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In vitro-studies on the anaerobic formation of ethanol by the larvae of Chironomus thummi thummi (Diptera)
Comp. Biochem. Physiol., Vol. 67B. pp. 239 to 242
0305-0491/80/0901-0239502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
I N V I T R O - S T U D I E S O N THE ANAEROBIC
FORMATION OF ETHANOL BY THE LARVAE OF C H I R O N O M U S T H U M M I T H U M M I (DIPTERA) H. WILPS* a n d U. SCH()TTLER Zoologisches Institut der Universitiit Miinster, Lehrstuhl fiir Tierphysiologie, Hindenburgplatz 55, D-4400 MOnster, Federal Republic of Germany (Received 11 February 1980) A b s t r a c t - - l . The anaerobic formation of ethanol by the larvae of Chironomus thummi thummi was investigated in homogenates and isolated mitochondria. 2. It was found that homogenates transform fructose-l,6-bisphosphate into ethanol and acetate. The accumulation of ethanol decreases substantially and the formation of acetate almost ceases when arsenite, an inhibitor of pyruvate dehydrogenase, is present in the incubation medium. 3. The cytosolic fraction of the homogenate was shown to degrade fructose-l,6-bisphosphate to pyruvate only. No ethanol could be detected, although there is high activity of alcohol dehydrogenase in the cytosol. 4. Isolated mitochondria produce large quantities of ethanol from pyruvate during anoxia. This ethanol production was shown to depend on the presence of NADH. It is deduced that this cosubstrate originates from intramitochondrial formation of acetate from pyruvate, which was always found to accumulate alongside ethanol.
Preparation and treatment of homogenates The larvae were homogenized in an equal volume of 20 mM Tris-HCl buffer, pH 7.5, containing 0.3 M sucrose and 5 mM EDTA by means of a Potter-Elvehjem type homogenizer with a loose-fitting Teflon pestle. 15 ml Warburg vessels were used for anaerobic incubation of the homogenates after gassing for 30 min with purified nitrogen. The incubation temperature was 25°C. Each vessel contained 2 mM MgC12, 4 mM KC1, 5 mM potassium phosphate, 2.SmM fructose-l,6-bisphosphate, 5 mM NAD and 3 mM ADP. To some vessels 10 mM arsenite was also added. The total volume of the incubation medium was 3 ml. At the end of the incubation 3 M perchloric acid was added (0.2 ml per ml incubation mixture) and the mixture homogenized with an Ultra-Turrax homogenizer at maximum speed and centrifuged for 10min at 50,0009. The supernatants were neutralized with 5 M potassium carbonate and the precipitated potassium perchlorate removed by centrifugation for 15 rain at 50,000 g.
INTRODUCTION
The larvae of the midge Chironomus thummi thummi live on the surface of the m u d d y b o t t o m sediments of eutrophic ponds, i.e. in a habitat where they may suffer from a lack of oxygen. One of the a d a p t a t i o n s to these conditions is their peculiar mode of glycogen degradation upon anaerobiosis giving rise to ethanol as main end-product. This metabolite is excreted in large quantities into the surrounding water. In addition, alanine, lactate, acetate a n d succinate have been found as m i n o r end-products of the anaerobic metabolism in these larvae (Wilps & Zebe, 1976). E t h a n o l can be formed in two different ways: (1) pyruvate is decarboxylated in the cytosol by pyruvate decarboxylase and the resulting acetaldehyde then functions as an acceptor of hydrogen from N A D H reduced in the oxydation of triose-phosphate. This is the well k n o w n alcoholic fermentation of yeasts. (2) In some bacteria pyruvate is transformed to acetyl-CoA by pyruvate dehydrogenase and subsequently this c o m p o u n d is reduced to ethanol by the c o m b i n e d actions of aldehyde dehydrogenase and alcohol dehydrogenase (Thauer et al., 1977). The objective of this paper was to find out how ethanol is formed by the C h i r o n o m u s larvae a n d to investigate the details of this process. MATERIALS AND METHODS Animals The larvae were reared in the laboratory and used in the second, third and fourth stage. * Present address: Institut fiir Biologie (Zoologie 1), Albertstr. 21a, D-7800 Freiburg i. Br., Federal Republic of Germany. Abbreviations used: ADH--alcohol dehydrogenase; EDTA---ethylenediamine tetraacetic acid; f.w.--fresh weight; GAPDH--glyceraldehyde-3-phosphate dehydrogenase; PDC--pyruvate decarboxylase; PGA--polyethyleneglycoll adipate; Prot--protein.
Preparation and treatment of mitochondria The larvae were homogenized as described above except that the 30-fold volume of the buffer was used. The homogenate was filtered through nylon tissue to remove coarse particles and the filtrate was centrifuged for 20min at 800 g. The supernatant was decanted cautiously, the sediment resuspended and centrifuged again at 800g. Both supernatants were mixed and centrifuged for 20min at 10,000 g. The resulting sediment was resuspended in a fifth of the previous volume and centrifuged again for 20 min at 800g. The supernatant was centrifuged for 15min at 10,000g and this procedure was repeated until all activity of GAPDH, a cytosolic enzyme, was removed. The final sediment was suspended in 150raM KC1 so that 1 ml of the mitochondrial preparation corresponded to 4 g of the larvae.
239
Assay of enzyme activities The activities of the following enzymes were estimated after the disruption of mitochondrial membranes by osmotic shock and homogenized with an Ultra-Turrax at maximum speed: GAPDH according to Biicher et al. (1964);
240
H. WILPS and U. SCHOTTLER
9
•]
Values before incubation
activity and metabolite concentrations were measured after destroying the cells by sonification. Protein determination Protein was estimated by means of a modified biuret method (Beisenherz et al., 1953).
~6 ::L 3
RESULTS I
= 0.
..... J
8
_
[
Arsenit"eadded Fig. 1. Formation of pyruvate, ethanol and acetate by homogenates of Chironomus larvae incubated for 4 hr anaerobically with fructose-l,6-bisphosphate. It was controlled previously, so that homogenates as well as mitochondria of these animals performed metabolites into ethanol proportional at incubation time. The data of this figure and the following ones represent the main values of three to five different experiments. ADH according to Bernt & Gutmann (1974); PDC according to Bergmeyer (1974) and Aldehyd-DH according to Stadtman & Burton (1956). Measurement of metabolites Pyruvate and ethanol were determined enzymatically by standard methods, pyruvate according to Czok & Lamprecht (1974) and ethanol according to Bernt & Gutmann (1974). Acetate was separated by steam distillation and estimated by gas-liquid chromatography (column: glass, length 3m, diameter 3.5mm; filled with Chromosorb AWA 60-80 mesh coated with 20~o PGA and 3~ phosphoric acid; temperatures: oven 140°C, injector 220°C, FID 250°C; carrier gas: N2 at 25 ml/min). Studies of microorganisms 0.1 ml of mitochondrial suspension was incubated on a malt-pepton-agar medium, pH 6.5, for 2 days at 30°C. Subsequently the colonies of microorganisms were incubated separately in Lyria-broth and then analyzed for ADH activity. The incubation procedure for the microorganisms was the same as that used for mitochondria containing 2.5 mM pyruvate or 17.5 mM ethanol as substrates. ADH
8
::L
Homogenates of Chironomus larvae were found to metabolize fructose-1,6-bisphosphate at constant rates for several hours. Therefore, in one experiment concentrated homogenate was incubated anaerobically with fructose-l,6-bisphosphate for 4 hr. As is shown in Fig. 1 large quantities of ethanol and acetate accumulate under these conditions. In the presence of arsenite the formation of ethanol is reduced and only traces of acetate appeared. Instead much pyruvate is found. In a second series of experiments a mitochondrial fraction and a soluble or supernatant fraction were prepared from the homogenate. The efficiency of the separation was monitored by assaying the activity of glyceraldehydephosphate dehydrogenase which occurs only in the cytosol and of alcohol dehydrogenase. As is evident from Fig. 2 the main product of fructose-l,6-bisphosphate metabolization by the supernatant is pyruvate. Only very small quantities of ethanol were found. On the other hand, the mitochondrial fraction transforms pyruvate, which is added as a substrate, mainly into ethanol. The metabolization of pyruvate by the mitochondria was analyzed in the next experiment. The results are shown in Fig. 3. In addition to ethanol large quantities of acetate accumulate. The production of both ethanol and acetate is inhibited completely by arsenite. When alcohol dehydrogenase and N A D H are present in the medium in addition to pyruvate, almost no influence of arsenite on the production of ethanol is observed, while the formation of acetate is suppressed. The activities of three enzymes possibly involved in the production of ethanol were measured in mitochondria which had been treated by osmotic shock.
-
4 =
2 -
Enzymeoctivity ( in % of 1"o'1-ol Qctivity)
o
>o
taJ
n
Cy'l'osol ADH GAPDH
76% 78%
[] ILl
Values b e f o r e Incubation
Mi?ochondria 20% 17%
Fig. 2. Formation of ethanol by the supernatant and mitochondrial fractions of homogenates incubated for 4hr anaerobically. Substrates: supernatant fraction: fructose-l,6-bisphosphate; mitochondrial fraction: pyruvate. The activities of alcohol dehydrogenase and glyceraldehyde dehydrogenase assayed in the fractions are indicated below. (The mitochondrial fraction was treated by osmotic shock prior to the measurem6nts.)
Ethanol formation by Chironomus larvae 2.7
~
.
•~
2.1
1.5
-6 E 09
0.3
Arsenite added
'
(a)
4£ O 1.5
"~ 0.9
_8
:3
8
<
03
Arsenite added"
(b)
Fig. 3. Formation of ethanol and acetate by isolated mitochondria incubated anaerobically for 4 hr. In Fig. 3a only pyruvate was added to the medium, while in Fig. 3b alcohol dehydrogenase and NADH in addition to pyruvate were present. Pyruvate decarboxylase activity was demonstrated to be high. Some alcohol dehydrogenase was also found to be present in the mitochondria, which most probably did not originate from the cytosol. No aldehyde dehydrogenase was detected. In addition to this, added Acetyl-CoA was not performed into ethanol, when the mitochondrial membranes had been made permeable for this metabolite. There is the possibility that symbiotic or parasitic microorganisms might have been present in the homogenates or in the mitochondrial preparations, which could be responsible for at least some of the results reported, especially for the formation of ethanol. Therefore, such preparations were checked for contaminating microorganisms. It was found that indeed there were bacteria averaging 10 5 germs per millilitre. Four different species of microorganism were isolated. However, only one of these contained alcohol dehydrogenase activity. When the microorganisms were incubated under exactly the same conditions as applied to the mitochondrial preparations, only small quantities of ethanol were found to have been formed from pyruvate. Using ethanol as a substrate instead of pyruvate resulted in the disappearance of a large part of this compound which apparently was metabolized. DISCUSSION
In the present paper further evidence is presented to show that ethanol is, in fact, the anaerobic endproduct of the catabolism of the midge larvae and does not arise secondarily by the metabolic activity of microorganisms. Although some bacteria were found to be present in the preparations, these either do not contain alcohol dehydrogenase and, therefore, cannot be responsible for the formation of ethanol, or they
241
metabolize ethanol instead of accumulating it as an end-product. The formation of ethanol from fructose-l,6bisphosphate was found to occur also in homogenates of Chironomus larvae which, in addition, produced large quantities of acetate. The accumulation of acetate was completely inhibited by arsenite, while the inhibitory effect on the production of ethanol was much smaller. This shows that pyruvate dehydrogenase must somehow be involved in the formation of both metabolites. Since some ethanol is still produced while the formation of acetate has ceased, ethanol cannot arise from acetyl CoA, i.e. the pathway of ethanol formation known to exist in some microorganisms is ruled out in Chironomus larvae. This is in accord with the finding that there is no aldehyde dehydrogenase detectable in the larvae. In yeast cells glucose is degraded to ethanol by enzymes of the cytosol. Evidently this does not hold true for the Chironomus larvae, for in the soluble fraction of the homogenate pyruvate is the endproduct of the breakdown of fructose-l,6bisphosphate, although most of the alcohol dehydrogenase activity was found to be present there. This may be explained by the observation, that decarboxylation of pyruvate does proceed only in the mitochondria. Therefore, the presence of these particles are absolutely necessary for the formation of ethanol. Furthermore, mitochondria of Chironomus larvae which are free of contaminating components of the cytosol accumulate ethanol when they are incubated with pyruvate, which means that they must contain alcohol dehydrogenase. The presence of this enzyme in the mitochondria could be demonstrated by the conventional optical test after the mitochondria membranes had been made permeable. The NADH required for the reduction of acetaldehyde to ethanol is supposedly supplied by the oxidative decarboxylation of pyruvate. Therefore, inhibition of pyruvate dehydrogenase affects the formation of ethanol, because it cuts off the source of NADH. When NADH is supplied in the medium together with alcohol dehydrogenase, arsenite does not have an inhibitory effect on the metabolization of pyruvate to ethanol as was shown experimentally. Pyruvate is apparently degraded to acetaldehyde by a decarboxylating activity originating in the mitochondria. Acetaldehyde, after diffusing into the medium, is reduced there to ethanol when alcohol dehydrogenase and NADH are present. The decarboxylating activity of the mitochondria was demonstrated directly. Although great caution is necessary in drawing conclusions from the results of in vitro experiments in order to explain metabolic patterns in vivo, it appears safe to state the formation of ethanol in the Chironomus larvae proceeds in a way which differs in detail from the alcoholic fermentation in yeast cells. In the insect larvae this metabolic speciality probably has developed rather recently. It seems obvious that, in addition, the conditions of anaerobic utilization of endogenous glycogen by a highly differentiated metazoan must be quite unlike those found in single cells which metabolize exogenous substrates. In spite of the low yield of ATP in the degradation of glycogen to ethanol, the production of this metab-
242
H. WILPS and U. SCHOTTLER
olite may prove advantageous when compared to other end-products, since ethanol easily permeates biological membranes. Therefore, it can be excreted without difficulty. This means that there is no danger of large quantities of end-products accumulating and causing an osmotic imbalance in an organism living in a hypoosmotic medium.
Acknowledgements--We are grateful to E. Zebe for his critical advice. This work was supported by Deutsche Forschungsgemeinschaft grant Gr 456/5 and 456/6.
REFERENCES
BEISENHERZG., BOLTZE H. J., BOCHERT., CZOK R., GARBADE K. n., MEYER-ARENDTE. & PFLEIDERERG. (1953) Diphosphofructose-Aldolase, PhosphoglycerinaldehydDehydrogenase, Milchs~iure-Dehydrogenase, Glycerophosphat-Dehydrogenase und Pyruvat-Kinase aus Kaninchenmuskel in einem Arbeitsgang. Z. Naturf. gb, 555-577. BERGMEYER H. U., GAWEHN K. 8£ GRABL M. (1974) Alkohol-Dehydrogenase. In Methoden der enzyrnatischen
Analyse, 3rd edn. (Edited by BERGMEYER H. U.) pp. 454-458. Verlag Chemie, Weinheim. BERNT E. & GUTMANN I. (1974) A,thanol. Bestimmung mit Alkohol-Dehydrogenase und NAD. In Methoden der enzymatischen Analyse, 3rd edn. (Edited by BERGMEYER H. U.) pp. 1545 1548. Verlag Chemie, Weinheim. BUCHER T., PETTE D. & LUH W. (1964) Einfache und zusammengesetzte optische Tests mit Pyridinnucleotiden. In Hoppe-Seyler/Thierfelder, Handbuch der physiologisch- und pathologisch-chemischen Analyse (Edited by LAND K. & LEHNARTZ E.) 10. Aufl., Bd. VI/A, pp. 292-339. Verlag Springer, Berlin. CZOK R. & LAMPRECHT W. (1974) Pyruvat, Phosphoenolpyruvat und D-Glycerat-2-phosphat. In Methoden der enzymatischen Analyse (Edited by BERGMEYERH. U.) pp. 1491-1496. Verlag Chemie, Weinheim. STADTMAN E. R. • BURTON R. M. (1955) Aldehyde Dehydrogenase from Clostridium kluyveri. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.) Vol. I, pp. 518-523. Academic Press, New York. THAUER R. K., JUNGERMANN K. 8~ DECKER K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bact. Rev. 41, 100-180. WlLPS H. & ZEBE E. (1976) The end-products of anaerobic carbohydrate metabolism in the larvae of Chironomus thumrni thummi. J. comp. Physiol. 112, 263-272.
0305-0491/80/0901-0239502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
I N V I T R O - S T U D I E S O N THE ANAEROBIC
FORMATION OF ETHANOL BY THE LARVAE OF C H I R O N O M U S T H U M M I T H U M M I (DIPTERA) H. WILPS* a n d U. SCH()TTLER Zoologisches Institut der Universitiit Miinster, Lehrstuhl fiir Tierphysiologie, Hindenburgplatz 55, D-4400 MOnster, Federal Republic of Germany (Received 11 February 1980) A b s t r a c t - - l . The anaerobic formation of ethanol by the larvae of Chironomus thummi thummi was investigated in homogenates and isolated mitochondria. 2. It was found that homogenates transform fructose-l,6-bisphosphate into ethanol and acetate. The accumulation of ethanol decreases substantially and the formation of acetate almost ceases when arsenite, an inhibitor of pyruvate dehydrogenase, is present in the incubation medium. 3. The cytosolic fraction of the homogenate was shown to degrade fructose-l,6-bisphosphate to pyruvate only. No ethanol could be detected, although there is high activity of alcohol dehydrogenase in the cytosol. 4. Isolated mitochondria produce large quantities of ethanol from pyruvate during anoxia. This ethanol production was shown to depend on the presence of NADH. It is deduced that this cosubstrate originates from intramitochondrial formation of acetate from pyruvate, which was always found to accumulate alongside ethanol.
Preparation and treatment of homogenates The larvae were homogenized in an equal volume of 20 mM Tris-HCl buffer, pH 7.5, containing 0.3 M sucrose and 5 mM EDTA by means of a Potter-Elvehjem type homogenizer with a loose-fitting Teflon pestle. 15 ml Warburg vessels were used for anaerobic incubation of the homogenates after gassing for 30 min with purified nitrogen. The incubation temperature was 25°C. Each vessel contained 2 mM MgC12, 4 mM KC1, 5 mM potassium phosphate, 2.SmM fructose-l,6-bisphosphate, 5 mM NAD and 3 mM ADP. To some vessels 10 mM arsenite was also added. The total volume of the incubation medium was 3 ml. At the end of the incubation 3 M perchloric acid was added (0.2 ml per ml incubation mixture) and the mixture homogenized with an Ultra-Turrax homogenizer at maximum speed and centrifuged for 10min at 50,0009. The supernatants were neutralized with 5 M potassium carbonate and the precipitated potassium perchlorate removed by centrifugation for 15 rain at 50,000 g.
INTRODUCTION
The larvae of the midge Chironomus thummi thummi live on the surface of the m u d d y b o t t o m sediments of eutrophic ponds, i.e. in a habitat where they may suffer from a lack of oxygen. One of the a d a p t a t i o n s to these conditions is their peculiar mode of glycogen degradation upon anaerobiosis giving rise to ethanol as main end-product. This metabolite is excreted in large quantities into the surrounding water. In addition, alanine, lactate, acetate a n d succinate have been found as m i n o r end-products of the anaerobic metabolism in these larvae (Wilps & Zebe, 1976). E t h a n o l can be formed in two different ways: (1) pyruvate is decarboxylated in the cytosol by pyruvate decarboxylase and the resulting acetaldehyde then functions as an acceptor of hydrogen from N A D H reduced in the oxydation of triose-phosphate. This is the well k n o w n alcoholic fermentation of yeasts. (2) In some bacteria pyruvate is transformed to acetyl-CoA by pyruvate dehydrogenase and subsequently this c o m p o u n d is reduced to ethanol by the c o m b i n e d actions of aldehyde dehydrogenase and alcohol dehydrogenase (Thauer et al., 1977). The objective of this paper was to find out how ethanol is formed by the C h i r o n o m u s larvae a n d to investigate the details of this process. MATERIALS AND METHODS Animals The larvae were reared in the laboratory and used in the second, third and fourth stage. * Present address: Institut fiir Biologie (Zoologie 1), Albertstr. 21a, D-7800 Freiburg i. Br., Federal Republic of Germany. Abbreviations used: ADH--alcohol dehydrogenase; EDTA---ethylenediamine tetraacetic acid; f.w.--fresh weight; GAPDH--glyceraldehyde-3-phosphate dehydrogenase; PDC--pyruvate decarboxylase; PGA--polyethyleneglycoll adipate; Prot--protein.
Preparation and treatment of mitochondria The larvae were homogenized as described above except that the 30-fold volume of the buffer was used. The homogenate was filtered through nylon tissue to remove coarse particles and the filtrate was centrifuged for 20min at 800 g. The supernatant was decanted cautiously, the sediment resuspended and centrifuged again at 800g. Both supernatants were mixed and centrifuged for 20min at 10,000 g. The resulting sediment was resuspended in a fifth of the previous volume and centrifuged again for 20 min at 800g. The supernatant was centrifuged for 15min at 10,000g and this procedure was repeated until all activity of GAPDH, a cytosolic enzyme, was removed. The final sediment was suspended in 150raM KC1 so that 1 ml of the mitochondrial preparation corresponded to 4 g of the larvae.
239
Assay of enzyme activities The activities of the following enzymes were estimated after the disruption of mitochondrial membranes by osmotic shock and homogenized with an Ultra-Turrax at maximum speed: GAPDH according to Biicher et al. (1964);
240
H. WILPS and U. SCHOTTLER
9
•]
Values before incubation
activity and metabolite concentrations were measured after destroying the cells by sonification. Protein determination Protein was estimated by means of a modified biuret method (Beisenherz et al., 1953).
~6 ::L 3
RESULTS I
= 0.
..... J
8
_
[
Arsenit"eadded Fig. 1. Formation of pyruvate, ethanol and acetate by homogenates of Chironomus larvae incubated for 4 hr anaerobically with fructose-l,6-bisphosphate. It was controlled previously, so that homogenates as well as mitochondria of these animals performed metabolites into ethanol proportional at incubation time. The data of this figure and the following ones represent the main values of three to five different experiments. ADH according to Bernt & Gutmann (1974); PDC according to Bergmeyer (1974) and Aldehyd-DH according to Stadtman & Burton (1956). Measurement of metabolites Pyruvate and ethanol were determined enzymatically by standard methods, pyruvate according to Czok & Lamprecht (1974) and ethanol according to Bernt & Gutmann (1974). Acetate was separated by steam distillation and estimated by gas-liquid chromatography (column: glass, length 3m, diameter 3.5mm; filled with Chromosorb AWA 60-80 mesh coated with 20~o PGA and 3~ phosphoric acid; temperatures: oven 140°C, injector 220°C, FID 250°C; carrier gas: N2 at 25 ml/min). Studies of microorganisms 0.1 ml of mitochondrial suspension was incubated on a malt-pepton-agar medium, pH 6.5, for 2 days at 30°C. Subsequently the colonies of microorganisms were incubated separately in Lyria-broth and then analyzed for ADH activity. The incubation procedure for the microorganisms was the same as that used for mitochondria containing 2.5 mM pyruvate or 17.5 mM ethanol as substrates. ADH
8
::L
Homogenates of Chironomus larvae were found to metabolize fructose-1,6-bisphosphate at constant rates for several hours. Therefore, in one experiment concentrated homogenate was incubated anaerobically with fructose-l,6-bisphosphate for 4 hr. As is shown in Fig. 1 large quantities of ethanol and acetate accumulate under these conditions. In the presence of arsenite the formation of ethanol is reduced and only traces of acetate appeared. Instead much pyruvate is found. In a second series of experiments a mitochondrial fraction and a soluble or supernatant fraction were prepared from the homogenate. The efficiency of the separation was monitored by assaying the activity of glyceraldehydephosphate dehydrogenase which occurs only in the cytosol and of alcohol dehydrogenase. As is evident from Fig. 2 the main product of fructose-l,6-bisphosphate metabolization by the supernatant is pyruvate. Only very small quantities of ethanol were found. On the other hand, the mitochondrial fraction transforms pyruvate, which is added as a substrate, mainly into ethanol. The metabolization of pyruvate by the mitochondria was analyzed in the next experiment. The results are shown in Fig. 3. In addition to ethanol large quantities of acetate accumulate. The production of both ethanol and acetate is inhibited completely by arsenite. When alcohol dehydrogenase and N A D H are present in the medium in addition to pyruvate, almost no influence of arsenite on the production of ethanol is observed, while the formation of acetate is suppressed. The activities of three enzymes possibly involved in the production of ethanol were measured in mitochondria which had been treated by osmotic shock.
-
4 =
2 -
Enzymeoctivity ( in % of 1"o'1-ol Qctivity)
o
>o
taJ
n
Cy'l'osol ADH GAPDH
76% 78%
[] ILl
Values b e f o r e Incubation
Mi?ochondria 20% 17%
Fig. 2. Formation of ethanol by the supernatant and mitochondrial fractions of homogenates incubated for 4hr anaerobically. Substrates: supernatant fraction: fructose-l,6-bisphosphate; mitochondrial fraction: pyruvate. The activities of alcohol dehydrogenase and glyceraldehyde dehydrogenase assayed in the fractions are indicated below. (The mitochondrial fraction was treated by osmotic shock prior to the measurem6nts.)
Ethanol formation by Chironomus larvae 2.7
~
.
•~
2.1
1.5
-6 E 09
0.3
Arsenite added
'
(a)
4£ O 1.5
"~ 0.9
_8
:3
8
<
03
Arsenite added"
(b)
Fig. 3. Formation of ethanol and acetate by isolated mitochondria incubated anaerobically for 4 hr. In Fig. 3a only pyruvate was added to the medium, while in Fig. 3b alcohol dehydrogenase and NADH in addition to pyruvate were present. Pyruvate decarboxylase activity was demonstrated to be high. Some alcohol dehydrogenase was also found to be present in the mitochondria, which most probably did not originate from the cytosol. No aldehyde dehydrogenase was detected. In addition to this, added Acetyl-CoA was not performed into ethanol, when the mitochondrial membranes had been made permeable for this metabolite. There is the possibility that symbiotic or parasitic microorganisms might have been present in the homogenates or in the mitochondrial preparations, which could be responsible for at least some of the results reported, especially for the formation of ethanol. Therefore, such preparations were checked for contaminating microorganisms. It was found that indeed there were bacteria averaging 10 5 germs per millilitre. Four different species of microorganism were isolated. However, only one of these contained alcohol dehydrogenase activity. When the microorganisms were incubated under exactly the same conditions as applied to the mitochondrial preparations, only small quantities of ethanol were found to have been formed from pyruvate. Using ethanol as a substrate instead of pyruvate resulted in the disappearance of a large part of this compound which apparently was metabolized. DISCUSSION
In the present paper further evidence is presented to show that ethanol is, in fact, the anaerobic endproduct of the catabolism of the midge larvae and does not arise secondarily by the metabolic activity of microorganisms. Although some bacteria were found to be present in the preparations, these either do not contain alcohol dehydrogenase and, therefore, cannot be responsible for the formation of ethanol, or they
241
metabolize ethanol instead of accumulating it as an end-product. The formation of ethanol from fructose-l,6bisphosphate was found to occur also in homogenates of Chironomus larvae which, in addition, produced large quantities of acetate. The accumulation of acetate was completely inhibited by arsenite, while the inhibitory effect on the production of ethanol was much smaller. This shows that pyruvate dehydrogenase must somehow be involved in the formation of both metabolites. Since some ethanol is still produced while the formation of acetate has ceased, ethanol cannot arise from acetyl CoA, i.e. the pathway of ethanol formation known to exist in some microorganisms is ruled out in Chironomus larvae. This is in accord with the finding that there is no aldehyde dehydrogenase detectable in the larvae. In yeast cells glucose is degraded to ethanol by enzymes of the cytosol. Evidently this does not hold true for the Chironomus larvae, for in the soluble fraction of the homogenate pyruvate is the endproduct of the breakdown of fructose-l,6bisphosphate, although most of the alcohol dehydrogenase activity was found to be present there. This may be explained by the observation, that decarboxylation of pyruvate does proceed only in the mitochondria. Therefore, the presence of these particles are absolutely necessary for the formation of ethanol. Furthermore, mitochondria of Chironomus larvae which are free of contaminating components of the cytosol accumulate ethanol when they are incubated with pyruvate, which means that they must contain alcohol dehydrogenase. The presence of this enzyme in the mitochondria could be demonstrated by the conventional optical test after the mitochondria membranes had been made permeable. The NADH required for the reduction of acetaldehyde to ethanol is supposedly supplied by the oxidative decarboxylation of pyruvate. Therefore, inhibition of pyruvate dehydrogenase affects the formation of ethanol, because it cuts off the source of NADH. When NADH is supplied in the medium together with alcohol dehydrogenase, arsenite does not have an inhibitory effect on the metabolization of pyruvate to ethanol as was shown experimentally. Pyruvate is apparently degraded to acetaldehyde by a decarboxylating activity originating in the mitochondria. Acetaldehyde, after diffusing into the medium, is reduced there to ethanol when alcohol dehydrogenase and NADH are present. The decarboxylating activity of the mitochondria was demonstrated directly. Although great caution is necessary in drawing conclusions from the results of in vitro experiments in order to explain metabolic patterns in vivo, it appears safe to state the formation of ethanol in the Chironomus larvae proceeds in a way which differs in detail from the alcoholic fermentation in yeast cells. In the insect larvae this metabolic speciality probably has developed rather recently. It seems obvious that, in addition, the conditions of anaerobic utilization of endogenous glycogen by a highly differentiated metazoan must be quite unlike those found in single cells which metabolize exogenous substrates. In spite of the low yield of ATP in the degradation of glycogen to ethanol, the production of this metab-
242
H. WILPS and U. SCHOTTLER
olite may prove advantageous when compared to other end-products, since ethanol easily permeates biological membranes. Therefore, it can be excreted without difficulty. This means that there is no danger of large quantities of end-products accumulating and causing an osmotic imbalance in an organism living in a hypoosmotic medium.
Acknowledgements--We are grateful to E. Zebe for his critical advice. This work was supported by Deutsche Forschungsgemeinschaft grant Gr 456/5 and 456/6.
REFERENCES
BEISENHERZG., BOLTZE H. J., BOCHERT., CZOK R., GARBADE K. n., MEYER-ARENDTE. & PFLEIDERERG. (1953) Diphosphofructose-Aldolase, PhosphoglycerinaldehydDehydrogenase, Milchs~iure-Dehydrogenase, Glycerophosphat-Dehydrogenase und Pyruvat-Kinase aus Kaninchenmuskel in einem Arbeitsgang. Z. Naturf. gb, 555-577. BERGMEYER H. U., GAWEHN K. 8£ GRABL M. (1974) Alkohol-Dehydrogenase. In Methoden der enzyrnatischen
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