BJ
Letters
is still useful, but, as Feeney et al. [6] rightly point out, the mutual effects of the two substrates are of interest and importance. In order to excavate this information there is no alternative to carrying out systematic measurements that extend to low concentrations of both substrates simultaneously, so that the true 'combined specificity constant', 1/0AB, may be discovered. The Krebs Institute is a Centre for Molecular Recognition Studies under the Molecular Recognition Initiative of S.E.R.C.
Paul C. ENGEL Krebs Institute for Biomolecular Research, Department of Molecular Biology, University of Sheffield, P.O. Box 549, Firth Court, Western Bank, Sheffield SlO 2UH, U.K. 1. Fersht, A. R. (1985) Enzyme Structure and Mechanism, pp. 105-106, W. H. Freeman, New York 2. Cornish-Bowden, A. & Wharton, C. W. (1988) Enzyme Kinetics, p. 6, IRL Press, Oxford 3. Dalziel, K. (1957) Acta Chem. Scand. 11, 1706-1723 4. Engel, P. C. (1981) Enzyme Kinetics: the Steady State Approach (2nd edn.) Chapman & Hall, London 5. Wilks, H. M., Hart, K. W., Feeney, R., Dunn, C. R., Muirhead, H., Chia, W. N., Barstow, D. A., Atkinson, T., Clarke, A. R. & Holbrook, J. J. (1988) Science 242, 1541-1544 6. Feeney, R., Clarke, A. R. & Holbrook, J. J. (1990) Biochem. Biophys. Res. Commun. 166, 667-672 Received 29 January 1992
An improved assay for pyruvate dehydrogenase in liver and heart In a recent paper Paxton & Sievert [1] describe a new method to correct for the contribution of BCDH complex to the measured activity of PDH complex in tissue extracts. Others have used antibodies to BCDH [2]; or a kinetic method involving BCDH complex activity measured at saturating concentrations of ketoleucine, the Km for pyruvate of BCDH complex (734 gM), and the ratio of Vm.. values for ketoleucine/pyruvate of 5:1 [3]. The method in [2] is referred to in their paper; the method in [3] is not, though the reference is cited. The new method employs 14CO2 production from [1-14C]pyruvate to assay PDH complex activity and DCA and ketoisoleucine to inhibit 14CO2 production from pyruvate by BCDH complex. The method is potentially helpful but the purpose of this Letter is to draw attention to some ambiguities, misleading statements and unaddressed anomalies necessitating further consideration. The new assay uses 50,u/M-pyruvate according to the methods section (p. 547 paragraph 5), 0.5 mM-pyruvate according to the summary, the legend to Fig. 1 and Table 2 and text p. 548, paragraph 8. The concentration used in Table 1 is not stated. On p. 547, paragraph 2 it is stated that "the ratio of total BCODH to total PDC is about 0.65" and on p. 548, paragraph 8 the opposite is stated. Neither ratio is likely to be correct as the concentration of PDH complex is much too low. In liver mitochondria the ratio of total BCDH/total PDH is 0.2 on 17 % protein diet and 0.35 on 22 % protein diet [3,4]. Assuming that the new assay [1] uses 0.5 mM-pyruvate the contribution of BCDH complex to pyruvate decarboxylation (computed from kinetic constants above, [3]) is 8 % of BCDH complex activity measured at saturating [ketoleucine]. The highest estimates of total and expressed BCDH complex in liver
605 with ketoleucine are approximately 1.1 unit/g [4,8] giving a computed activity with 0.5 mM-pyruvate of 88 munits/g [expressed activity refers to the active (dephospho-) form of BCDH and PDH complexes and total activity to the sum of active and inactive forms. In livers of rats fed 22 % protein diets the two activities are virtually identical.] The total PDH complex activity of rat liver in [1] (Table 2) at 0.5 mM-pyruvate was 970 munits/g. Thus the computed contribution of BCDH complex to measured total PDH complex in Table 2 is 9 % and to expressed PDH complex activity (143 munits/g) is 61 % (a 6.8-fold difference). The values observed in Table 2 were 21 % and 27 % respectively. In an earlier paper reporting the antibody method [2] no effect of antibodies to BCDH on total PDH complex activity of liver was detected, even at 5 mM-pyruvate. There is no reference to or attempt to explain these apparent anomalies. In Table 2 of the paper by Paxton & Sievert [1] total PDH activities with 0.5 mM-pyruvate are given as 0.97 and 1.43 units/g (approximating to V.ax.) in livers of fed and starved rats respectively. These values are low for extracts prepared with 1 % Triton and protease inhibitors. Under comparable conditions of extraction (given in [4]) the value in this laboratory is 3.6 units/g fresh liver (fed) for total PDH with citrate synthase at 11 units/g (P. M. Stace & P. J. Randle, unpublished work). Walajtys-Rode & Williamson [5] measured 12.2 units PDH/g dry wt following extraction of hepatocytes with Lubrol (equivalent to 2.9 units/g wet wt liver). Table 2 of their paper also shows the effect of DCA/ ketoisoleucine to decrease 14CO2 production from [114C]pyruvate in liver. This decrement should presumably represent BCDH activity with pyruvate as substrate. The results show that the decrements for expressed and total BCDH activities were 33 and 169 munits/g respectively (fed) and 38 and 120 munits/g respectively (starved). It is well established that expressed and total activities of BCDH complex are essentially identical in livers of rats fed 22 % protein diets [4,8] (this diet was fed in [1]) i.e. the decrements for expressed and total activities should be the same. No explanation is offered for the 3-5 fold differences in the present study. If these unexplained discrepancies are due to errors inherent in the method then the method is clearly subject to substantial error and unacceptable. The data on rat heart in Table 2 shows even more striking discrepancies. The decrement in 14CO2 production from [1-_4C]pyruvate with DCA/ketoisoleucine was equivalent to 1 unit/g wet wt of BCDH complex measured with 0.5 mM-pyruvate as substrate for either total or expressed activity (fed rats). This is equivalent to 12.3 units BCDH complex/g wet wt with saturating ketoleucine (see paragraph 2 above). However measured total activities of BCDH complex in rat heart with ketoleucine as substrate are only 0.12 [19], 0.57 [20] and 0.3 units/g [21] with 48 % [19] or 9 % [20] or 18 % [21] of BCDH complex in the active form. These calculations indicate either that the decrement in 14CO2 production with DCA/ketoisoleucine is not due solely to inhibition of BCDH complex or that the errors inherent in the method are substantial. Table 2 in their paper also reports that 75 % of PDH is in the active form in hearts of fed rats, a result at variance with the 25-35 % found by others in hearts removed under pentobarbital anaesthesia. In paragraph 3 of Results and discussion the authors cite three references to work by others, which they claim shows that 60-80 % of PDH is active in hearts of fed rats. One of these [9] contains no measurements of % PDHa in heart; only measurements of % PDHa in heart mitochondria. Another [10] (from Dr. M. C. Sugden's laboratory) records a value of 59.4 %,
Abbreviations used: BCDH, branched chain 2-oxoacid dehydrogenase; PDH, pyruvate dehydrogenase; ketoleucine, 4-methyl-2-oxopentanoate; ketoisoleucine, 3-methyl-2-oxopentanoate; DCA, dichloroacetate. Vol. 284
606
but in seven other publications from the same laboratory [7,11-16], which are not mentioned here, values of 13.2%, 31.4%, and 32% were recorded in three and values of 18%, 27 %, 28 % and 29 % can be computed from values of expressed PDHa in munits/unit citrate synthase in four others (using a ratio of citrate synthase/total PDH of 14.2: 1 [1]). Three of these lower values are eventually mentioned in the penultimate paragraph of the paper. The most likely explanation for the high value for % PDHa in hearts of fed (and starved rats) obtained by Paxton & Sievert is their use of decapitation before removing hearts. This leads to tonic contraction of body muscles (which delays removal of tissues), haemorrhage (with hypovolaemia and likelihood of adrenaline discharge) and ischaemia, circumstances which are all known to increase % PDHa. This cannot be tested in the U.K. because decapitation is not recognized as a method of humane killing by the licensing authorities. However it has been shown [17] that cervical dislocation or 20 s of delay in removing hearts under pentobarbitone anaesthesia increase % PDHA. It is difficult to see the basis for their contention that values measured in tissues from decapitated animals may reflect "true in vivo values"; (third paragraph from end of [1]). The authors also conclude from data in Table 2, and contrary to the findings of others, that starvation decreases total PDH complex in heart. In isolated heart (or liver) mitochondria, in which total PDH complex is more easily measured than in whole tissue, total complex activity is also not altered by starvation of the rat [3,4,22] and neither is citrate synthase. According to my calculation the alleged effect of starvation on total PDC/CS in heart in [1] (last line of Table 2) is not statistically significant by the unpaired t-test (P = 0.11) even without taking into account the adverse effect of the highly significant difference in variance between the two groups. In the absence of an adequate explanation for these discrepancies, the new method is suspect. If antisera cannot be produced then a much simpler and seemingly more reliable method of achieving these objectives is to measure BCDH activity with [14C]ketoleucine and then to determine the equivalent PDH activity by calculation as in [3].
Philip J. RANDLE Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford OX3 9DU, U.K. 1. Paxton, R. & Sievert, L. M. (1991) Biochem. J. 277, 547-551 2. Goodwin, G. W., Paxton, R., Gillim, S. E. & Harris, R. A. (1986) Biochem. J. 236, 111-114 3. Fatania, H. R., Vary, T. C. & Randle, P. J. (1986) Biochem. J. 234, 233-236 4. Beggs, M. & Randle, P. J. (1988) Biochem. J. 256, 929-934 5. Walajtys-Rode, E. & Williamson, J. R. (1980) J. Biol. Chem. 255, 413-418 6. Caterson, 1. D., Fuller, S. J. & Randle, P. J. (1982) Biochem. J. 208, 53-60 7. French, T. J., Goode, A. W., Holness, M. J., MacLennan, P. A. & Sugden, M. C. (1988) Biochem. J. 256, 935-939 8. Harris, R. A., Powell, S. M., Paxton, R., Gillim, S. E. & Nagae, H. (1985) Arch. Biochem. Biophys. 243, 542-555 9. Hutson, N. J., Kerbey, A. L., Sugden, P. H. & Randle, P. J. (1978) Biochem. J. 173, 669-680 10. Holness, M. J., Palmer, T. N. & Sugden, M. C. (1985) Biochem. J. 232, 255-259 11. Sugden, M. C. & Holness, M. J. (1990) Biochem. Soc. Trans. 18, 847-850 12. Holness, M. J., Liu, T. C. & Sugden, M. C. (1989) Biochem. J. 264, 771-776 13. Holness, M. J. & Sugden, M. C. (1989) Biochem. J. 262, 321-325 14. Holness, M. J. & Sugden, M. C. (1989) Biochem. J. 258, 529-533 15. Sugden, M. C. & Holness, M. J. (1989) Biochem. J. 262, 669-672
BJ Letters 16. French, T. J., MacLennan, P. A., Holness, M. J. & Sugden, M. C. (1988) Biochem. J. 250, 773-779 17. French, T. J., Goode, A. W. & Sugden, M. C. (1986) Biochem. Int. 13, 843-852 19. Gillim, S. E., Paxton, R., Cook, G. A. & Harris, R. (1983) Biochem. Biophys. Res. Commun. 111, 74-81 20. Patston, P. A., Espinal, J. & Randle, P. J. (1984) Biochem. J. 222, 711-719 21. Solomon, M., Cook, G. & Yeaman, S. J. (1987) Biochim. Biophys. Acta 931, 335-338 22. McCormack, J. G., Edgell, N. J. & Denton, R. M. (1982) Biochem. J. 202, 419-427
Received 29 August 1991
An improved assay for pyruvate dehydrogenase: reply The assay for pyruvate dehydrogenase (PDC) activity in tissue extracts was improved [1] by removing branched-chain 2-oxoacid dehydrogenase's (BCODH) contribution to pyruvate decarboxylation with readily available reagents. The method eliminates pyruvate decarboxylation by BCODH without inhibiting PDC, was applicable to extracts of rat tissues and gave similar results to the only other proven method [2], based on antibodies inhibitory to BCODH, that eliminates BCODH's interference in PDC assays. Application of this improved PDC assay to rat liver and heart resulted in PDC activities similar in magnitude and in % active form to those previously reported (see [1] and below) and revealed overestimation of PDC by BCODH in liver, in expressed PDC activity in starved rat hearts and in total PDC activity in fed and starved rat hearts. The described ([3], paragraph 1) 'ambiguities', 'misleading statements' and 'unaddressed anomalies' are difficult to address because they are based on unpublished observations, misleading statements, errors in fact or inappropriate conclusions (see below). The first misleading statement ([3] paragraphs 1 and 3) is that a published kinetic method exists for determining BCODH contribution to pyruvate decarboxylation. The cited study [4] contains a description of undefined enzyme preparations studied by unpublished protocols that lead the authors to conclude without experimental proof that maximum total PDC activity in rat liver attributable to BCODH with 1 mM-pyruvate is 2 %. This method is not applied in that study or later ones [4-7]. A more recent estimate by the same author ([3] paragraph 3), based on the same observations, for maximum BCODH contribution to pyruvate decarboxylation with 0.5 mM-pyruvate is 8 % of total BCODH activity measured with ketoleucine. However, based on the average of published kinetic constants [8-12] the percent BCODH activity measured with ketoleucine and ketovaline, respectively, that could contribute to pyruvate decarboxylation at 0.5 mM-pyruvate are 14 % and 9.8 % and at 1 mM-pyruvate are 20 % and 14 %. It is difficult to impossible to estimate mean BCODH activity from the mean PDC activities given in Table 2 [1], as suggested ([3], paragraphs 3 and 5), because the assay was designed to measure the activity of PDC and not BCODH. The assay minimized BCODH activity by using a very low concentration, 0.5 mm (only 60 % of the Km value), of a relatively poor substrate, pyruvate (see [1]). The differences between the modified and unmodified assay can be attributed to BCODH activity (see Table I and Fig. 1 [1]) but should not, as suggested ([3], paragraphs 3 and 5), be taken as an accurate estimate of BCODH activity. However, even with these large uncertainties the estimated range of PDC overestimation in liver by BCODH 1992
Letters
is still useful, but, as Feeney et al. [6] rightly point out, the mutual effects of the two substrates are of interest and importance. In order to excavate this information there is no alternative to carrying out systematic measurements that extend to low concentrations of both substrates simultaneously, so that the true 'combined specificity constant', 1/0AB, may be discovered. The Krebs Institute is a Centre for Molecular Recognition Studies under the Molecular Recognition Initiative of S.E.R.C.
Paul C. ENGEL Krebs Institute for Biomolecular Research, Department of Molecular Biology, University of Sheffield, P.O. Box 549, Firth Court, Western Bank, Sheffield SlO 2UH, U.K. 1. Fersht, A. R. (1985) Enzyme Structure and Mechanism, pp. 105-106, W. H. Freeman, New York 2. Cornish-Bowden, A. & Wharton, C. W. (1988) Enzyme Kinetics, p. 6, IRL Press, Oxford 3. Dalziel, K. (1957) Acta Chem. Scand. 11, 1706-1723 4. Engel, P. C. (1981) Enzyme Kinetics: the Steady State Approach (2nd edn.) Chapman & Hall, London 5. Wilks, H. M., Hart, K. W., Feeney, R., Dunn, C. R., Muirhead, H., Chia, W. N., Barstow, D. A., Atkinson, T., Clarke, A. R. & Holbrook, J. J. (1988) Science 242, 1541-1544 6. Feeney, R., Clarke, A. R. & Holbrook, J. J. (1990) Biochem. Biophys. Res. Commun. 166, 667-672 Received 29 January 1992
An improved assay for pyruvate dehydrogenase in liver and heart In a recent paper Paxton & Sievert [1] describe a new method to correct for the contribution of BCDH complex to the measured activity of PDH complex in tissue extracts. Others have used antibodies to BCDH [2]; or a kinetic method involving BCDH complex activity measured at saturating concentrations of ketoleucine, the Km for pyruvate of BCDH complex (734 gM), and the ratio of Vm.. values for ketoleucine/pyruvate of 5:1 [3]. The method in [2] is referred to in their paper; the method in [3] is not, though the reference is cited. The new method employs 14CO2 production from [1-14C]pyruvate to assay PDH complex activity and DCA and ketoisoleucine to inhibit 14CO2 production from pyruvate by BCDH complex. The method is potentially helpful but the purpose of this Letter is to draw attention to some ambiguities, misleading statements and unaddressed anomalies necessitating further consideration. The new assay uses 50,u/M-pyruvate according to the methods section (p. 547 paragraph 5), 0.5 mM-pyruvate according to the summary, the legend to Fig. 1 and Table 2 and text p. 548, paragraph 8. The concentration used in Table 1 is not stated. On p. 547, paragraph 2 it is stated that "the ratio of total BCODH to total PDC is about 0.65" and on p. 548, paragraph 8 the opposite is stated. Neither ratio is likely to be correct as the concentration of PDH complex is much too low. In liver mitochondria the ratio of total BCDH/total PDH is 0.2 on 17 % protein diet and 0.35 on 22 % protein diet [3,4]. Assuming that the new assay [1] uses 0.5 mM-pyruvate the contribution of BCDH complex to pyruvate decarboxylation (computed from kinetic constants above, [3]) is 8 % of BCDH complex activity measured at saturating [ketoleucine]. The highest estimates of total and expressed BCDH complex in liver
605 with ketoleucine are approximately 1.1 unit/g [4,8] giving a computed activity with 0.5 mM-pyruvate of 88 munits/g [expressed activity refers to the active (dephospho-) form of BCDH and PDH complexes and total activity to the sum of active and inactive forms. In livers of rats fed 22 % protein diets the two activities are virtually identical.] The total PDH complex activity of rat liver in [1] (Table 2) at 0.5 mM-pyruvate was 970 munits/g. Thus the computed contribution of BCDH complex to measured total PDH complex in Table 2 is 9 % and to expressed PDH complex activity (143 munits/g) is 61 % (a 6.8-fold difference). The values observed in Table 2 were 21 % and 27 % respectively. In an earlier paper reporting the antibody method [2] no effect of antibodies to BCDH on total PDH complex activity of liver was detected, even at 5 mM-pyruvate. There is no reference to or attempt to explain these apparent anomalies. In Table 2 of the paper by Paxton & Sievert [1] total PDH activities with 0.5 mM-pyruvate are given as 0.97 and 1.43 units/g (approximating to V.ax.) in livers of fed and starved rats respectively. These values are low for extracts prepared with 1 % Triton and protease inhibitors. Under comparable conditions of extraction (given in [4]) the value in this laboratory is 3.6 units/g fresh liver (fed) for total PDH with citrate synthase at 11 units/g (P. M. Stace & P. J. Randle, unpublished work). Walajtys-Rode & Williamson [5] measured 12.2 units PDH/g dry wt following extraction of hepatocytes with Lubrol (equivalent to 2.9 units/g wet wt liver). Table 2 of their paper also shows the effect of DCA/ ketoisoleucine to decrease 14CO2 production from [114C]pyruvate in liver. This decrement should presumably represent BCDH activity with pyruvate as substrate. The results show that the decrements for expressed and total BCDH activities were 33 and 169 munits/g respectively (fed) and 38 and 120 munits/g respectively (starved). It is well established that expressed and total activities of BCDH complex are essentially identical in livers of rats fed 22 % protein diets [4,8] (this diet was fed in [1]) i.e. the decrements for expressed and total activities should be the same. No explanation is offered for the 3-5 fold differences in the present study. If these unexplained discrepancies are due to errors inherent in the method then the method is clearly subject to substantial error and unacceptable. The data on rat heart in Table 2 shows even more striking discrepancies. The decrement in 14CO2 production from [1-_4C]pyruvate with DCA/ketoisoleucine was equivalent to 1 unit/g wet wt of BCDH complex measured with 0.5 mM-pyruvate as substrate for either total or expressed activity (fed rats). This is equivalent to 12.3 units BCDH complex/g wet wt with saturating ketoleucine (see paragraph 2 above). However measured total activities of BCDH complex in rat heart with ketoleucine as substrate are only 0.12 [19], 0.57 [20] and 0.3 units/g [21] with 48 % [19] or 9 % [20] or 18 % [21] of BCDH complex in the active form. These calculations indicate either that the decrement in 14CO2 production with DCA/ketoisoleucine is not due solely to inhibition of BCDH complex or that the errors inherent in the method are substantial. Table 2 in their paper also reports that 75 % of PDH is in the active form in hearts of fed rats, a result at variance with the 25-35 % found by others in hearts removed under pentobarbital anaesthesia. In paragraph 3 of Results and discussion the authors cite three references to work by others, which they claim shows that 60-80 % of PDH is active in hearts of fed rats. One of these [9] contains no measurements of % PDHa in heart; only measurements of % PDHa in heart mitochondria. Another [10] (from Dr. M. C. Sugden's laboratory) records a value of 59.4 %,
Abbreviations used: BCDH, branched chain 2-oxoacid dehydrogenase; PDH, pyruvate dehydrogenase; ketoleucine, 4-methyl-2-oxopentanoate; ketoisoleucine, 3-methyl-2-oxopentanoate; DCA, dichloroacetate. Vol. 284
606
but in seven other publications from the same laboratory [7,11-16], which are not mentioned here, values of 13.2%, 31.4%, and 32% were recorded in three and values of 18%, 27 %, 28 % and 29 % can be computed from values of expressed PDHa in munits/unit citrate synthase in four others (using a ratio of citrate synthase/total PDH of 14.2: 1 [1]). Three of these lower values are eventually mentioned in the penultimate paragraph of the paper. The most likely explanation for the high value for % PDHa in hearts of fed (and starved rats) obtained by Paxton & Sievert is their use of decapitation before removing hearts. This leads to tonic contraction of body muscles (which delays removal of tissues), haemorrhage (with hypovolaemia and likelihood of adrenaline discharge) and ischaemia, circumstances which are all known to increase % PDHa. This cannot be tested in the U.K. because decapitation is not recognized as a method of humane killing by the licensing authorities. However it has been shown [17] that cervical dislocation or 20 s of delay in removing hearts under pentobarbitone anaesthesia increase % PDHA. It is difficult to see the basis for their contention that values measured in tissues from decapitated animals may reflect "true in vivo values"; (third paragraph from end of [1]). The authors also conclude from data in Table 2, and contrary to the findings of others, that starvation decreases total PDH complex in heart. In isolated heart (or liver) mitochondria, in which total PDH complex is more easily measured than in whole tissue, total complex activity is also not altered by starvation of the rat [3,4,22] and neither is citrate synthase. According to my calculation the alleged effect of starvation on total PDC/CS in heart in [1] (last line of Table 2) is not statistically significant by the unpaired t-test (P = 0.11) even without taking into account the adverse effect of the highly significant difference in variance between the two groups. In the absence of an adequate explanation for these discrepancies, the new method is suspect. If antisera cannot be produced then a much simpler and seemingly more reliable method of achieving these objectives is to measure BCDH activity with [14C]ketoleucine and then to determine the equivalent PDH activity by calculation as in [3].
Philip J. RANDLE Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford OX3 9DU, U.K. 1. Paxton, R. & Sievert, L. M. (1991) Biochem. J. 277, 547-551 2. Goodwin, G. W., Paxton, R., Gillim, S. E. & Harris, R. A. (1986) Biochem. J. 236, 111-114 3. Fatania, H. R., Vary, T. C. & Randle, P. J. (1986) Biochem. J. 234, 233-236 4. Beggs, M. & Randle, P. J. (1988) Biochem. J. 256, 929-934 5. Walajtys-Rode, E. & Williamson, J. R. (1980) J. Biol. Chem. 255, 413-418 6. Caterson, 1. D., Fuller, S. J. & Randle, P. J. (1982) Biochem. J. 208, 53-60 7. French, T. J., Goode, A. W., Holness, M. J., MacLennan, P. A. & Sugden, M. C. (1988) Biochem. J. 256, 935-939 8. Harris, R. A., Powell, S. M., Paxton, R., Gillim, S. E. & Nagae, H. (1985) Arch. Biochem. Biophys. 243, 542-555 9. Hutson, N. J., Kerbey, A. L., Sugden, P. H. & Randle, P. J. (1978) Biochem. J. 173, 669-680 10. Holness, M. J., Palmer, T. N. & Sugden, M. C. (1985) Biochem. J. 232, 255-259 11. Sugden, M. C. & Holness, M. J. (1990) Biochem. Soc. Trans. 18, 847-850 12. Holness, M. J., Liu, T. C. & Sugden, M. C. (1989) Biochem. J. 264, 771-776 13. Holness, M. J. & Sugden, M. C. (1989) Biochem. J. 262, 321-325 14. Holness, M. J. & Sugden, M. C. (1989) Biochem. J. 258, 529-533 15. Sugden, M. C. & Holness, M. J. (1989) Biochem. J. 262, 669-672
BJ Letters 16. French, T. J., MacLennan, P. A., Holness, M. J. & Sugden, M. C. (1988) Biochem. J. 250, 773-779 17. French, T. J., Goode, A. W. & Sugden, M. C. (1986) Biochem. Int. 13, 843-852 19. Gillim, S. E., Paxton, R., Cook, G. A. & Harris, R. (1983) Biochem. Biophys. Res. Commun. 111, 74-81 20. Patston, P. A., Espinal, J. & Randle, P. J. (1984) Biochem. J. 222, 711-719 21. Solomon, M., Cook, G. & Yeaman, S. J. (1987) Biochim. Biophys. Acta 931, 335-338 22. McCormack, J. G., Edgell, N. J. & Denton, R. M. (1982) Biochem. J. 202, 419-427
Received 29 August 1991
An improved assay for pyruvate dehydrogenase: reply The assay for pyruvate dehydrogenase (PDC) activity in tissue extracts was improved [1] by removing branched-chain 2-oxoacid dehydrogenase's (BCODH) contribution to pyruvate decarboxylation with readily available reagents. The method eliminates pyruvate decarboxylation by BCODH without inhibiting PDC, was applicable to extracts of rat tissues and gave similar results to the only other proven method [2], based on antibodies inhibitory to BCODH, that eliminates BCODH's interference in PDC assays. Application of this improved PDC assay to rat liver and heart resulted in PDC activities similar in magnitude and in % active form to those previously reported (see [1] and below) and revealed overestimation of PDC by BCODH in liver, in expressed PDC activity in starved rat hearts and in total PDC activity in fed and starved rat hearts. The described ([3], paragraph 1) 'ambiguities', 'misleading statements' and 'unaddressed anomalies' are difficult to address because they are based on unpublished observations, misleading statements, errors in fact or inappropriate conclusions (see below). The first misleading statement ([3] paragraphs 1 and 3) is that a published kinetic method exists for determining BCODH contribution to pyruvate decarboxylation. The cited study [4] contains a description of undefined enzyme preparations studied by unpublished protocols that lead the authors to conclude without experimental proof that maximum total PDC activity in rat liver attributable to BCODH with 1 mM-pyruvate is 2 %. This method is not applied in that study or later ones [4-7]. A more recent estimate by the same author ([3] paragraph 3), based on the same observations, for maximum BCODH contribution to pyruvate decarboxylation with 0.5 mM-pyruvate is 8 % of total BCODH activity measured with ketoleucine. However, based on the average of published kinetic constants [8-12] the percent BCODH activity measured with ketoleucine and ketovaline, respectively, that could contribute to pyruvate decarboxylation at 0.5 mM-pyruvate are 14 % and 9.8 % and at 1 mM-pyruvate are 20 % and 14 %. It is difficult to impossible to estimate mean BCODH activity from the mean PDC activities given in Table 2 [1], as suggested ([3], paragraphs 3 and 5), because the assay was designed to measure the activity of PDC and not BCODH. The assay minimized BCODH activity by using a very low concentration, 0.5 mm (only 60 % of the Km value), of a relatively poor substrate, pyruvate (see [1]). The differences between the modified and unmodified assay can be attributed to BCODH activity (see Table I and Fig. 1 [1]) but should not, as suggested ([3], paragraphs 3 and 5), be taken as an accurate estimate of BCODH activity. However, even with these large uncertainties the estimated range of PDC overestimation in liver by BCODH 1992
