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Improved α-glycerophosphate dehydrogenase assay system suitable for continuous recording
ANALYTICAL
123, 170- 173 (1982)
BIOCHEMISTRY
Improved
a-Glycerophosphate Dehydrogenase Assay System Suitable for Continuous Recording BRIAN C. W. HUMMEL
Thyroid Research Laboratory, Sinai Hospital, Toronto,
AND
PAUL G. WALFISH'
Harold Tanenbaum Department of Research, and Endocrine Division, Mount and Department of Medicine, University of Toronto School of Medicine, Toronto, Ontario, Canada Received
December
29, 198 I
Improvements have been made in the Lee and Lardy assay for mitochondrial ol-glycerophosphate dehydrogenase. Sonication of mitochondria in 1% Triton X-100 assures uniform dispersion of the particles and prevents precipitation of formazan, thus allowing continuous recording of the progress of the reaction.
In studying the relationship of age to hepatic metabolism of thyroxine and cu-glycerophosphate dehydrogenase (a-GPDH)’ levels in rats, we found that an assay system which was simpler, more rapid, and more suitable for kinetic measurements than that of Lee and Lardy ( 1) (a modification of the method of Nachlus et al. (2)) was preferred. Recent investigations on the mechanism of triiodothyronine control of hepatic enzyme levels have necessitated measurement of (YGPDH (3-8). We have accordingly modified the Lee and Lardy method to allow continuous photometric recording of the course of reduction and developed a procedure more satisfactory for the assessment of changes in rat hepatic enzyme activity. EXPERIMENTAL Equipment. Zeiss spectrophotometer MQ3, with automatic cell changer PMQII, photometer system PMQ3, and thermostated ’ To whom correspondence and requests for reprints should be addressed at Mount Sinai Hospital, Suite #639-640. 600 University Ave., Toronto, Ontario M5G 1X5, Canada. ’ Abbreviations used: wGPDH, o-glycerophosphate dehydrogenase; INT, 2-p-iodophenyl-3-p-nitrophenyl5-phenyl-tetrazolium chloride; PES, 5-ethyl phenazium ethyl sulfate; PMS, 5-methyl phenazinium methyl sulfate. 0003-2697/82/090170-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
cell holder (Carl Zeiss, Oberkocken, West Germany); Servogor potentiometric recorder, 10 or 20 mV full scale (Goerz Electro, GmbH, Vienna, Austria); bath with circulating pump and thermoregulator; ultrasonic disintegrator PGlOO, 150 W with titanium microprobe (MSE Scientific Instruments, Crawley, Sussex, England). Materials. Chemicals were obtained from the following sources: Sodium cY-glycerophosphate. 6H20 and INT (Sigma Chemical Co., St. Louis, MO.); PES (ICN Pharmaceuticals, Cleveland, Ohio); Triton X- 100 (Amersham Corp., Arlington Heights, Ill.); KCN, KH2P04 (Fisher Scientific Company, Fairlawn, N. J.). Reagents. A stock substrate solution sufficient for 30 assays was prepared as follows: 4.5 ml buffer (250 mM potassium phosphate, 10 mM KCN, pH 7.5), 4.5 ml substrate (300 mM sodium a-glycerophosphate, pH 7.5) 9.0 ml PES (45.0 mg in 2.0 ml phosphate/ KCN buffer, pH 7.5, plus 8.0 ml HzO),3 and 9.0 ml INT (13.2 mg in 2.0 ml phosphate/ KCN buffer, pH 7.5, plus 8.0 ml H*O). Preparation of mitochondria. To 2.0 ml of mitochondrial suspension in 0.25 M su3 Suspended material ution kept in the dark
170
removed as much
by centrifugation; as possible.
sol-
IMPROVED
171
a-GPDH ASSAY
tivity units per milligram protein. The sample volume may be varied and the volume of water altered equally while maintaining a total volume in the reaction mixture of 1.5 ml. Protein assay. The modification of the method of Lowry et al. (9) by Hartree (10) was used. Samples containing particulate material were first hydrolyzed in 0.75 N NaOH for several hours at room temperature or for 45 min at 95°C.
16
I
I
14
12
INCUBATION
10
I
I
I
8
6
4
2
TIME,
MIN,
1
RESULTS
I
0
AT 30"
FIG. 1. Tracing of continuous recording of absorbance increase at 500 nm of mitochondrial suspension (100 ~1) sonicated in 1% Triton X-100, mixed with 1.4 ml buffered substrate-dye mixture, and incubated at 30°C.
crose/O.l M Tris-HCl, pH 7.6, which had been stored at -20°C and thawed rapidly at 3O”C, were added 222 ~1 of 10% aqueous Triton X- 100. The mixture was cooled to O5°C in an ice bath and sonicated for two periods of 30 s with the amplitude set at approximately 12 pm. The mixture was cooled in the ice bath for 1 min between sonications and stored on ice. Enzyme assay procedure. To each of five photometer cells (l-cm light path) were added 0.90 ml of stock substrate plus 0.50 ml H20, and the cells were allowed to warm to 30°C in the spectrophotometer chamber for 4 min. A sixth (reference) cell contained 900 ~1 stock substrate plus 0.60 ml H,O. To start the reaction 100 ~1 of mitochondrial preparation, prewarmed to 30°C and containing 20-60 pg of protein, was added to each cell and mixed. Absorbance was determined in each cuvette in turn at 2-min intervals at 500 nm using the automatic sample changer and recorded for 0.5 to 20 min with a full-scale sensitivity of 0.5 absorbance units. One unit of enzyme activity (U) is defined as an increase in Asoo of 10-3/min. Specific activity is expressed in terms of ac-
Frozen mitochondrial suspensions yielded large clumps of precipitated material upon thawing. Difficulties in homogenization without loss of material were avoided by sonication, which produced an opalescent dispersion in the presence of 1% Triton X-100. Continuous recording of the course of the reaction gave a curve sufficiently smooth to allow the rate to be determined (Fig. l), whereas with homogenized, but nonsonicated, mitochondria this was not possible. The reference cuvette showed negligible change in A500 when measured against water over a 20-min period. In the absence of the substrate cu-glycerophosphate, the rate of reaction, as expected, was also negligible. A typical recording is shown in Fig. 1. Reaction rate was found to be a linear function of mitochondrial sample volume over the range O-0.40 units (Fig. 2) corresponding to O-52 pg of mitochondrial protein. The data of Fig. 2, calculated on the basis of 100 ~1 of sonicated sample, have a mean value of 43.2 enzyme units, a variance of 16.3, standard deviation of 4.04, standard error of the mean of 1.17, and coefficient of variation of 9.35. DISCUSSION
The present assay system for CY-GPDH is derived from that of Lee and Lardy (l), which in turn is based on the assay for succinic dehydrogenase developed by Nachlus et al. (2), and depends upon the reduction
172
HUMMEL
VOLUME
AND WALFISH
I
I
1
I
25
50
75
100
MITOCHONDRIAL
SUSPENSION,
OF
pl
FIG. 2. Relationship between mitochondrial concentration (microliter suspension per 1.5 ml reaction mixture) and rate of formazan formation as measured at 500 nm and 30°C.
of a tetrazolium salt to formazan using phenazinium salt as an intermediate carrier accepting electrons directly from the dehydrogenase (I I). In the currently used method (I), the reduction is stopped after a suitable period of incubation by the addition of trichloroacetic acid and ethanol, and the extracted formazan is estimated colorimetritally after centrifuging the mixture. We sought to overcome certain aspects of the procedure which are unsatisfactory. First, dye reduction takes place on or within the mitochondrial membrane rather than in free solution, a situation in which the rate of reaction is dependent upon the rate of diffusion of substrate and dyes to the catalytic surface. Kinetic analysis in this situation differs from that in obtaining free solution ( 14,15). Mitochondrial suspensions which have been frozen and then thawed undergo almost complete aggregation of the particulate material, necessitating effective and reproducible dispersion without loss of mitochondria to avoid sampling errors, sedimentation during incubation, and variable rates of diffusion of reactants and products. Second, since waterinsoluble red formazan accumulates mainly
within the mitochondria rather than in the buffer, there is some uncertainty as to whether extraction by an organic solvent is quantitative. Third, valuable kinetic information is lost because this procedure is not amenable to continuous measurement during the course of reduction. We have accordingly introduced the following modifications of the Lee and Lardy procedure ( 1) to permit continuous automatic recording to five mitochondrial (YGPDH assays simultaneously: (a) employment of Triton X- 100, which has been shown to solubilize this enzyme from the bound form in mitochondria (12) and which we have found to retain formazan in solution during the assay period; (b) sonication of mitochondria in the presence of Triton X100, yielding an almost clear dispersion and thus greatly reducing irregularities in absorbance recording caused by sedimentation; (c) the use of PES, which is more stable than PMS (13). The assay system described here has proven satisfactory in our investigations into the changes in hepatic enzyme activity during aging in the rat.
IMPROVED
ACKNOWLEDGMENTS The excellent technical assistance of Mr. Paul Fung and the secretarial expertise of Ms. Diane Cartwright are greatly appreciated.
REFERENCES 1. Lee, Y.-P., and Lardy, H. A. (1965) J. Biol. Chem. 240, 1427. 2. Nachlus, M. M., Margulies, S. I., and Seligman, A. M. (1960) J. Biol. Chem. 235, 490. 3. Oppenheimer, J. H., Silva, E., Schwartz, H. L., and Surks, M. I. (1977) J. Cfin. Invest. 59, 517. 4. Oppenheimer, J. W., Coulombe, P., Schwartz, H. L., and Gutfeld, N. W. (1977) J. Clin. Invest. 61, 987. 5. Dillmann, W. H., Schwartz, H. L., and Oppenheimer, J. H. (1978) Biochem. Biophys. Rex Commun. 80, 259. 6. Schwartz, H. L., Forciea, M. A., Mariash, C. N.,
ol-GPDH
173
ASSAY
and Oppenheimer, J. H. (1974) Endocrinology 105, 41. 7. Simat, B. M., Towle, A. C., Schwartz, H. L., and Oppenheimer, J, H. (1980) Endocrinology 107, 1338. 8. Oppenheimer, J. H., and Schwartz, H. L. (1980) Endocrinology 107, 1460. 9. Lowry, 0. H., Rosenbrough, A., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 10. Hartree, E. F. (1972) Anal. Biochem. 48, 422. Il. Singer, T. P., and Kearney, E. B. (1954) Biochim. Biophys. Acta 15, 151. 12. Dawson, them.
A. P., and Thorne, J. Ill, 27.
C. J. R. (1969)
Bio-
13. Bernofsky, C., and Swan, M. (1973) Anal. Biothem. 53, 452. 14. Regan, D. L., Lilly, M. D., and Dunnill, P. (1974) Biotechnol. Bioeng. 16, 108 1. 15. Narinesingh, D., Ngo, T. T., and Laidler, (1975) Canad. J. Biochem. 53, 1061.
U. J.
123, 170- 173 (1982)
BIOCHEMISTRY
Improved
a-Glycerophosphate Dehydrogenase Assay System Suitable for Continuous Recording BRIAN C. W. HUMMEL
Thyroid Research Laboratory, Sinai Hospital, Toronto,
AND
PAUL G. WALFISH'
Harold Tanenbaum Department of Research, and Endocrine Division, Mount and Department of Medicine, University of Toronto School of Medicine, Toronto, Ontario, Canada Received
December
29, 198 I
Improvements have been made in the Lee and Lardy assay for mitochondrial ol-glycerophosphate dehydrogenase. Sonication of mitochondria in 1% Triton X-100 assures uniform dispersion of the particles and prevents precipitation of formazan, thus allowing continuous recording of the progress of the reaction.
In studying the relationship of age to hepatic metabolism of thyroxine and cu-glycerophosphate dehydrogenase (a-GPDH)’ levels in rats, we found that an assay system which was simpler, more rapid, and more suitable for kinetic measurements than that of Lee and Lardy ( 1) (a modification of the method of Nachlus et al. (2)) was preferred. Recent investigations on the mechanism of triiodothyronine control of hepatic enzyme levels have necessitated measurement of (YGPDH (3-8). We have accordingly modified the Lee and Lardy method to allow continuous photometric recording of the course of reduction and developed a procedure more satisfactory for the assessment of changes in rat hepatic enzyme activity. EXPERIMENTAL Equipment. Zeiss spectrophotometer MQ3, with automatic cell changer PMQII, photometer system PMQ3, and thermostated ’ To whom correspondence and requests for reprints should be addressed at Mount Sinai Hospital, Suite #639-640. 600 University Ave., Toronto, Ontario M5G 1X5, Canada. ’ Abbreviations used: wGPDH, o-glycerophosphate dehydrogenase; INT, 2-p-iodophenyl-3-p-nitrophenyl5-phenyl-tetrazolium chloride; PES, 5-ethyl phenazium ethyl sulfate; PMS, 5-methyl phenazinium methyl sulfate. 0003-2697/82/090170-04$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
cell holder (Carl Zeiss, Oberkocken, West Germany); Servogor potentiometric recorder, 10 or 20 mV full scale (Goerz Electro, GmbH, Vienna, Austria); bath with circulating pump and thermoregulator; ultrasonic disintegrator PGlOO, 150 W with titanium microprobe (MSE Scientific Instruments, Crawley, Sussex, England). Materials. Chemicals were obtained from the following sources: Sodium cY-glycerophosphate. 6H20 and INT (Sigma Chemical Co., St. Louis, MO.); PES (ICN Pharmaceuticals, Cleveland, Ohio); Triton X- 100 (Amersham Corp., Arlington Heights, Ill.); KCN, KH2P04 (Fisher Scientific Company, Fairlawn, N. J.). Reagents. A stock substrate solution sufficient for 30 assays was prepared as follows: 4.5 ml buffer (250 mM potassium phosphate, 10 mM KCN, pH 7.5), 4.5 ml substrate (300 mM sodium a-glycerophosphate, pH 7.5) 9.0 ml PES (45.0 mg in 2.0 ml phosphate/ KCN buffer, pH 7.5, plus 8.0 ml HzO),3 and 9.0 ml INT (13.2 mg in 2.0 ml phosphate/ KCN buffer, pH 7.5, plus 8.0 ml H*O). Preparation of mitochondria. To 2.0 ml of mitochondrial suspension in 0.25 M su3 Suspended material ution kept in the dark
170
removed as much
by centrifugation; as possible.
sol-
IMPROVED
171
a-GPDH ASSAY
tivity units per milligram protein. The sample volume may be varied and the volume of water altered equally while maintaining a total volume in the reaction mixture of 1.5 ml. Protein assay. The modification of the method of Lowry et al. (9) by Hartree (10) was used. Samples containing particulate material were first hydrolyzed in 0.75 N NaOH for several hours at room temperature or for 45 min at 95°C.
16
I
I
14
12
INCUBATION
10
I
I
I
8
6
4
2
TIME,
MIN,
1
RESULTS
I
0
AT 30"
FIG. 1. Tracing of continuous recording of absorbance increase at 500 nm of mitochondrial suspension (100 ~1) sonicated in 1% Triton X-100, mixed with 1.4 ml buffered substrate-dye mixture, and incubated at 30°C.
crose/O.l M Tris-HCl, pH 7.6, which had been stored at -20°C and thawed rapidly at 3O”C, were added 222 ~1 of 10% aqueous Triton X- 100. The mixture was cooled to O5°C in an ice bath and sonicated for two periods of 30 s with the amplitude set at approximately 12 pm. The mixture was cooled in the ice bath for 1 min between sonications and stored on ice. Enzyme assay procedure. To each of five photometer cells (l-cm light path) were added 0.90 ml of stock substrate plus 0.50 ml H20, and the cells were allowed to warm to 30°C in the spectrophotometer chamber for 4 min. A sixth (reference) cell contained 900 ~1 stock substrate plus 0.60 ml H,O. To start the reaction 100 ~1 of mitochondrial preparation, prewarmed to 30°C and containing 20-60 pg of protein, was added to each cell and mixed. Absorbance was determined in each cuvette in turn at 2-min intervals at 500 nm using the automatic sample changer and recorded for 0.5 to 20 min with a full-scale sensitivity of 0.5 absorbance units. One unit of enzyme activity (U) is defined as an increase in Asoo of 10-3/min. Specific activity is expressed in terms of ac-
Frozen mitochondrial suspensions yielded large clumps of precipitated material upon thawing. Difficulties in homogenization without loss of material were avoided by sonication, which produced an opalescent dispersion in the presence of 1% Triton X-100. Continuous recording of the course of the reaction gave a curve sufficiently smooth to allow the rate to be determined (Fig. l), whereas with homogenized, but nonsonicated, mitochondria this was not possible. The reference cuvette showed negligible change in A500 when measured against water over a 20-min period. In the absence of the substrate cu-glycerophosphate, the rate of reaction, as expected, was also negligible. A typical recording is shown in Fig. 1. Reaction rate was found to be a linear function of mitochondrial sample volume over the range O-0.40 units (Fig. 2) corresponding to O-52 pg of mitochondrial protein. The data of Fig. 2, calculated on the basis of 100 ~1 of sonicated sample, have a mean value of 43.2 enzyme units, a variance of 16.3, standard deviation of 4.04, standard error of the mean of 1.17, and coefficient of variation of 9.35. DISCUSSION
The present assay system for CY-GPDH is derived from that of Lee and Lardy (l), which in turn is based on the assay for succinic dehydrogenase developed by Nachlus et al. (2), and depends upon the reduction
172
HUMMEL
VOLUME
AND WALFISH
I
I
1
I
25
50
75
100
MITOCHONDRIAL
SUSPENSION,
OF
pl
FIG. 2. Relationship between mitochondrial concentration (microliter suspension per 1.5 ml reaction mixture) and rate of formazan formation as measured at 500 nm and 30°C.
of a tetrazolium salt to formazan using phenazinium salt as an intermediate carrier accepting electrons directly from the dehydrogenase (I I). In the currently used method (I), the reduction is stopped after a suitable period of incubation by the addition of trichloroacetic acid and ethanol, and the extracted formazan is estimated colorimetritally after centrifuging the mixture. We sought to overcome certain aspects of the procedure which are unsatisfactory. First, dye reduction takes place on or within the mitochondrial membrane rather than in free solution, a situation in which the rate of reaction is dependent upon the rate of diffusion of substrate and dyes to the catalytic surface. Kinetic analysis in this situation differs from that in obtaining free solution ( 14,15). Mitochondrial suspensions which have been frozen and then thawed undergo almost complete aggregation of the particulate material, necessitating effective and reproducible dispersion without loss of mitochondria to avoid sampling errors, sedimentation during incubation, and variable rates of diffusion of reactants and products. Second, since waterinsoluble red formazan accumulates mainly
within the mitochondria rather than in the buffer, there is some uncertainty as to whether extraction by an organic solvent is quantitative. Third, valuable kinetic information is lost because this procedure is not amenable to continuous measurement during the course of reduction. We have accordingly introduced the following modifications of the Lee and Lardy procedure ( 1) to permit continuous automatic recording to five mitochondrial (YGPDH assays simultaneously: (a) employment of Triton X- 100, which has been shown to solubilize this enzyme from the bound form in mitochondria (12) and which we have found to retain formazan in solution during the assay period; (b) sonication of mitochondria in the presence of Triton X100, yielding an almost clear dispersion and thus greatly reducing irregularities in absorbance recording caused by sedimentation; (c) the use of PES, which is more stable than PMS (13). The assay system described here has proven satisfactory in our investigations into the changes in hepatic enzyme activity during aging in the rat.
IMPROVED
ACKNOWLEDGMENTS The excellent technical assistance of Mr. Paul Fung and the secretarial expertise of Ms. Diane Cartwright are greatly appreciated.
REFERENCES 1. Lee, Y.-P., and Lardy, H. A. (1965) J. Biol. Chem. 240, 1427. 2. Nachlus, M. M., Margulies, S. I., and Seligman, A. M. (1960) J. Biol. Chem. 235, 490. 3. Oppenheimer, J. H., Silva, E., Schwartz, H. L., and Surks, M. I. (1977) J. Cfin. Invest. 59, 517. 4. Oppenheimer, J. W., Coulombe, P., Schwartz, H. L., and Gutfeld, N. W. (1977) J. Clin. Invest. 61, 987. 5. Dillmann, W. H., Schwartz, H. L., and Oppenheimer, J. H. (1978) Biochem. Biophys. Rex Commun. 80, 259. 6. Schwartz, H. L., Forciea, M. A., Mariash, C. N.,
ol-GPDH
173
ASSAY
and Oppenheimer, J. H. (1974) Endocrinology 105, 41. 7. Simat, B. M., Towle, A. C., Schwartz, H. L., and Oppenheimer, J, H. (1980) Endocrinology 107, 1338. 8. Oppenheimer, J. H., and Schwartz, H. L. (1980) Endocrinology 107, 1460. 9. Lowry, 0. H., Rosenbrough, A., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 10. Hartree, E. F. (1972) Anal. Biochem. 48, 422. Il. Singer, T. P., and Kearney, E. B. (1954) Biochim. Biophys. Acta 15, 151. 12. Dawson, them.
A. P., and Thorne, J. Ill, 27.
C. J. R. (1969)
Bio-
13. Bernofsky, C., and Swan, M. (1973) Anal. Biothem. 53, 452. 14. Regan, D. L., Lilly, M. D., and Dunnill, P. (1974) Biotechnol. Bioeng. 16, 108 1. 15. Narinesingh, D., Ngo, T. T., and Laidler, (1975) Canad. J. Biochem. 53, 1061.
U. J.