 0.69 ~1 of Our first aim was to check the relationship HCl (0.25 N). Then 0.69 ~1 of the following between the concentration of phosphoenol-
MONIKA
378
pyruvate present in the coupled luminomettic assayused for the activity determination of PEPCK and the resulting light signal. To do this, increasing amounts of phosphoenolpyruvate were incubated in the first step reaction mixture as described under Materials and Methods and subsequently subjected to the complete three-step procedure. As is shown in Fig. 1, there was a linear relationship between luminescence and the amount of added phosphoenolpyruvate in the range of 1 X lo-i3 and 75 X 10-l’ mol of pyruvate. For comparison, the same reaction was performed adding known concentrations of ATP in an additional seriesof experiments. The yield of signal was almost identical for ATP and phosphoenolpyruvate. IDP is also known to be a substrate for pyruvate kinase activity (11). To exclude a conversion of phosphoenolpyruvate without simultaneous formation of ATP the following experiment was performed. Increasing amounts of phosphoenolpyruvate and in a second seriesphosphoenolpyruvate plus IDP (identical concentrations) were added to the reaction mixture of step 1 and the conversion of phosphoenolpyruvate into ATP was
determined by running the samplesthrough steps 2 and 3. The resulting signals were nearly identical for both series of experiments (Fig. 2), thus proving that there is no loss of formed phosphoenolpyruvate by an IDP-dependent side reaction. In the following this assaysystemwas ap plied to liver homogenates from a normal rat and from a diabetic rat expected to contain high PEPCK activity. To prove the specificity of the PEPCK reaction step 1.04 X lo-’ g liver homogenate was incubated in the presence of 3-mercaptopicolinic acid for increasing incubation times. This substance,a specific inhibitor of PEPCK, had no influence on steps2 and 3 of the assayand produced no increase in background luminescence. In the presence of 3mercaptopicolinic acid, there was no detectable formation of phosphoenolpyruvate and all measured values corresponded to the signals registrated as tissueblanks. This held for all examined incubation times (5 to 60 min). The time dependence of phosphoenolpyruvate formation is shown in Fig. 3. Identical amounts ( 1.04 X lo-’ g) of liver homogenates of the normal (C) and diabetic (D) rats
mV
FIG. 1. Dependence of the bioluminescence signal (mV) on the concentration of ATP (v) and of PEP (0) added to the first step mixture.. The values are presented as mean * standard deviation (n = 5). Blank value, 168 -+ 7 mV. Regression analysis shows a linear correlation (ATP, r = 0.998; PEP, r = 0.998).
PHOSPHOENOLPYRUVATE mV
t
PEP+
IDP
pm01
FIG. 2. Dependence of the bioluminescence signal (mV) on the concentration of PEP (a) and on the concentration of PEP in the presence of IDP (v) added to the first step reaction mixture. The values are presented as mean + standard deviation (n = 5). Blank value, 115 f 4 mV. Regression analysis shows a strong linear correlation (PEP, r = 0.998; PEP + IDP, r = 0.998).
CARBOXYKINASE
ACTIVITY
379
was proportional to incubation time for at least up to 60 min. In Fig. 3 this signal was converted to equivalence of substrate tumover as calculated from the phosphoenolpyruvate standard. In the measurement performed with the homogenate of a nondiabetic rat a linear progress with time was observed only between 15 and 60 min of reaction time. The measurements obtained during the first 15 min were only 20% different from the blank values and therefore the occurrence of the lag phase is most likely due to the limitations in resolving the PEPCK reaction signal from the signal of the tissue blank. The relationship between the amount of product formed in the PEPCK reaction within 25 min and the enzyme concentration corresponding to a given amount of tissue is shown in Fig. 4 for the livers of normal (C) and diabetic (D) rats. Both series of experiments showed a linear relation of the mea-
were incubated for increasing times. For the extract of the diabetic animal containing high PEPCK activity the luminescence signal
mol
x10-6 I
PEP/g YD
FIG. 3. Dependence of product (PEP) formation by liver tissue (1.04 X IO-’ g) in moles/gram on the incubation time. The values are presented as mean f standard deviation (n = 5). C, Normal rat liver; D, diabetic rat liver. Regression analysis shows a strong linear correlation (C, r = 0.977; D, r = 0.998).
RG. 4. Dependence of PEP formation (pmol/min) on the amount of liver tissue i.e., on the amount of PEPCK present in the assay. All samples were incubated for 25 min. The values am presented as mean + standard deviation (n = 5). C, Normal rat liver; D, diabetic rat liver. Regression analysis shows a linear correlation for the normal rat liver up to 4 X lOA7 g (r = 0.992) and up to 3 X lo-’ g for the diabetic rat liver (r = 0.996).
380
MONIKA
WIMMER
tinely used in this laboratory for the determination of activities in 50- to lOO-ng weight samples from liver. The assay relies on the formation of PEP and IDP from oxaloacetate and ITP in the PEPCK reaction. It has been previously reported that the activity of the enzyme in the DISCUSSION presence of ITP is about twice that measured PEPCK takes part in the conversion of py- with GTP as a substrate ( 17). The use of ITP ruvate into glucose. It catalyzes an irreversalso has the benefit that in the presence of ible step in gluconeogenesis and is one of the ADP, IDP in low concentrations is only an regulatory elements of gluconeogenesis. The inferior substrate of pyruvate kinase and, activities of PEPCK in liver and kidney are therefore, only ATP is formed in; the second influenced by hormones like glucocorticoids reaction step as shown in Fig. 2. The formaand insulin ( 12,13). Low concentrations of tion of ITP in this reaction would lead into a insulin as found under diabetic conditions dead end, since the third step is absolutely derepress the PEPCK gene (14) and thus in- specific for ATP ( 18). The described test is absolutely specific for crease the amount of enzyme. However, the PEPCK activity. 3-Mercaptopicolinic there is evidence that the changes in various cells within the organs are different dependacid added to the first reaction step coming on the functional location within the pletely prevented the conversion of oxaloaceliver lobules (1516) or the nephron. Hortate and ITP into phosphoenolpyruvate plus monal influences on the cellular level, there- IDP. 3-Mercaptopicolinic acid is known to infore, require extremely sensitive methods of be a specific and efficient noncompetitive hibitor of PEPCK (10). activity determination. The high sensitivity of this assay is attained Burch et al. (4) used a 7-step fluorometric assay in which PEP formed in the PEPCK by the use of firefly bioluminescence. Firefly reaction was determined in a pyruvate ki- luciferase catalyzes the ATP-dependent oxidation of D-luciferin with simultaneous nase/lactate dehydrogenase system coupled with enzymatic cycling as described by emission of light, which is directly proportional to the concentration of ATP (I 8). This Lowry and Passonneau (8). The sophisticated, time-consuming procedure requires a reaction is characterized by a high quantum high level of experimental effort and espe- yield ( 19) and its absolute specificity for cially the cycling procedure is extremely sen- ATP, thus excluding many possible interferences. The assay is run step by step thus prositive to trace impurities present in commerviding the possibility to arrange optimal cial enzymes used for the auxiliary reaction. The radioactive assay procedure used by conditions for each reaction. Substances inhibitory to the firefly luciferase are largely Vandewalle et al. (5) for kidney and Miethke et al. (2) for liver tissue requires the separa- excluded by the high dilution used in the last tion of the radioactive product from the sub- step. Possible analytical interferences by the strate as well as a trap for removing the auxiliary reaction or by any side reactions are formed phosphoenolpyruvate to reduce the controlled by the use of internal standards (phosphoenolpyruvate/ATP) which are prointerference by endogenous pyruvate kinase. A further disadvantage is the low sensitivity cessed the same way as the tissue samples. The applicability of the luminometric of this method. The three step procedure described in this report provides an easy to per- assay device for activity determination in tissues is demonstrated by the use of liver samform and reliable method for the determination of PEPCK activities. It has been rou- ples with normal PEPCK activity and with sured enzyme activity in the presence of low tissue enzyme concentrations. A linear increase of the measured PEPCK activity was observed up to 400 ng of normal and up to 250 ng of diabetic liver.
PHOSPHOENOLPYRUVATE
enhanced activity. For both cases the assay is linear with respect to time and concentration of tissue provided that the product formation is in the range of 2.5 to 100 pmol. SUMMARY
The luminometric procedure used for the determination of PEPCK activity is highly specific and sensitive. The test is easy to perform and allows measurements with short incubation in very small tissue samples or in samples containing very low activity. The incubation times, however, may be prolonged if necessary without a significant increase in blank values. ACKNOWLEDGMENTS I thank Dr. Frank J. Tiano, Smith Kline and French Laboratories (Philadelphia, PA), for the generous gift of 3-mercaptopicolinic acid. I am also indebted to Ms. Monika Lachat for typing the manuscript and Ms. Barbara Wilmering for the preparation of the figures.
REFERENCES 1. Fleig, W. E., Noether-Fleig, G., Roeben, H., and Ditschuneit, H. (1984) Arch. Biochem. Biophys. 229,368-378.
Miethke, H., Wittig, B., Nath, A., Zierz, S., and Jungermann, K. (1985) Biol. Chem. HoppeSeyler 366,493~50 1. 3. Guder, W. G., and Schmidt, U. (1976) HoppeSeylers Z. Physiol. Chem. 357, 1793- 1800. 4. Burch, H. B., Narins, R. G., Chu, C., Fagioli, S., 2.
CARBOXYKINASE
5.
6. 7. 8.
9.
ACTIVITY
381
Choi, S., McCarthy, W., and Lowry, 0. H. (1978) Amer. Physiol. Sot. 235(3), F246-F253. Vandewalle, A., Wirthensohn, G., Heidrich, H. G., and Guder, W. G. (198 1) Amer. J. Physiol. 240, F492-F5CQ. Walsh, D. A., and Chen, L.-J. (1971) Biochem. Biophys. Rex Commun. 45,669-675. Chen, V., and Ianuzzo, C. D. (1982) Can. J. Physiol. Pharmacol. 60, 1251-1256. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York/London. Fink, H., and Pette, D. (1983) Anal. Biochem. 133, 220-225.
10. Jomain-Baum, M., Schramm, V. L., and Hanson, R. W. (1976) J. Biof. Chem. 251(l), 37-44. 11. Carbonell, J., Feliu, J. E., Marco, R., and Sols, A. (1973) Eur. J. Biochem. 37, 148-l 56. 12. Oliver, I. T., Edwards, A. M., and Pitot, H. C. (1978) Eur. J. Biochem. 87,221-227. 13. Iynedjian, P. B., and Salavert, A. (1984) Eur. J. Biochem. 145,489-497. 14. Granner, D., Andreone, T., Sasaki, K., and Beale, E. (1983) Nufure 305,549-55 1. 15. Matsumura, T., Kashiwagi, T., Meren, H., and Thurman, R. G. (1984) Eur. J. Biochem. 144, 409-415. 16. Kinguasa, A., and Thurman, R. G. (1986) Biochem. J. 236,425-430. 17. Nordlie, R. C., and Lardy, H. A. (1963) J. Biol. Chem. 2438,2259-2263. 18. DeLuca, M. (1976) in Advances in Enzymology (A. Meister, Ed.), Vol. 44, pp. 37-68, Interscience, New York. 19. Seliger, H. H., and McElroy, W. D. ( 1959) Biochem. Biophys. Res. Commun. 1,2 l-24.
A Bioluminescent Assay for the Determination of Phosphoenolpyruvate Carboxykinase Activity in Nanogram-Sized Tissue Samples MONIKA
WIMMER
Department of Anatomy, University of Bask Pestalozzistrasse 20, CH-4056 Basel, Switzerland Received October 26, 1987 A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels. @ 1988 Academic Press,Inc. KBY WORDS: phosphoenolpyruvate carboxykinase; bioluminescence method; picomole range.
Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) catalyzes the irreversible conversion of oxaloacetate to phosphoenolpyruvate. The expression of the enzyme is subjected to hormonal control (l), and it is generally supposed to be one of the control points in gluconeogenesis. In liver and kidney, i.e., the primary sites of gluconeogenesis, however, the enzyme exhibits different activity in various cells depending on their localization (2-5). Extremely sensitive methods of enzyme activity determination are required to measure the distribution of the PEPCK’ activity within these tissues. Up to now, two different methods have been used to determine the activity of the enzyme in nanogram-sized tissue samples: (i) determination of the phosphoenolpyruvate formed in the PEPCK reaction by a coupled fluorometric method (4), and (ii) determination of yp2P incorporation in phosphoenolpyruvate from [T-~*P]GTP, which is used by the enzyme as a phosphate donor (2,3,5). Method (i) is highly sensitive (detection limit
ca. 5 X lo-‘* mol) but complicated and time-consuming. Method (ii) is fairly simple but less sensitive (detection limit lo-‘* mol) (6) than the fluorometric method. This report describes a third method providing a highly sensitive and convenient means for the determination of PEPCK activity in small tissue samples. The assay relies on the conversion of oxaloacetate to phosphoenolpyruvate and CO2 by PEPCK in the presence of ITP. In a second step ATP and pyruvate are formed from ADP and phosphoenolpyruvate in the presence of pyruvate kinase. The resulting concentration of ATP is quantified by a luminometric method which employs the firefly luciferin/luciferase system. MATERIALS
Substrates, streptozotocin, and ATP bioluminescence CLS kit were supplied by Boehringer (Mannheim, FRG). Adenosine diphosphate (A 4386), pyruvate kinase (P 7889), oxaloacetate, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). The other chemicals were supplied by Merck (Darmstadt, FRG).
’ Abbreviations used: PEPCK, phosphoenolpyruvate carboxykinase; PEP, phosphoenolpyruvate; Hepes, 4(2-hydroxyethyl)-I-piperazineethanesulfonic acid. 0003-2697188 $3.00 Copyrigbt Q 1988 by Academic PISS, Inc. All rights of reproduction in any form resewed.
AND METHODS
376
PHOSPHOENOLPYRUVATE
CARBOXYIUNASE
ACTIVITY
371
(50 3-Mercaptopicolinic acid was a generous gift media was added: K2HP04/KH2P04 mM, pH 7.0), ADP (2 mM), KC1 (2.5 mM), from Smith Kline and French, Inc. (Philadelphia, PA). MgC12 (5 mM), pyruvate kinase (0.05 IU), Male Wistar rats were used. Diabetes was and bovine serum albumin (0.05%). The reaction was terminated after 45 min of incuinduced by a single intravenous application bation at 25°C by the addition of 0.69 ~1 of of streptozotocin (7 mg/ 100 g body wt, disNaOH (0.7 N). solved in 50 mM citrate buffer, pH 4.5) (7). Third step: Luminometric determination Control animals received an injection of 0.1 ml/ 100 g body wt of 0.9% NaCl. The onset of ofATP. One microliter of the assay mixture diabetes was controlled by the increase of was transferred into a cuvette using a Hamilblood glucose values from 8.42 (kO.7) mM in ton Microlab 1000 and diluted with 199 ~1 of the control group to 17.16 (k2.5) mM in the redistilled water. Then 50 ~1 of the reagent streptozotocin-treated group. Blood glucose solution from the ATP luminescence CLS kit concentrations were determined by the glu- (Boehringer) was added and the peak signal of emitted light was recorded by an LKBcose oxidase method using the GOD-Perid Wallac 125 1 luminometer. Different lots of kit from Boehringer. Homogenates. For the preparation of ho- the luminescence kit gave variable yields of signal. Therefore, the effimogenates, liver samples frozen in liquid ni- luminescence trogen and kept at -80°C were weighed and ciency of each reagent kit was standardized immediately homogenized in 9 vol of ice- with known concentrations of phosphoenolcold 50 mM Hepes’ buffer, pH 7.5, by using a pyruvate and ATP. Standards. The reaction was standardized glass homogenizer fitted with a Teflon pestel. The PEPCK activity was determined by a with phosphoenolpyruvate as well as with three step luminometric procedure. Steps 1 ATP of known concentrations added to the and 2 were performed under oil following the first step reaction mixture. Blanks and controls. Blank reactions were oil-well technique of Lowry and Passonneau (8). Volumes less than 5 ~1 were pipetted performed in the presence of the reaction with the equipment described by Fink and mixture incubated with and without substrate. Samples incubated without substrate Pette (9). were used as tissue blanks. First step: Formation of phosphoenolpyruThe blank values did not change over a vate in the PEPCK reaction. The assays ( 1.04 ~1) contained potassium phosphate buffer period of 2 h. Tissue blanks were variable (50 mM, pH 7.5) MgS04 (10 mM), MnSO, presumably depending on the remaining ATP content. The typical standard deviation (0.1 mM) EGTA (0.1 mM), mercaptoethanol of a series of tissue blank measurements of (1 mM), oxaloacetate (2.5 mM), and bovine serum albumin (0.05%). the same sample was less than 10% of the The reaction was initiated by the addition mean value. of 0.69 ~1 inosine-5’-triphosphate (5 mM). Specificity test. The specificity of the The samples were incubated at 25°C for 20 PEPCK reaction was controlled by the addimin unless otherwise indicated. The reaction tion of 3-mercaptopicolinic acid (0.5 mrvr) to was terminated by the addition of 0.69 ~1 the assay mixture used for the first step. This Na3P04/K2HP04 (250 mM, pH 12.0) and concentration effectively inhibited the subsequent heating to 70°C for 10 min. PEPCK reaction ( 10). Second step: Formation of A TP from phosphoenolpyruvate and ADP. After terminaRESULTS tion of the first step the samples were brought to neutral pH by the addition of 0.69 ~1 of Our first aim was to check the relationship HCl (0.25 N). Then 0.69 ~1 of the following between the concentration of phosphoenol-
MONIKA
378
pyruvate present in the coupled luminomettic assayused for the activity determination of PEPCK and the resulting light signal. To do this, increasing amounts of phosphoenolpyruvate were incubated in the first step reaction mixture as described under Materials and Methods and subsequently subjected to the complete three-step procedure. As is shown in Fig. 1, there was a linear relationship between luminescence and the amount of added phosphoenolpyruvate in the range of 1 X lo-i3 and 75 X 10-l’ mol of pyruvate. For comparison, the same reaction was performed adding known concentrations of ATP in an additional seriesof experiments. The yield of signal was almost identical for ATP and phosphoenolpyruvate. IDP is also known to be a substrate for pyruvate kinase activity (11). To exclude a conversion of phosphoenolpyruvate without simultaneous formation of ATP the following experiment was performed. Increasing amounts of phosphoenolpyruvate and in a second seriesphosphoenolpyruvate plus IDP (identical concentrations) were added to the reaction mixture of step 1 and the conversion of phosphoenolpyruvate into ATP was
determined by running the samplesthrough steps 2 and 3. The resulting signals were nearly identical for both series of experiments (Fig. 2), thus proving that there is no loss of formed phosphoenolpyruvate by an IDP-dependent side reaction. In the following this assaysystemwas ap plied to liver homogenates from a normal rat and from a diabetic rat expected to contain high PEPCK activity. To prove the specificity of the PEPCK reaction step 1.04 X lo-’ g liver homogenate was incubated in the presence of 3-mercaptopicolinic acid for increasing incubation times. This substance,a specific inhibitor of PEPCK, had no influence on steps2 and 3 of the assayand produced no increase in background luminescence. In the presence of 3mercaptopicolinic acid, there was no detectable formation of phosphoenolpyruvate and all measured values corresponded to the signals registrated as tissueblanks. This held for all examined incubation times (5 to 60 min). The time dependence of phosphoenolpyruvate formation is shown in Fig. 3. Identical amounts ( 1.04 X lo-’ g) of liver homogenates of the normal (C) and diabetic (D) rats
mV
FIG. 1. Dependence of the bioluminescence signal (mV) on the concentration of ATP (v) and of PEP (0) added to the first step mixture.. The values are presented as mean * standard deviation (n = 5). Blank value, 168 -+ 7 mV. Regression analysis shows a linear correlation (ATP, r = 0.998; PEP, r = 0.998).
PHOSPHOENOLPYRUVATE mV
t
PEP+
IDP
pm01
FIG. 2. Dependence of the bioluminescence signal (mV) on the concentration of PEP (a) and on the concentration of PEP in the presence of IDP (v) added to the first step reaction mixture. The values are presented as mean + standard deviation (n = 5). Blank value, 115 f 4 mV. Regression analysis shows a strong linear correlation (PEP, r = 0.998; PEP + IDP, r = 0.998).
CARBOXYKINASE
ACTIVITY
379
was proportional to incubation time for at least up to 60 min. In Fig. 3 this signal was converted to equivalence of substrate tumover as calculated from the phosphoenolpyruvate standard. In the measurement performed with the homogenate of a nondiabetic rat a linear progress with time was observed only between 15 and 60 min of reaction time. The measurements obtained during the first 15 min were only 20% different from the blank values and therefore the occurrence of the lag phase is most likely due to the limitations in resolving the PEPCK reaction signal from the signal of the tissue blank. The relationship between the amount of product formed in the PEPCK reaction within 25 min and the enzyme concentration corresponding to a given amount of tissue is shown in Fig. 4 for the livers of normal (C) and diabetic (D) rats. Both series of experiments showed a linear relation of the mea-
were incubated for increasing times. For the extract of the diabetic animal containing high PEPCK activity the luminescence signal
mol
x10-6 I
PEP/g YD
FIG. 3. Dependence of product (PEP) formation by liver tissue (1.04 X IO-’ g) in moles/gram on the incubation time. The values are presented as mean f standard deviation (n = 5). C, Normal rat liver; D, diabetic rat liver. Regression analysis shows a strong linear correlation (C, r = 0.977; D, r = 0.998).
RG. 4. Dependence of PEP formation (pmol/min) on the amount of liver tissue i.e., on the amount of PEPCK present in the assay. All samples were incubated for 25 min. The values am presented as mean + standard deviation (n = 5). C, Normal rat liver; D, diabetic rat liver. Regression analysis shows a linear correlation for the normal rat liver up to 4 X lOA7 g (r = 0.992) and up to 3 X lo-’ g for the diabetic rat liver (r = 0.996).
380
MONIKA
WIMMER
tinely used in this laboratory for the determination of activities in 50- to lOO-ng weight samples from liver. The assay relies on the formation of PEP and IDP from oxaloacetate and ITP in the PEPCK reaction. It has been previously reported that the activity of the enzyme in the DISCUSSION presence of ITP is about twice that measured PEPCK takes part in the conversion of py- with GTP as a substrate ( 17). The use of ITP ruvate into glucose. It catalyzes an irreversalso has the benefit that in the presence of ible step in gluconeogenesis and is one of the ADP, IDP in low concentrations is only an regulatory elements of gluconeogenesis. The inferior substrate of pyruvate kinase and, activities of PEPCK in liver and kidney are therefore, only ATP is formed in; the second influenced by hormones like glucocorticoids reaction step as shown in Fig. 2. The formaand insulin ( 12,13). Low concentrations of tion of ITP in this reaction would lead into a insulin as found under diabetic conditions dead end, since the third step is absolutely derepress the PEPCK gene (14) and thus in- specific for ATP ( 18). The described test is absolutely specific for crease the amount of enzyme. However, the PEPCK activity. 3-Mercaptopicolinic there is evidence that the changes in various cells within the organs are different dependacid added to the first reaction step coming on the functional location within the pletely prevented the conversion of oxaloaceliver lobules (1516) or the nephron. Hortate and ITP into phosphoenolpyruvate plus monal influences on the cellular level, there- IDP. 3-Mercaptopicolinic acid is known to infore, require extremely sensitive methods of be a specific and efficient noncompetitive hibitor of PEPCK (10). activity determination. The high sensitivity of this assay is attained Burch et al. (4) used a 7-step fluorometric assay in which PEP formed in the PEPCK by the use of firefly bioluminescence. Firefly reaction was determined in a pyruvate ki- luciferase catalyzes the ATP-dependent oxidation of D-luciferin with simultaneous nase/lactate dehydrogenase system coupled with enzymatic cycling as described by emission of light, which is directly proportional to the concentration of ATP (I 8). This Lowry and Passonneau (8). The sophisticated, time-consuming procedure requires a reaction is characterized by a high quantum high level of experimental effort and espe- yield ( 19) and its absolute specificity for cially the cycling procedure is extremely sen- ATP, thus excluding many possible interferences. The assay is run step by step thus prositive to trace impurities present in commerviding the possibility to arrange optimal cial enzymes used for the auxiliary reaction. The radioactive assay procedure used by conditions for each reaction. Substances inhibitory to the firefly luciferase are largely Vandewalle et al. (5) for kidney and Miethke et al. (2) for liver tissue requires the separa- excluded by the high dilution used in the last tion of the radioactive product from the sub- step. Possible analytical interferences by the strate as well as a trap for removing the auxiliary reaction or by any side reactions are formed phosphoenolpyruvate to reduce the controlled by the use of internal standards (phosphoenolpyruvate/ATP) which are prointerference by endogenous pyruvate kinase. A further disadvantage is the low sensitivity cessed the same way as the tissue samples. The applicability of the luminometric of this method. The three step procedure described in this report provides an easy to per- assay device for activity determination in tissues is demonstrated by the use of liver samform and reliable method for the determination of PEPCK activities. It has been rou- ples with normal PEPCK activity and with sured enzyme activity in the presence of low tissue enzyme concentrations. A linear increase of the measured PEPCK activity was observed up to 400 ng of normal and up to 250 ng of diabetic liver.
PHOSPHOENOLPYRUVATE
enhanced activity. For both cases the assay is linear with respect to time and concentration of tissue provided that the product formation is in the range of 2.5 to 100 pmol. SUMMARY
The luminometric procedure used for the determination of PEPCK activity is highly specific and sensitive. The test is easy to perform and allows measurements with short incubation in very small tissue samples or in samples containing very low activity. The incubation times, however, may be prolonged if necessary without a significant increase in blank values. ACKNOWLEDGMENTS I thank Dr. Frank J. Tiano, Smith Kline and French Laboratories (Philadelphia, PA), for the generous gift of 3-mercaptopicolinic acid. I am also indebted to Ms. Monika Lachat for typing the manuscript and Ms. Barbara Wilmering for the preparation of the figures.
REFERENCES 1. Fleig, W. E., Noether-Fleig, G., Roeben, H., and Ditschuneit, H. (1984) Arch. Biochem. Biophys. 229,368-378.
Miethke, H., Wittig, B., Nath, A., Zierz, S., and Jungermann, K. (1985) Biol. Chem. HoppeSeyler 366,493~50 1. 3. Guder, W. G., and Schmidt, U. (1976) HoppeSeylers Z. Physiol. Chem. 357, 1793- 1800. 4. Burch, H. B., Narins, R. G., Chu, C., Fagioli, S., 2.
CARBOXYKINASE
5.
6. 7. 8.
9.
ACTIVITY
381
Choi, S., McCarthy, W., and Lowry, 0. H. (1978) Amer. Physiol. Sot. 235(3), F246-F253. Vandewalle, A., Wirthensohn, G., Heidrich, H. G., and Guder, W. G. (198 1) Amer. J. Physiol. 240, F492-F5CQ. Walsh, D. A., and Chen, L.-J. (1971) Biochem. Biophys. Rex Commun. 45,669-675. Chen, V., and Ianuzzo, C. D. (1982) Can. J. Physiol. Pharmacol. 60, 1251-1256. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York/London. Fink, H., and Pette, D. (1983) Anal. Biochem. 133, 220-225.
10. Jomain-Baum, M., Schramm, V. L., and Hanson, R. W. (1976) J. Biof. Chem. 251(l), 37-44. 11. Carbonell, J., Feliu, J. E., Marco, R., and Sols, A. (1973) Eur. J. Biochem. 37, 148-l 56. 12. Oliver, I. T., Edwards, A. M., and Pitot, H. C. (1978) Eur. J. Biochem. 87,221-227. 13. Iynedjian, P. B., and Salavert, A. (1984) Eur. J. Biochem. 145,489-497. 14. Granner, D., Andreone, T., Sasaki, K., and Beale, E. (1983) Nufure 305,549-55 1. 15. Matsumura, T., Kashiwagi, T., Meren, H., and Thurman, R. G. (1984) Eur. J. Biochem. 144, 409-415. 16. Kinguasa, A., and Thurman, R. G. (1986) Biochem. J. 236,425-430. 17. Nordlie, R. C., and Lardy, H. A. (1963) J. Biol. Chem. 2438,2259-2263. 18. DeLuca, M. (1976) in Advances in Enzymology (A. Meister, Ed.), Vol. 44, pp. 37-68, Interscience, New York. 19. Seliger, H. H., and McElroy, W. D. ( 1959) Biochem. Biophys. Res. Commun. 1,2 l-24.