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Reliability of the enzymatic determination of adenosine phosphates in crude extracts of marine bivalves
Comp. Biochem. Physiol.,
1977, Vol. 58B, p. 313. Pergamon Press. Printed
in Great Britain
SHORT COMMUNICATION RELIABILITY OF THE ENZYMATIC DETERMINATION OF ADENOSINE PHOSPHATES IN CRUDE EXTRACTS OF MARINE BIVALVES THEODORUS C. M. WIJSMAN* Laboratory of Chemical Animal Physiology, 8 Padualaan, Utrecht, The Netherlands (Received 21 March 1977)
Recently the validity of enzymatic assay of adenosine phosphates in crude extracts of some marine bivalves has been questioned (Zs.-Nagy, 1977). The paper of Wijsman (1976a) was used as an example of this doubtful method, as adenosine phosphate concentrations were calculated from the change in absorbance (extrapolated to the starting point of the reaction) and the extinction coefficient of NADH. Zs.Nagy (1977) demonstrated that crude TCA or PCA extracts of Mytilus galloprovincialis show a direct oxidative effect on NADH in the test solutions for enzymatic assay of adenosine phosphates (Jaworek et al., 1970a,b), before the necessary enzymes are added. He suggests that this pseudo-reaction may contribute to a very high extent to the ATP or ADP concentrations, measured as a decrease of NADH. The direct oxidative effect on NADH has been observed by Wijsman too (1976a, b). Samples of 100 #1 TCA tissue extract of Mytilus edulis, corresponding to 25 mg wet weight, involved the oxidation of maximal 0.2 nmole NADH/min per ml test solution used for the ATP and ADP/AMP determinations (initial NADH concentration was 141 nmole/ml). When the reaction was initiated with phosphoglycerate kinase, pyruvate kinase or myokinase a rapid decrease in absorbance was noticed corresponding to the oxidation of about 10-60 nmole NADH under the test conditions used. Within 10 min, however, this exponential decrease passed into a linear one with a slope equal to that of the pre-incubation reaction, indicating that oxidation of NADH due to the adenosine phosphates was finished. That is just the reason why the amount of oxidized NADH, due to the relative adenosine phosphate, was determined after extrapolation to the starting point of the reaction, and not by a simple subtraction of the absorption before and after the reaction at any time, which is possible if no pseudo-reaction takes place. This extrapolationmethod is in general use for enzymatic substrate determinations (Bergmeyer, 1974). Therefore the so-called pseudo-reaction cannot explain the great discrepancy between the ATP and ADP concentrations obtained by Zs.-Nagy & Ermini (1972), and Zs.-Nagy (1973) and those of other authors (Beis & Newsholme, 1975; Wijsman, 1976a, b) * Present address: Department of Biology, Free University, Amsterdam, The Netherlands. 313
for marine bivalves. It is not the reason why the ATP content of PCA-extraets was much lower than that of the TCA-extracts in the experiments of Wijsman (1976a). Moreover, this author reports also that ADP concentrations were increased, corresponding to the loss of ATP when PCA-extracts were used. Finally, the decreased ATP content of Mytilus edulis after anoxia (Wijsman, 1976a, b) cannot be caused by a decreased oxidative effect of the TCA extract. Moreover, constant ATP levels in Mytilus edulis during anoxia, assumed by Zs.-Nagy, would contradict his own results with Mytilus galloprovincialis (Zs.-Nagy & Ermini, 1972) and Anodonta cygnea (Zs.-Nagy, 1973).
REFERENCES
BEISI. & NEWSHOLMEE. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem. J. 152, 23-32. BERGMEYER H. U. (1974) Methoden der Enzymatischen Analyse. Vol. 1, p. 112. Verlag Chemic, Weinheim. JAWOREK D., GRUBER W. 8/: BERGMEYER H. U. (1970a) Adenosin-5'-diphosphat und Adenosin-5'-monophosphat. In Methoden der Enzymatischen Analyse (Edited by BERGMEYERH. U.), Vol. 2, pp. 2051-2055. Verlag Chemic, Weinheim. JAWOREK D., GRUBER W. ~¢, BERGMEYER H. U. (1970b). Adenosin-5'-triphosphat. In Methoden der Enzymatischen Analyse (Edited by BERGMEYER H. U.), Vol. 2, pp. 2020-2024. Verlag Chemie, Weinheim. WUSMAN T. C. M. (1976a). Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions. J. comp. Physiol. 107, 129-140. WIJSMAN T. C. M. (1976b). ATP content and mortality in Mytilus edulis L. from different habitats in relation to anaerobiosis. Neth. J. Sea Res. 10, 140-148. Zs.-NAoY I. (1973) Adenosine phosphate concentrations and carbohydrate consumption in the tissues of Anodonta cygnea L. (Mollusca, Pelecypoda) under normal and anoxic conditions. Acta Biochim. Biophys. Acad. Sci. Hung. 8, 143-151. Zs.-NAGY I. (1977) On the validity of enzymatic assay of adenosine phosphates in crude extracts of some marine bivalves. Comp. Biochem. Physiol. 56B, 271-272. Zs.-NAGY I. 86 EgmNI M. (1972) ATP production in the tissues of the bivalve Mytilus galloprovincialis (Pelecypoda) under normal and anoxic conditions. Comp. Biochem. Physiol. 43B, 593-600.
1977, Vol. 58B, p. 313. Pergamon Press. Printed
in Great Britain
SHORT COMMUNICATION RELIABILITY OF THE ENZYMATIC DETERMINATION OF ADENOSINE PHOSPHATES IN CRUDE EXTRACTS OF MARINE BIVALVES THEODORUS C. M. WIJSMAN* Laboratory of Chemical Animal Physiology, 8 Padualaan, Utrecht, The Netherlands (Received 21 March 1977)
Recently the validity of enzymatic assay of adenosine phosphates in crude extracts of some marine bivalves has been questioned (Zs.-Nagy, 1977). The paper of Wijsman (1976a) was used as an example of this doubtful method, as adenosine phosphate concentrations were calculated from the change in absorbance (extrapolated to the starting point of the reaction) and the extinction coefficient of NADH. Zs.Nagy (1977) demonstrated that crude TCA or PCA extracts of Mytilus galloprovincialis show a direct oxidative effect on NADH in the test solutions for enzymatic assay of adenosine phosphates (Jaworek et al., 1970a,b), before the necessary enzymes are added. He suggests that this pseudo-reaction may contribute to a very high extent to the ATP or ADP concentrations, measured as a decrease of NADH. The direct oxidative effect on NADH has been observed by Wijsman too (1976a, b). Samples of 100 #1 TCA tissue extract of Mytilus edulis, corresponding to 25 mg wet weight, involved the oxidation of maximal 0.2 nmole NADH/min per ml test solution used for the ATP and ADP/AMP determinations (initial NADH concentration was 141 nmole/ml). When the reaction was initiated with phosphoglycerate kinase, pyruvate kinase or myokinase a rapid decrease in absorbance was noticed corresponding to the oxidation of about 10-60 nmole NADH under the test conditions used. Within 10 min, however, this exponential decrease passed into a linear one with a slope equal to that of the pre-incubation reaction, indicating that oxidation of NADH due to the adenosine phosphates was finished. That is just the reason why the amount of oxidized NADH, due to the relative adenosine phosphate, was determined after extrapolation to the starting point of the reaction, and not by a simple subtraction of the absorption before and after the reaction at any time, which is possible if no pseudo-reaction takes place. This extrapolationmethod is in general use for enzymatic substrate determinations (Bergmeyer, 1974). Therefore the so-called pseudo-reaction cannot explain the great discrepancy between the ATP and ADP concentrations obtained by Zs.-Nagy & Ermini (1972), and Zs.-Nagy (1973) and those of other authors (Beis & Newsholme, 1975; Wijsman, 1976a, b) * Present address: Department of Biology, Free University, Amsterdam, The Netherlands. 313
for marine bivalves. It is not the reason why the ATP content of PCA-extraets was much lower than that of the TCA-extracts in the experiments of Wijsman (1976a). Moreover, this author reports also that ADP concentrations were increased, corresponding to the loss of ATP when PCA-extracts were used. Finally, the decreased ATP content of Mytilus edulis after anoxia (Wijsman, 1976a, b) cannot be caused by a decreased oxidative effect of the TCA extract. Moreover, constant ATP levels in Mytilus edulis during anoxia, assumed by Zs.-Nagy, would contradict his own results with Mytilus galloprovincialis (Zs.-Nagy & Ermini, 1972) and Anodonta cygnea (Zs.-Nagy, 1973).
REFERENCES
BEISI. & NEWSHOLMEE. A. (1975) The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem. J. 152, 23-32. BERGMEYER H. U. (1974) Methoden der Enzymatischen Analyse. Vol. 1, p. 112. Verlag Chemic, Weinheim. JAWOREK D., GRUBER W. 8/: BERGMEYER H. U. (1970a) Adenosin-5'-diphosphat und Adenosin-5'-monophosphat. In Methoden der Enzymatischen Analyse (Edited by BERGMEYERH. U.), Vol. 2, pp. 2051-2055. Verlag Chemic, Weinheim. JAWOREK D., GRUBER W. ~¢, BERGMEYER H. U. (1970b). Adenosin-5'-triphosphat. In Methoden der Enzymatischen Analyse (Edited by BERGMEYER H. U.), Vol. 2, pp. 2020-2024. Verlag Chemie, Weinheim. WUSMAN T. C. M. (1976a). Adenosine phosphates and energy charge in different tissues of Mytilus edulis L. under aerobic and anaerobic conditions. J. comp. Physiol. 107, 129-140. WIJSMAN T. C. M. (1976b). ATP content and mortality in Mytilus edulis L. from different habitats in relation to anaerobiosis. Neth. J. Sea Res. 10, 140-148. Zs.-NAoY I. (1973) Adenosine phosphate concentrations and carbohydrate consumption in the tissues of Anodonta cygnea L. (Mollusca, Pelecypoda) under normal and anoxic conditions. Acta Biochim. Biophys. Acad. Sci. Hung. 8, 143-151. Zs.-NAGY I. (1977) On the validity of enzymatic assay of adenosine phosphates in crude extracts of some marine bivalves. Comp. Biochem. Physiol. 56B, 271-272. Zs.-NAGY I. 86 EgmNI M. (1972) ATP production in the tissues of the bivalve Mytilus galloprovincialis (Pelecypoda) under normal and anoxic conditions. Comp. Biochem. Physiol. 43B, 593-600.