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Studies on the anaerobic energy metabolism in the foot muscle of marine gastropod Patella caerulea (L.)
Comp. Biochem. Physiol. Vol. 95B, No. 3, pp. 493-500, 1990 Printed in Great Britain
0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc
STUDIES ON THE ANAEROBIC ENERGY METABOLISM IN THE FOOT MUSCLE OF MARINE GASTROPOD P A T E L L A C A E R U L E A (L.) BASILE MICHAELIDIS and ISIDOROSBEIS* Laboratory of Animal Physiology, Dept. of Zoology, School of Science, University of Thessaloniki, Thessaloniki 54006, Greece (Received 4 July 1989)
Abstract--1. The foot muscle of P. caerulea has a complete sequence of glycolytic enzymes. The low activity of hexokinase, in comparison with the activities of glycogen phosphorylase and phosphofructokinase, indicate that glycogen is the main fuel oxidized. 2. The reduction of aspartate content in combination with the accumulation of alanine and the presence of considerable activities of glutamate-oxaloacetate transaminase and glutamate~pyruvate transaminase indicates a coupled metabolism of glycogen and aspartate during exposure to air. 3. From the changes in the concentration of the metabolites during exposure to air it appears that up to the second hour of anaerobiosis alanine, lactate and glutamate are the end-products which accumulate in the foot muscle of P. caerulea, whereas from the second to the fourth hour only succinate and alanine accumulate. 4. The low activities of the Krebs cycleenzymesas wellas the absence of ~t-ketoglutaratedehydrogenase activity suggest that the Krebs cycle is not in operation. 5. The absence of opine dehydrogenases shows that the end products octopine, alanopine and strombine are not accumulated in the foot muscle under anaerobiosis. 6. The high activity of malate dehydrogenase in the direction of malate formation in combination with the low activity of ~-lactate dehydrogenase and the absence of opine dehydrogenases suggests that the former dehydrogenase is coupled 1: 1 to glyceraldehyde-3-phosphatedehydrogenase.
INTRODUCTION
It is widely observed that intertidal molluscs display impressive anaerobic capacities and that they can sustain prolonged anoxia exposures. The most widely investigated group, relating to the overall energy metabolism as well as to the mode of energy production during anaerobiosis, is the bivalve molluscs. In these animals, glycogen and aspartate serve as the main source for energy production (ATP) and instead of lactate, other end-products are accumulated in their tissues during anaerobiosis, the most important being succinate, alanine, octopine, alanopine, strombine and the volatile acids propionate and acetate (de Zwaan, 1983). In contrast to vertebrates, anaerobiosis does not cause any marked increase in the rate of glycogen utilization (no Pasteur effect) in the tissues of bivalve molluscs. The lack of Pasteur effect is mainly due to the drastic drop of energy demand which is observed in their tissues during anoxia (Ebberink et aL, 1979; de Zwaan and Putzer, 1985). This, in combination with the higher energetic efficiency due to the accumulation of succinate and propionate, which is the main energy yielding mechanism during anoxia (Livingstone, 1982), enables them to withstand long periods of exposure to air. As in bivalves, anaerobiosis also occurs in intertidal gastropods (Livingstone and de Zwaan, 1983). *Author to whom correspondence should be addressed.
In these animals a number of anaerobic end-products have been detected, namely lactate, octopine, alanine, succinate, acetate and propionate (de Zwaan, 1983). In contrast to well documented studies of bivalves, however, the knowledge on the overall energy metabolism of marine gastropods is rather limited and the mechanisms controlling the accumulation of end-products in marine gastropods are far from clear. The accumulation of different end-products in marine gastropods during anaerobiosis is variable among species and it appears that it is dependent on their pattern of intertidal zonation (Wieser, 1980; Kooijman et al., 1982). In an effort to study the overall metabolism as well as the anaerobic energy metabolism in the tissues of marine gastropods, we estimated the maximum activity of the enzymes involved in the glycolytic sequence and the Krebs cycle, as well as the enzymes related to the overall energy metabolism in the foot muscle of the Mediterranean intertidal gastropod, Patella caerulea. In addition, in order to find out which metabolic routes operate and hence which end-products accumulate in the foot muscle of P. caerulea during anaerobiosis, we studied the changes in the concentrations of glycolytic and Krebs cycle metabolites as well as the free amino acids, aspartate, alanine and glutamate, in the foot muscle during different periods (0, 1, 2, 4, 8 and 16 hr) ofP. caerulea exposure to air.
493
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BASILEMICHAELIDIS and ISIDOROSBE1S MATERIALS AND METHODS
Animals
Limpets (Patella caerulea) were collected from the shores of Chalkidiki Peninsula, near Thessaloniki and were kept in circulating sea water at about 17°C. They were used 48-72 hr after arrival. Chemicals and enzymes
The substrates, enzymes and coenzymes were purchased from Sigma Chemical (St. Louis, USA). All other chemicals were purchased from Serva (Heidelberg, FRG). Preparation o f homogenates
Muscles were dissected as quickly as possible and were cut into small pieces before homogenization. Muscles were homogenized in a ground glass homogenizer with 5 ~ 0 volumes of extraction medium. The extraction medium for all enzymes except for phosphorylase, phosphofructokinase (PFK), citrate synthetase, NAD+(P) isocitrate dehydrogenase, ~-ketoglutarate dehydrogenase, creatine and arginine phosphokinase and myofibrillar ATPase, contained 50 mM triethanolamine, 1 mM EDTA, 2 mM MgC12 and 30raM mercaptoethanol adjusted to pH 7.4 with KOH (Crabtree et al., 1979). For phosphorylase the extraction medium consisted of 35 mM glycerol-2-phosphate, 20 mM NaF, 1 mM EDTA and 30 mM mercaptoethanol at pH 6.2 (Cornblath et al., 1963); that for PFK contained 70mM Tris-HC1, 1 mM EDTA and 5 mM MgSO 4 at pH 8.2 (Opie and Newsholme, 1967); that for citrate synthetase contained 25 mM Tris-HC1 and 1 mM EDTA at pH 7.4 (Alp et al., 1976); that for NAD + isocitrate dehydrogenase contained 50 mM triethanolamine, l mM EDTA, 2 mM ADP, 3 mM MgC12 and 30 mM mercaptoethanol at pH 7.4; for NADP isocitrate dehydrogenase the same extraction medium was used except that it did not contain ADP (Alp et al., 1976). The extraction medium for c~-ketoglutarate dehydrogenase contained 50 mM triethanolamine, 2 mM EDTA, 30 mM mercaptoethanol, 2 mM glycerol and 1 mM ~-ketoglutarate at pH 7.4 (Read et al., 1977). For creatine and arginine phosphokinase the extraction medium contained 50mM Pipes, 4 m M dithiothreitol and 1 mM EDTA at pH 7.0 (Zammit and Newsholme, 1976). For myofibrillar ATPase activity the preparation of homogenate was according to the method of Szent-Gy6rgyi et al. (1971). Homogenates were centrifuged briefly at low speed (600g) to sediment cell debris, in order to minimize turbidity in the cuvette. After the homogenates were sonicated for two 15-sec periods with the microprobe of an MSH conicator operating at an amplitude of 6/~m, the following enzymes were assayed: all enzymes of the Krebs cycle, PEP-carboxykinase, glutamate-oxaloacetate, transaminase (GOT), glutamate pyruvate transaminase (GPT), nucleoside diphosphate kinase, glutamate dehydrogenase and malic enzyme. Assay of enzyme activities
All enzymes were measured at 25°C using a Gilford recording spectrophotometer by following the change in O.D. caused by the oxidation or reduction of NAD+(P) (H). The methods by which glycolytic enzymes, fructose-1,6-bisphosphate (E.C. 3.1.3.11), glucose-6-phosphate dehydrogenase (E.C. 1.11.1.6), gluconate-6-phosphate dehydrogenase (E.C.I. 1.I.44) and PEP-carboxykinase (E.C. 4.1.1.32) were measured were similar to those described by Barrett and Beis (1973). Pyruvate carboxylase (E.C. 6.4.1.1) was measured according to Martin and Denton (1970); citrate synthetase (E.C. 4.2.1.3) according to Srere et al. (1963); aconitase (E.C. 4.2.1.3) according to Fansler and Lowenstein (1969); NAD + isocitrate dehydrogenase (E.C. 1.1.1.41)
and NADP isocitrate dehydrogenase according to Alp et al. (1976); ~-ketoglutarate dehydrogenase (E.C. 1.2.4.2) according to Read et al. (1977); succinyl-CoA synthetase (E.C. 6.2.1.4) according to Cha (1969); succinate dehydrogenase (E.C. 1.3.99.1) according to Slater and Bonner (1952); fumarase (E.C. 4.2.1.2) according to Racker (1950); malate dehydrogenase (E.C. 1.1.1.37) according to Shonk and Boxer (1964); malic enzyme (E.C. 1.1.1.38) according to Ochoa (1955); octopine dehydrogenase (E.C. 1.5.11) according to Zammit and Newsholme (1976); strombine dehydrogenase (E.C. 1.5.1.x) and alanopine dehydrogenase (E.C. 1.5.1 .x) according to de Zwaan and Zurburg (1981); arginine phosphokinase (E.C. 2.7.3.2) according to Beenakkers (1969); creatine phosphokinase (E.C. 2.7.3.2) was assayed in the same way as arginine phosphokinase except that creatine was used instead of arginine; glutamate dehydrogenase (E.C. 1.4.1.3) according to Schmidt (1963); GOT (E.C. 2.6.1.1) and GPT (E.C. 2.6.1.2) according to Bergmeyer and Bernt (1974a,b); nucleoside diphosphate kinase (E.C. 2.7.4.6) according to Parks and Agarwal (1973); myofibrillar ATPase (E.C. 3.6.1.3) according to Szent-Gy6rgi et al. (1971). Experimental procedure o f anaerobiosis and preparation q f tissue extracts
Groups of six individuals were exposed to air for 1, 2, 4, 8 and 16 hr at 15-18°C. After each period of exposure to air the foot muscles were rapidly dissected and immediately freeze clamped with the aid of aluminium tongs cooled in liquid nitrogen. From dissecting to freezing the muscles, the procedure was completed in 8-10 sec. The frozen muscles were separated in a percussion mortar at -70°C and the powdered muscles were extracted by adding 4~5 volumes of frozen HC104 (10% w/v). The precipitated protein was removed by centrifugation at 4000g for 10min and the supernatants were neutralized with 3 M KHCO 3. The precipitated potassium perchlorite was removed by centrifugation as above and the supernatants were taken for determination of metabolite concentrations. Determination o f metabolites
All metabolites were measured spectrophotometrically by using enzymatic systems coupled to NAD+(H) or NADP(H). (i) Metabolites o f glycolysis. Glucose was estimated by the method of Kaplan (1957); glucose-6-phosphate and fructose-6-phosphate were estimated in the same cuvette by the method of Hohorst (1963); fructose-l,6-diphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate were estimated in the same cuvette by the method of Bucher and Hohorst (1963); 1,3-phosphoglycerate by the method of Negelein (1974); 3-phosphoglycerate by the method of Czok (1974); pyruvate, phosphoenolpyruvate (PEP) and 2-phosphoglycerate were estimated in the same cuvette by the method of Bucher et al. (1963); D-lactate by the method of Gutman and Wahlefed (1974a) by using D-LDH. (ii) Metabolites o f the Krebs cycle. Malate was determined by the method of Gutman and Wahlefeld (1974b); oxaloacetate by the method of Wahlefeld (1974); succinate by the method of Michal et al. (1976); a-ketoglutarate by the method of Bergmeyer and Bernt (1974c). (iii) Free amino acids. Aspartate was measured by the method of Bergmeyer et al. (1974); alanine by the method of Williamson (1974); glutamate by the method of Bergmeyer and Bernt (1974d). (iv) Adenosines phosphates. Adenosine triphosphate (ATP) was estimated by the method of Lamprecht and Trautschold (1963); adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were estimated in the same cuvette by the method of Adam (1963).
A n a e r o b i c m e t a b o l i s m in Patella muscle RESULTS
Table 2. The maximum activities of enzymes of the Krebs cycle in the foot muscle of P. caerulea
Enzyme activities The activities of all enzymes studied are presented in Tables 1-3. Noteworthy is the very low activity of hexokinase (0.47 ___0.05/~mol/min/g fresh wt) compared to that of phosphorylase (3.48 +0.23 #mol/min/g fresh wt) and PFK (7.93+0.65 /~mol/min/g fresh wt) and the presence of both PK and PEP-carboxykinase. The activity of D-LDH measured was very low (Table 1), whereas no activity of octopine, alanopine and strombine dehydrogenase could be detected (Table 3). The activities of the enzymes of the Krebs cycle, with the exception of malate dehydrogenase measured in the direction of oxaloacetate reduction, are low (Table 2). In addition, no activity of ct-ketaglutarate dehydrogenase could be detected. In Table 3 the activities of some enzymes related to the glycolytic pathway and the overall energy metabolism are given. From the measured enzymes nucleoside diphosphate kinase (in the direction of oxaloacetate reduction) gave the highest activity, whereas no activities of creatine phosphokinase and pyruvate carboxylase were detectable. Changes in the concentrations of metabolites during anaerobiosis The changes in the concentrations of different metabolites in the foot muscle of P. caerulea during various periods of anaerobiosis are given in Figs 1 and 2 for glycolytic metabolites, Fig. 3 for Krebs cycle metabolites, Fig. 4 for amino acids and Fig. 5 for adenosine phosphates. (i) Glycolytic metabolites. During anaerobiosis the concentrations of all glycolytic metabolites, except those of glucose, PEP and D-lactate, were altered in about the same way: they increased up to the fourth hour of exposure to air and then fell until the eighth hour of exposure. From then, up to the sixteenth hour of anaerobiosis metabolite concentrations were stabilized at/or below control level (0 hr anaerobiosis) (Figs 1 and 2). The concentration of glucose remained almost constant at all stages of anaerobiosis (Fig. 1a), whereas that of PEP decreased in the first two hours and after a slight increase at four hours of anaerobiosis remained almost constant below control level (Fig. 2d). D-Lactate concentration showed a slight Table 1. The maximum activities of glycolytic enzymes in the foot muscle of P. caerulea Enzyme Hexokinase Pbosphorylase Phosphoglucomutase Phosphoglucose isomerase Phosphofructokinase Aldolase Triosephosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Phosphogiycerate kinase Phosphoglyceromutase Enolase Pyruvate kinase D-Lactate dehydrogenase
495
Activity 0.47 _+ 0.50 (10) 3.48 ± 0.23 (7) 15.32 ± 0.90 (8) 30.93 ± 1.67 (10) 7.93 ± 0.65 (7) 13.65 ± 0.86 (9) 158.53 ± 3.83 (6) 39.58 ± 3.26 (10) 12.58 ± 3.26 (9) 15.23 ± 1.34 (8) 9.55 ± 0.75 (10) 11.13 4- 0.73 (6) 1.01 ± 0.20 (10)
The enzyme activities are expressed as #mol/min/g fresh wt at 25°C and are given as means _+ SEM. The number of assays performed for each enzyme is shown in parentheses.
Enzyme Citrate synthetase Aconitase NAD + isocitrate dehydrogenase NADP isocitrate dehydrogenase ~t-Ketoglutarate dehydrogenase Succinyl-CoA synthetase Succinate dehydrogenase Eumarase Malate dehydrogenase (a) Oxaloacetate to malate (b) Malate to oxaloacetate
Activity 1.22 ± 0.10 (10) 0.83 _ 0.06 (10)
0.13 _+0.01 (8) 1.17 _+0.10 (9) Not detectable 0.13 + 0.01 (5) 0.26 _4-_0.01 (4) 5.60 + 0.60 (5) 74.03 + 2.22 (10) 14.06 +__0.52 (8)
The enzyme activities are expressed as #mol/min/g fresh wt at 25°C and are given as means ± SEM. The number of assays performed for each enzyme is shown in parentheses.
increase at the second hour of anaerobiosis reaching control levels thereafter (Fig. 2f). (ii) Krebs cycle metabolites. As can be seen in Fig. 3 the concentration of citrate decreased in the first two hours of anaerobiosis and then remained constant (Fig. 3a). A decrease is also observed in the concentration of oxaloacetate and ct-ketoglutarate at all stages of anaerobiosis (Fig. 3b, d respectively). In contrast, the concentration of malate increased from the first hour of anaerobiosis (Fig. 3c), while that of succinate increased in the period between the second and fourth hour (Fig. 3e). After the fourth hour of anaerobiosis the concentration of malate remained almost constant while that of succinate showed a small decrease. (iii) Amino acids. Compared to all metabolites measured, the free amino acids have the highest concentrations in the foot muscle of P. caerulea at rest. The concentrations of aspartate and alanine, however, changed in an opposite manner during anaerobiosis; that is, aspartate decreased and alanine increased (Fig. 4a, b respectively). The concentration of glutamate increased like that of alanine, but only in the first two hours of anaerobiosis, reaching control levels thereafter (Fig. 4c). (iv) Adenosine phosphates. During exposure of P. caerulea to air the concentration of ATP in the foot muscle decreased in the first two hours (Fig. 5a) and after a slight increase at the fourth hour, Table 3. The maximum activities of enzymes relating to energy metabolism in the foot muscle of P. caerulea Enzyme Glutamate dehydrogenase Malic enzyme Arginine phosphokinase Creatine phosphokinase Pyruvate carboxylase Glucose-6-phosphate dehydrogenase Gluconate-6-phosphate dehydrogenase Nucleoside diphosphate kinase PEP-carboxykinase Octopine dehydrogenase Alanopine dehydrogenase Strombine dehydrogenase Fructose- 1,6-bisphosphatase Glutamate-oxaloacetate transaminase Glutamate-pyruvate transaminase Myofibrillar ATPase
Activity 0.69 ± 0.03 (10) 0.24 ± 0.02 (10) 20.84 _+ 1.72 (9) N.D.* N.D. 1.10 + 0.10(10) 0.40 ± 0.05 (7) 42.86 _+ 3.53 (9) 0.13 + 0.02 (10) N.D. N.D. N.D. 0.08 ± 0.01 (8) 7.06 + 0.27 (10) 3.23 + 0.26 (10) 14.49 _+ 1.20 (6)
*Not detectable, The activities are expressed as #mol/min/g fresh wt at 25°C and are given as means _+ SEM. The number of assays performed for each enzyme is shown in parentheses.
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Fig. I. Changes in the concentrations of glycolytic metabolites in the foot muscle of P. caerulea during exposure to air. (a) Glucose; (b) glucose-6-phosphate; (c) fructose-6-phosphate; (d) fructose-l,6-bisphosphate; (e) dihydroxyacetone phosphate; (f) glyceraldehyde-3-phosphate. Each point represents the mean of six determinations. remained almost constant. A D P and A M P concentrations followed similar but reciprocal patterns to that of ATP, showing an initial increase in the first two hours of anaerobiosis and a decrease at the fourth hour (Fig. 5b, c respectively). However, the sum of adenosine phosphates content remained fairly constant at all stages of anaerobiosis. DISCUSSION
From the results of the present study it appears that the foot muscle of P. caerulea has a complete
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sequence of glycolytic enzymes. The high activity of phosphorylase (3.48 _+ 0.23 #mol/min/g fresh wt) in comparison to hexokinase activity (0.47_+ 0.05 pmol/min/g fresh wt) and the considerable activity of P F K (7.93 + 0.65/~mol/min/g fresh wt) support the suggestion that glycogen is the main fuel oxidized. A similar pattern of activities was found by Zammit and Newsholme (1976) in a number of muscles from other marine invertebrates. The increased concentrations of glycolytic intermediates in the foot muscle of P. caerulea in the first four hours of anaerobiosis (Figs 1 and 2) indicate an increased rate of glycolysis.
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duration of anaerobiosis h Fig. 2. Changes in the concentrations of glycolytic metabolites in the foot muscle of P, caerulea during exposure to air. (a) 1,3-Diphosphoglycerate; (b) 3-phosphoglycerate; (c) 2-phosphoglycerate; (d) phosphoenolpyruvate; (e) pyruvat¢; (f) D-lactate. Each point represents the mean of six determinations.
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Fig. 3. Changes in the concentrations of Krebs cycle metabolites in the foot muscle of P. caerulea during exposure to air. (a) Citrate; (b) oxaloacetate; (c) malate; (d) ~-ketoglutarate; (e) succinate. Each point represents the mean of six determinations. However, the reduction of aspartate content in the same period of anaerobiosis (Fig. 4a), in combination with the accumulation of alanine (Fig. 4b) and the presence of considerable activities of GOT (7.06+0.27#mol/min/g fresh wt) and GPT
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(3.23 -I- 0.26 #mol/min/g fresh wt), indicate a coupled metabolism of glycogen and aspartate in the foot muscle of P. caerulea during exposure to air. Glycogen and aspartate are considered as the major sources for energy production in the tissues of marine gastropods (Livingstone and de Zwaan, 1983) and bivalves (de Zwaan, 1983) under anoxia leading to accumulation of various end-products including succinate, alanine, octopine, strombine, alanopine, D-lactate, acetate and propionate. Succinate, glutamate and to a lesser extent Dlactate are also accumulated in the foot muscle of P. caerulea during exposure to air. However, their accumulation depends on the stage of anaerobiosis. Specifically, in the first two hours, alanine, glutamate and o-lactate are accumulated (Figs 4b, c and 2f respectively), while in the next two hours succinate and alanine levels are increased (Figs 3e and 4b respectively). This indicates that, in the formation of these end-products, separate metabolic routes are involved after the step of PEP, possibly by the presence of the enzymes PEP-carboxykinase and PK as in the bivalves (de Zwaan, 1977). The slight increase of D-lactate and the accumulation of alanine in the first two hours of anaerobiosis show that a small amount of pyruvate is converted to o-lactate but most of it is transaminated to alanine with aspartate serving as the source of amino groups. The small accumulation of D-lactate is consistent with the low maximal activity of D-LDH measured in the foot muscle of P. caerulea (Table 1). It has been found that o-lactate is mainly accumulated in land molluscs during anaerobiosis (Wieser, 1980; Long et al., 1979), while the transamination of pyruvate to alanine at the expense of aspartate is the main metabolic fate of pyruvate in some marine gastropods (Livingstone and de Zwaan, 1983) and in most marine bivalves (de Zwaan, 1983).
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Fig. 5. Changes in the concentrations of adenosine phosphates and energy charge values (E.C.) in the foot muscle of P. caerulea during exposure to air. (a) ATP; (b) ADP; (c) AMP; (d) energy charge. Each point represents the mean of six determinations. In contrast to bivalves (de Zwaan, 1983), the absence of opine dehydrogenases in the foot muscle excludes the case of pyruvate conversion to octopine, strombine and alanopine in this muscle during anaerobiosis and it is in agreement with the data reported for P. vulgata as regards the existence of these dehydrogenases in the foot muscle (Livingstone et al., 1983). Opine dehydrogenases seem to be present especially in muscles with high energy demand, replacing LDH to reoxidize glycolytic N A D H (Gfide and Grieshaber, 1986). From the examined dehydrogenases in the foot muscle of P. caerulea only malate dehydrogenase measured in the direction of oxaloacetate reduction gave the highest maximal activity (Table 2), showing that reoxidation of glycolytic N A D H by the reaction catalysed by the malate dehydrogenase can supply the demanded N A D ÷ for the maximum glycolytic rate. The accumulation of glutamate in the foot muscle of P. caerulea in the first two hours of anaerobiosis (Fig. 4c) shows that an amount of pyruvate is also converted to glutamate. The presence of citrate synthetase, aconitase, NAD+(P) isocitrate dehydrogenase (Table 2) and glutamate dehydrogenase, which is distributed in both cytosolic and mitochondrial compartments in the foot muscle of P. caerulea (Michaelidis, 1984), in combination with the decrease of citrate concentration in the foot muscle in the same period which glutamate rises, indicates that glutamate is formed by the reactions catalysed by the enzymes of the first part of the Krebs cycle and glutamate dehydrogenase. This metabolic route for glutamate formation and a slight accumulation of glutamate had been observed in the tissues of some marine molluscs during anaerobiosis (de Zwaan and van Marrewijk, 1973; de Zwaan et al., 1981; Widdows et al., 1979). It has been suggested that the role of glutamate dehydrogenase is related to the reoxidation of cytosolic N A D H at the onset of anaerobiosis if insufficient substrates are available for other cytosolic dehydrogenases (de Zwaan, 1983).
No accumulation of o-lactate (Fig. 2f) and glutamate (Fig. 4c) is observed in the foot muscle in the period between the second and fourth hour of anaerobiosis. In addition, during the same period the concentration of pyruvate remains constant (Fig. 2e), while that of PEP and succinate increases (Figs 2d and 3e respectively). These observations indicate that PK activity is inhibited after the second hour of anaerobiosis. It has been demonstrated that inhibition of PK in the foot muscle is caused by the phosphorylation of enzyme (Michaelidis et al., 1988) and the inhibitors [H ÷] and alanine (Michaelidis et al., 1985). In particular, after the second hour of P. caerulea exposure to air, the degree of phosphorylation of PK from the foot muscle increased, resulting in a less active form as regards the substrate PEP and a more sensitive form with respect to inhibition by alanine (Michaelidis et al., 1988). Similar to the present results, Storey et al. (1989) have found that the glycolytic rate is reduced in the foot of the marine whelk Busycotypus canaliculatum in the first two hours of anoxia and that phosphorylation of PK plays a key role in depression of glycolytic rate. Although PK is inhibited after the second hour of anaerobiosis, the concentration of alanine continues to increase (Fig. 4b), while that of aspartate decreases (Fig. 4a). In addition, the concentration of succinate increases after this period of anaerobiosis (Fig. 3e). At this point the question of how alanine is produced at the expense of aspartate after PK has been inhibited arises, de Zwaan et al. (1983) postulated a modified reaction scheme of the classical aspartatemalate cycle in order to explain the accumulation of alanine and succinate at the expense of aspartate in the tissues of Mytilus edulis under anoxia. In this reaction scheme malate derived from the reduction of oxaloacetate can follow two different metabolic routes in mitochondria. Firstly, it can be decarboxylated to pyruvate by malic enzyme and then to alanine by GPT. Secondly, it can be converted to succinate by the first part of the Krebs cycle operating in the
Anaerobic metabolism in Patella muscle opposite direction. Both malic enzyme and GPT are located in the mitochondria of P. caerulea (Michaelidis, 1984), indicating that such a mechanism of alanine and succinate production at the expense of aspartate could operate in this muscle after the inhibition of PK. An alternative route of succinate production in the foot muscle of P. caerulea could be the conversion of PEP to oxaloacetate by PEP-carboxykinase and consequently to succinate via the first part of the Krebs cycle as has been reported above. The reaction catalysed by PEP-carboxykinase results in the production of an extra high energy nucleotide (possible ITP) which is converted to ATP by the nucleoside diphosphate kinase (de Zwaan, 1977). Both PEP-carboxykinase and nucleoside diphosphate kinase are present in the foot muscle of P. caerulea, showing the existence of the above described metabolic route of succinate production. PEP-carboxykinase displays optimum activity at low pH values (pH 6.9) (Michaelidis, 1984) compared to that of PK (pH 7.5) (Michaelidis et al., 1985). So, after the first two hours of anaerobiosis, phos° phorylation of PK and the increased concentrations of alanine and [H ÷] could result in inhibition of enzyme activity. This would enable PEP- carboxykinase to compete with PK for the substrate PEP, although the former enzyme has a lower maximal acitivity (Table 3) compared to the latter (Table 1). Prolonged anoxia results in conversion of succinate to propionate in bivalves (Schulz et al., 1982). In the present work the case of succinate conversion to propionate was not studied. As far as the rest of the enzymes of Table 3 are concerned, it is shown that gluconeogenesis does not take place in this muscle, since pyruvate carboxylase is not detectable and because of the very low activity of fructose-l,6-bisphosphatase. The presence of glucose-6-phosphate and gluconate-6-phosphate dehydrogenase indicate that the pentose phosphate pathway could operate in this muscle. However, the amount of phosphoexose residues which are metabolized through this pathway is unknown at present and is under investigation. The presence of arginine phosphokinase instead or creatine phosphokinase is in accordance with results obtained from other muscles of marine molluscs (Zammit and Newsholme, 1976), indicating that arginine phosphate reserves are oxidized during the first stages of muscle contraction for ATP supply. The low concentration of adenosine phosphates measured in P. caerulea are consistent with the low rate of energy expenditure by these muscles during muscular contraction (Riiegg, 1971; Baguet and Gilles, 1968). The increase of energy charge, however, in the period between the second and fourth hours of anaerobiosis (Fig. 5d) indicates that during this period an ATP producing metabolic process is activated. This, in combination with the increase of succinate concentration during the same period, indicates that the succinate pathway must be responsible for the observed increase of ATP concentration in the foot muscle of P. caerulea from the second to the fourth hour of anaerobiosis.
499 REFERENCES
Adam H. (1963) Adenosine-5'-diphosphate and adenosine5'-monophosphate. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 573-577. Academic Press, New York. Alp P. R., Newsholme E. A. and Zammit V. (1976) Activities of citrate synthetase, NAD + and NADP-linked isocitrate dehydrogenase in muscles from vertebrates and invertebrates. Biochem. J. 154, 689-700. Baguet F. and Gilles M. J. (1968) Energy cost of tonic contraction in a lamellibranch catch muscle. J. Physiol. (Lond.) 198, 127-143. Barrett J. and Beis I. (1973) Studies on glycolysis in the muscle tissue of Ascaris lumbricoides (Nematode). Comp. Biochem. Physiol. 44B, 751-761. Beenakkers A. M. T. (1969) Carbohydrate and fat as a fuel for insect flight. A comparative study. J. Insect Physiol. 15, 353-361. Bergmeyer H. U. and Bernt E. (1974a) Glutamateoxaloacetate transaminase. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 727-733. Academic Press, New York. Bergmeyer H. U. and Beret E. (1974b) Glutamateoxaloacetate transaminase. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 752-758. Academic Press, New York. Bergmeyer H. U. and Bernt E. (1974c) 2-Oxoglutarate. UV-spectrophotometric determination. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 1577-1580. Academic Press, New York. BergrneyerH. U. and Bernt E. (1974d) L-Glutamate. UV-spectrophotometric assay with glutamate dehydrogenase and NAD. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 1704-1708. Academic Press, New York. Bergmeyer H. U., Bernt E., Mrllering H. and Phleidere G. (1974) L-Aspartate and L-asparagine. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 1696-1700. Academic Press, New York. B/icher T. and Hohorst H. J. (1963) Dihydroxyacetone phosphate, fructose-l,6-diphosphate and D-glyceraldehyde-3-phosphate. Determination with glycerol-l-phosphate dehydrogenase, aldolase and triosephosphate isomerase. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 246-252. Academic Press, New York. Bucher T., Czok R., Lamprecht W. and Latzko E. (1963) Pyruvate. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 253-259. Academic Press, New York. Cha S. (1969) Succinate thiokinase from pig heart. In Methods in Enzymology (Edited by Lowenstein J. M.), Vol. XIII, pp. 62-64. Academic Press, New York. Cornblath M., Rantle P. J., Parmagiani A. and Morgan H. E. (1963) Regulation of glyceogenolysis in muscle. J. biol. Chem. 238, 1592-1597. Crabtree B., Leech A. R. and Newsholme E. A. (1979) Measurement of enzyme activities in crude extracts of tissues. In Techniques in Metabolic Research, Vol. B 211, pp. 1-37. Elsevier/North Holland, Amsterdam. Czok R. (1974) o-Glycerate-3-phosphate. In Methods in Enzyamtic Analysis (Edited by Bergmeyer H. U.), pp. 1424-1427. Academic Press, New York. de Zwaan A. (1977) Anaerobic energy metabolism in bivalve molluscs. Oceanogr. mar. Biol. Ann. Rev. 15, 103-187. de Zwaan A. (1983) Carbohydrate catabolism in bivalves. In The Mollusca (Edited by Wilbur K. M.), Vol. 1, pp. 137-175. Academic Press, New York. de Zwaan A. and Putzer V. (1985) Metabolic adaptations of intertidal invertebrates to environmental hypoxia (a comparison of environmental anoxia to exercise anoxia). In Physiological Adaptations of Marine Animals (Edited by Lawerack M. S.), Vol. 39, pp. 33~2. Cambridge University Press.
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de Zwaan A. and van Marrewijk W. J. A. (1973) Anaerobic glucose degradation in the sea mussel Mytilus edulis L. Comp. Biochem. Physiol. 44B, 429-439. de Zwaan A. and Zurburg W. (1981) The formation of strombine in the adductor muscle of the sea mussle Mytilus edulis L. Mar. Biol. Lett. 2, 179 192. de Zwaan A., Holwerda D. A. and Veenhof P. R. (1981) Anaerobic malate metabolism in mitochondria of the sea mussle Mytilus edulis L. Mar. Biol. Lett. 2, 131-140. de Zwaan A., de Bont A. M. T. and Hemelrad J. (1983) The role of phosphoenolpyruvate carboxykinase in the anaerobic metabolism of the sea mussel Mytilus edulis L. J. comp. Physiol. 153, 267-274. Ebberink R. H. M., Zurburg W. and Zandee D. I. (1979) The energy demand of posterior adductor muscle of Mytilus edulis in catch during exposure to air. Mar. Biol. Lett. 1, 23-31. Fansler B. and Lowenstein J. M. (1969) Aconitase from pig heart. In Methods in Enzymology (Edited by Lowenstein J. M.), Vol. XIII, pp. 26-30. Academic Press, New York. G/ide G. and Grieshaber M. K. (1986) Pyruvate reductases catalyze the formation of lactate and opines in anaerobic invertebrates. Comp. Biochem. Physiol. 83B, 255-272. Gutman I. and Wahlefeld W. A. (1974a) L-(+)-Lactate. In Methods' in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 1464-1468. Academic Press, New York. Gutman 1. and Wahlefeld W. A. (1974b) L-Malate determination with malate dehydrogenase and NAD ÷ . In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 1585 1589. Academic Press, New York. Hohorst H. J. (1963) D-Glucose-6-phosphate and D-fructose-6-phosphate. Determination with glucose-6-phosphate dehydrogenase and phosphoglucose isomerase. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 134 138. Academic Press, New York. Kaplan N. O. (1957) Enzymatic determination of free sugars. In Methods in Enzymology (Edited by Colowick S, P. and Kaplan N. O.), Vol. III, pp. 109 110. Academic Press, New York. Kooijman D., van Zoonen H., Zurburg W. and Kluytmans J. H. (1982) On the aerobic and anaerobic energy metabolism of Littoria species in relation to the pattern of intertidal zonation. In Exogenous Influences on Metabolic and Neural Control (Edited by Addink A. D. F. and Sponk N.), Vol. 2, pp, 134-135. Abstr. 3rd Congr. Eur. Soc. Comp. Physiol. Biochem., Noordwijkerhout, The Netherlands. Pergamon Press, Oxford. Lamprecht W. and Trautschold I. (1963) Adenosine-Ytriphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In Methods in Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 543-551. Academic Press, New York. Livingstone D. R. (1982) Energy production in the muscle tissues of different kinds of molluscs. In Exogenous and Endogenous Influences on Metabolic and Neural Control (Edited by Addink A. D. E.), Vol. 1, pp. 257-274. Invited lectures, Proc. 3rd Congr. Eur. Soc. Comp. Physiol. Biochem., Noordwijkerhout, The Netherlands. Pergamon Press, Oxford. Livingstone D. R. and de Zwaan A. (1983) Carbohydrate metabolism of gastropods. In The Mollusca (Edited by Wilbur K. M.), Vol. 1, pp. 177-242. Academic Press, New York. Livingstone D. R., de Zwaan A., Leopold M. and Marteijn E. (1983) Studies on the phylogenetic distribution of pyruvate oxidoreductases, Biochem. Syst. Ecol. 11, 415-425. Long G. L., Ellington W. R. and Duda T. F. (1979) Comparative enzymology and physiological role of Dlactate dehydrogenase from the foot muscle of two gastropod molluscs. J. exp. Zool. 207, 237-248. Martin B. R. and Denton R. M. (1970) The intracellular localization of enzymes in white adipose tissue fat cells
and permeability properties of fat cell mitochondria. Biochem. J. 117, 861-877. Michaelidis B. (1984) Study of energy metabolism in the foot of the sea mollusc Patella caerulea (L.). Doctorate Thesis, University of Thessaloniki, Thessaloniki, Greece. Michaelidis B., Lazou A, and Beis I. (1985) Purification, catalytic and regulatory properties of pyruvate kinase from the foot of Patella caerulea (L.) Comp. Biochem. Physiol. 82B, 405-412. Michaelidis B., Gaitanaki C. and Beis I. (1988) Modification of pyruvate kinase from the foot muscle of Patella caerulea (L) during anaerobiosis. J. exp. Zool. 248, 264-271. Michal G., Beutlet H. O., Lang G. and Guentnes U. (1976) Enzymatic determination of succinic acid in food stuffs. Z. Analyt. Chem. 279, 137-138. Negelein E. (1974) D-Glycerate-l,3-diphosphate. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U,), pp. 1429-1431. Academic Press, New York. Ochoa S. (1955) Malic enzyme. In Methods in Enzymology (Edited by Colowick S. P. and Kaplan N. O.), Vol. I, pp. 739-753. Academic Press, New York. Opie L. H. and Newsholme E. A. (1967) The activities of fructose- 1,6-diphosphase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white and red muscle. Biochem. J. 103, 391-399. Parks R. E. and Agarwal R. P. (1973) Nucleoside diphosphokinases. In The Enzymes (Edited by Boyer P. D.), Vol. VIII, pp. 307-333. Academic Press, New York. Racker E, (1950) Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids. Biochim. biophys. Acta 4, 211-214. Read G., Crabtree B. and Smith G. H. (1977) The activities of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in hearts and mammary glands from ruminants and non-ruminants. Biochem. J. 164, 349-355. R/iegg J. (1971) Smooth muscle tone. Physiol. Rev. 51, 201-248. Schulz T. K. F., Kluytmans J. H. and Zandee D. F. (1982) In vitro production of propionate by mantle mitochondria of the sea mussel Mytilus edulis L. Overall mechanism. Comp. Biochem. Physiol. 73B, 673-680. Schmidt E. (1963) Glutamate dehydrogenase. In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), pp. 152 156. Academic Press, New York. Shonk C. E. and Boxer G. E. (1964) Enzyme patterns in human tissues I. Methods for the determination of glycolytic enzymes. Cancer Res. 24, 709-721. Slater E. C. and Bonner W. D. (1952) The effect of fluorite on the succinic oxidase system. J. Biochem. 52, 185-196. Srere P. A., Brazil H. and Gonen L. (1963) The citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta chem. scand. 17, 129-134. Storey B. K., Kelly A. D., Duncan J. A. and Storey M. J. (1989) Anaerobiosis and organ-specific regulation of glycolysis in a whelk. J. comp. Physiol. (submitted). Szent-Gy6rgyi A. G., Cohen C. and Kendrick-Jones J. (1971) Paramyosin and thin filaments of molluscan catch muscles. II. Native filaments isolation and characterization. J. molec. Biol. 56, 239 258. Widdows J., Bayne B. L. and Livingstone D. R. (1979) Physiological and biochemical responses of bivalve molluscs to exposure to air. Comp. Biochem. Physiol. 62A, 301-308. Wieser W. (1980) Metabolic end products in three species of marine gastropods. J. mar. Biol. Assoc. UK 60, 175-180. Zammit V. A, and Newsholme E. A. (1976) The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenase, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphate kinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates. Biochem. J. 160, 447-462,
0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc
STUDIES ON THE ANAEROBIC ENERGY METABOLISM IN THE FOOT MUSCLE OF MARINE GASTROPOD P A T E L L A C A E R U L E A (L.) BASILE MICHAELIDIS and ISIDOROSBEIS* Laboratory of Animal Physiology, Dept. of Zoology, School of Science, University of Thessaloniki, Thessaloniki 54006, Greece (Received 4 July 1989)
Abstract--1. The foot muscle of P. caerulea has a complete sequence of glycolytic enzymes. The low activity of hexokinase, in comparison with the activities of glycogen phosphorylase and phosphofructokinase, indicate that glycogen is the main fuel oxidized. 2. The reduction of aspartate content in combination with the accumulation of alanine and the presence of considerable activities of glutamate-oxaloacetate transaminase and glutamate~pyruvate transaminase indicates a coupled metabolism of glycogen and aspartate during exposure to air. 3. From the changes in the concentration of the metabolites during exposure to air it appears that up to the second hour of anaerobiosis alanine, lactate and glutamate are the end-products which accumulate in the foot muscle of P. caerulea, whereas from the second to the fourth hour only succinate and alanine accumulate. 4. The low activities of the Krebs cycleenzymesas wellas the absence of ~t-ketoglutaratedehydrogenase activity suggest that the Krebs cycle is not in operation. 5. The absence of opine dehydrogenases shows that the end products octopine, alanopine and strombine are not accumulated in the foot muscle under anaerobiosis. 6. The high activity of malate dehydrogenase in the direction of malate formation in combination with the low activity of ~-lactate dehydrogenase and the absence of opine dehydrogenases suggests that the former dehydrogenase is coupled 1: 1 to glyceraldehyde-3-phosphatedehydrogenase.
INTRODUCTION
It is widely observed that intertidal molluscs display impressive anaerobic capacities and that they can sustain prolonged anoxia exposures. The most widely investigated group, relating to the overall energy metabolism as well as to the mode of energy production during anaerobiosis, is the bivalve molluscs. In these animals, glycogen and aspartate serve as the main source for energy production (ATP) and instead of lactate, other end-products are accumulated in their tissues during anaerobiosis, the most important being succinate, alanine, octopine, alanopine, strombine and the volatile acids propionate and acetate (de Zwaan, 1983). In contrast to vertebrates, anaerobiosis does not cause any marked increase in the rate of glycogen utilization (no Pasteur effect) in the tissues of bivalve molluscs. The lack of Pasteur effect is mainly due to the drastic drop of energy demand which is observed in their tissues during anoxia (Ebberink et aL, 1979; de Zwaan and Putzer, 1985). This, in combination with the higher energetic efficiency due to the accumulation of succinate and propionate, which is the main energy yielding mechanism during anoxia (Livingstone, 1982), enables them to withstand long periods of exposure to air. As in bivalves, anaerobiosis also occurs in intertidal gastropods (Livingstone and de Zwaan, 1983). *Author to whom correspondence should be addressed.
In these animals a number of anaerobic end-products have been detected, namely lactate, octopine, alanine, succinate, acetate and propionate (de Zwaan, 1983). In contrast to well documented studies of bivalves, however, the knowledge on the overall energy metabolism of marine gastropods is rather limited and the mechanisms controlling the accumulation of end-products in marine gastropods are far from clear. The accumulation of different end-products in marine gastropods during anaerobiosis is variable among species and it appears that it is dependent on their pattern of intertidal zonation (Wieser, 1980; Kooijman et al., 1982). In an effort to study the overall metabolism as well as the anaerobic energy metabolism in the tissues of marine gastropods, we estimated the maximum activity of the enzymes involved in the glycolytic sequence and the Krebs cycle, as well as the enzymes related to the overall energy metabolism in the foot muscle of the Mediterranean intertidal gastropod, Patella caerulea. In addition, in order to find out which metabolic routes operate and hence which end-products accumulate in the foot muscle of P. caerulea during anaerobiosis, we studied the changes in the concentrations of glycolytic and Krebs cycle metabolites as well as the free amino acids, aspartate, alanine and glutamate, in the foot muscle during different periods (0, 1, 2, 4, 8 and 16 hr) ofP. caerulea exposure to air.
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BASILEMICHAELIDIS and ISIDOROSBE1S MATERIALS AND METHODS
Animals
Limpets (Patella caerulea) were collected from the shores of Chalkidiki Peninsula, near Thessaloniki and were kept in circulating sea water at about 17°C. They were used 48-72 hr after arrival. Chemicals and enzymes
The substrates, enzymes and coenzymes were purchased from Sigma Chemical (St. Louis, USA). All other chemicals were purchased from Serva (Heidelberg, FRG). Preparation o f homogenates
Muscles were dissected as quickly as possible and were cut into small pieces before homogenization. Muscles were homogenized in a ground glass homogenizer with 5 ~ 0 volumes of extraction medium. The extraction medium for all enzymes except for phosphorylase, phosphofructokinase (PFK), citrate synthetase, NAD+(P) isocitrate dehydrogenase, ~-ketoglutarate dehydrogenase, creatine and arginine phosphokinase and myofibrillar ATPase, contained 50 mM triethanolamine, 1 mM EDTA, 2 mM MgC12 and 30raM mercaptoethanol adjusted to pH 7.4 with KOH (Crabtree et al., 1979). For phosphorylase the extraction medium consisted of 35 mM glycerol-2-phosphate, 20 mM NaF, 1 mM EDTA and 30 mM mercaptoethanol at pH 6.2 (Cornblath et al., 1963); that for PFK contained 70mM Tris-HC1, 1 mM EDTA and 5 mM MgSO 4 at pH 8.2 (Opie and Newsholme, 1967); that for citrate synthetase contained 25 mM Tris-HC1 and 1 mM EDTA at pH 7.4 (Alp et al., 1976); that for NAD + isocitrate dehydrogenase contained 50 mM triethanolamine, l mM EDTA, 2 mM ADP, 3 mM MgC12 and 30 mM mercaptoethanol at pH 7.4; for NADP isocitrate dehydrogenase the same extraction medium was used except that it did not contain ADP (Alp et al., 1976). The extraction medium for c~-ketoglutarate dehydrogenase contained 50 mM triethanolamine, 2 mM EDTA, 30 mM mercaptoethanol, 2 mM glycerol and 1 mM ~-ketoglutarate at pH 7.4 (Read et al., 1977). For creatine and arginine phosphokinase the extraction medium contained 50mM Pipes, 4 m M dithiothreitol and 1 mM EDTA at pH 7.0 (Zammit and Newsholme, 1976). For myofibrillar ATPase activity the preparation of homogenate was according to the method of Szent-Gy6rgyi et al. (1971). Homogenates were centrifuged briefly at low speed (600g) to sediment cell debris, in order to minimize turbidity in the cuvette. After the homogenates were sonicated for two 15-sec periods with the microprobe of an MSH conicator operating at an amplitude of 6/~m, the following enzymes were assayed: all enzymes of the Krebs cycle, PEP-carboxykinase, glutamate-oxaloacetate, transaminase (GOT), glutamate pyruvate transaminase (GPT), nucleoside diphosphate kinase, glutamate dehydrogenase and malic enzyme. Assay of enzyme activities
All enzymes were measured at 25°C using a Gilford recording spectrophotometer by following the change in O.D. caused by the oxidation or reduction of NAD+(P) (H). The methods by which glycolytic enzymes, fructose-1,6-bisphosphate (E.C. 3.1.3.11), glucose-6-phosphate dehydrogenase (E.C. 1.11.1.6), gluconate-6-phosphate dehydrogenase (E.C.I. 1.I.44) and PEP-carboxykinase (E.C. 4.1.1.32) were measured were similar to those described by Barrett and Beis (1973). Pyruvate carboxylase (E.C. 6.4.1.1) was measured according to Martin and Denton (1970); citrate synthetase (E.C. 4.2.1.3) according to Srere et al. (1963); aconitase (E.C. 4.2.1.3) according to Fansler and Lowenstein (1969); NAD + isocitrate dehydrogenase (E.C. 1.1.1.41)
and NADP isocitrate dehydrogenase according to Alp et al. (1976); ~-ketoglutarate dehydrogenase (E.C. 1.2.4.2) according to Read et al. (1977); succinyl-CoA synthetase (E.C. 6.2.1.4) according to Cha (1969); succinate dehydrogenase (E.C. 1.3.99.1) according to Slater and Bonner (1952); fumarase (E.C. 4.2.1.2) according to Racker (1950); malate dehydrogenase (E.C. 1.1.1.37) according to Shonk and Boxer (1964); malic enzyme (E.C. 1.1.1.38) according to Ochoa (1955); octopine dehydrogenase (E.C. 1.5.11) according to Zammit and Newsholme (1976); strombine dehydrogenase (E.C. 1.5.1.x) and alanopine dehydrogenase (E.C. 1.5.1 .x) according to de Zwaan and Zurburg (1981); arginine phosphokinase (E.C. 2.7.3.2) according to Beenakkers (1969); creatine phosphokinase (E.C. 2.7.3.2) was assayed in the same way as arginine phosphokinase except that creatine was used instead of arginine; glutamate dehydrogenase (E.C. 1.4.1.3) according to Schmidt (1963); GOT (E.C. 2.6.1.1) and GPT (E.C. 2.6.1.2) according to Bergmeyer and Bernt (1974a,b); nucleoside diphosphate kinase (E.C. 2.7.4.6) according to Parks and Agarwal (1973); myofibrillar ATPase (E.C. 3.6.1.3) according to Szent-Gy6rgi et al. (1971). Experimental procedure o f anaerobiosis and preparation q f tissue extracts
Groups of six individuals were exposed to air for 1, 2, 4, 8 and 16 hr at 15-18°C. After each period of exposure to air the foot muscles were rapidly dissected and immediately freeze clamped with the aid of aluminium tongs cooled in liquid nitrogen. From dissecting to freezing the muscles, the procedure was completed in 8-10 sec. The frozen muscles were separated in a percussion mortar at -70°C and the powdered muscles were extracted by adding 4~5 volumes of frozen HC104 (10% w/v). The precipitated protein was removed by centrifugation at 4000g for 10min and the supernatants were neutralized with 3 M KHCO 3. The precipitated potassium perchlorite was removed by centrifugation as above and the supernatants were taken for determination of metabolite concentrations. Determination o f metabolites
All metabolites were measured spectrophotometrically by using enzymatic systems coupled to NAD+(H) or NADP(H). (i) Metabolites o f glycolysis. Glucose was estimated by the method of Kaplan (1957); glucose-6-phosphate and fructose-6-phosphate were estimated in the same cuvette by the method of Hohorst (1963); fructose-l,6-diphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate were estimated in the same cuvette by the method of Bucher and Hohorst (1963); 1,3-phosphoglycerate by the method of Negelein (1974); 3-phosphoglycerate by the method of Czok (1974); pyruvate, phosphoenolpyruvate (PEP) and 2-phosphoglycerate were estimated in the same cuvette by the method of Bucher et al. (1963); D-lactate by the method of Gutman and Wahlefed (1974a) by using D-LDH. (ii) Metabolites o f the Krebs cycle. Malate was determined by the method of Gutman and Wahlefeld (1974b); oxaloacetate by the method of Wahlefeld (1974); succinate by the method of Michal et al. (1976); a-ketoglutarate by the method of Bergmeyer and Bernt (1974c). (iii) Free amino acids. Aspartate was measured by the method of Bergmeyer et al. (1974); alanine by the method of Williamson (1974); glutamate by the method of Bergmeyer and Bernt (1974d). (iv) Adenosines phosphates. Adenosine triphosphate (ATP) was estimated by the method of Lamprecht and Trautschold (1963); adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were estimated in the same cuvette by the method of Adam (1963).
A n a e r o b i c m e t a b o l i s m in Patella muscle RESULTS
Table 2. The maximum activities of enzymes of the Krebs cycle in the foot muscle of P. caerulea
Enzyme activities The activities of all enzymes studied are presented in Tables 1-3. Noteworthy is the very low activity of hexokinase (0.47 ___0.05/~mol/min/g fresh wt) compared to that of phosphorylase (3.48 +0.23 #mol/min/g fresh wt) and PFK (7.93+0.65 /~mol/min/g fresh wt) and the presence of both PK and PEP-carboxykinase. The activity of D-LDH measured was very low (Table 1), whereas no activity of octopine, alanopine and strombine dehydrogenase could be detected (Table 3). The activities of the enzymes of the Krebs cycle, with the exception of malate dehydrogenase measured in the direction of oxaloacetate reduction, are low (Table 2). In addition, no activity of ct-ketaglutarate dehydrogenase could be detected. In Table 3 the activities of some enzymes related to the glycolytic pathway and the overall energy metabolism are given. From the measured enzymes nucleoside diphosphate kinase (in the direction of oxaloacetate reduction) gave the highest activity, whereas no activities of creatine phosphokinase and pyruvate carboxylase were detectable. Changes in the concentrations of metabolites during anaerobiosis The changes in the concentrations of different metabolites in the foot muscle of P. caerulea during various periods of anaerobiosis are given in Figs 1 and 2 for glycolytic metabolites, Fig. 3 for Krebs cycle metabolites, Fig. 4 for amino acids and Fig. 5 for adenosine phosphates. (i) Glycolytic metabolites. During anaerobiosis the concentrations of all glycolytic metabolites, except those of glucose, PEP and D-lactate, were altered in about the same way: they increased up to the fourth hour of exposure to air and then fell until the eighth hour of exposure. From then, up to the sixteenth hour of anaerobiosis metabolite concentrations were stabilized at/or below control level (0 hr anaerobiosis) (Figs 1 and 2). The concentration of glucose remained almost constant at all stages of anaerobiosis (Fig. 1a), whereas that of PEP decreased in the first two hours and after a slight increase at four hours of anaerobiosis remained almost constant below control level (Fig. 2d). D-Lactate concentration showed a slight Table 1. The maximum activities of glycolytic enzymes in the foot muscle of P. caerulea Enzyme Hexokinase Pbosphorylase Phosphoglucomutase Phosphoglucose isomerase Phosphofructokinase Aldolase Triosephosphate isomerase Glyceraldehyde-3-phosphate dehydrogenase Phosphogiycerate kinase Phosphoglyceromutase Enolase Pyruvate kinase D-Lactate dehydrogenase
495
Activity 0.47 _+ 0.50 (10) 3.48 ± 0.23 (7) 15.32 ± 0.90 (8) 30.93 ± 1.67 (10) 7.93 ± 0.65 (7) 13.65 ± 0.86 (9) 158.53 ± 3.83 (6) 39.58 ± 3.26 (10) 12.58 ± 3.26 (9) 15.23 ± 1.34 (8) 9.55 ± 0.75 (10) 11.13 4- 0.73 (6) 1.01 ± 0.20 (10)
The enzyme activities are expressed as #mol/min/g fresh wt at 25°C and are given as means _+ SEM. The number of assays performed for each enzyme is shown in parentheses.
Enzyme Citrate synthetase Aconitase NAD + isocitrate dehydrogenase NADP isocitrate dehydrogenase ~t-Ketoglutarate dehydrogenase Succinyl-CoA synthetase Succinate dehydrogenase Eumarase Malate dehydrogenase (a) Oxaloacetate to malate (b) Malate to oxaloacetate
Activity 1.22 ± 0.10 (10) 0.83 _ 0.06 (10)
0.13 _+0.01 (8) 1.17 _+0.10 (9) Not detectable 0.13 + 0.01 (5) 0.26 _4-_0.01 (4) 5.60 + 0.60 (5) 74.03 + 2.22 (10) 14.06 +__0.52 (8)
The enzyme activities are expressed as #mol/min/g fresh wt at 25°C and are given as means ± SEM. The number of assays performed for each enzyme is shown in parentheses.
increase at the second hour of anaerobiosis reaching control levels thereafter (Fig. 2f). (ii) Krebs cycle metabolites. As can be seen in Fig. 3 the concentration of citrate decreased in the first two hours of anaerobiosis and then remained constant (Fig. 3a). A decrease is also observed in the concentration of oxaloacetate and ct-ketoglutarate at all stages of anaerobiosis (Fig. 3b, d respectively). In contrast, the concentration of malate increased from the first hour of anaerobiosis (Fig. 3c), while that of succinate increased in the period between the second and fourth hour (Fig. 3e). After the fourth hour of anaerobiosis the concentration of malate remained almost constant while that of succinate showed a small decrease. (iii) Amino acids. Compared to all metabolites measured, the free amino acids have the highest concentrations in the foot muscle of P. caerulea at rest. The concentrations of aspartate and alanine, however, changed in an opposite manner during anaerobiosis; that is, aspartate decreased and alanine increased (Fig. 4a, b respectively). The concentration of glutamate increased like that of alanine, but only in the first two hours of anaerobiosis, reaching control levels thereafter (Fig. 4c). (iv) Adenosine phosphates. During exposure of P. caerulea to air the concentration of ATP in the foot muscle decreased in the first two hours (Fig. 5a) and after a slight increase at the fourth hour, Table 3. The maximum activities of enzymes relating to energy metabolism in the foot muscle of P. caerulea Enzyme Glutamate dehydrogenase Malic enzyme Arginine phosphokinase Creatine phosphokinase Pyruvate carboxylase Glucose-6-phosphate dehydrogenase Gluconate-6-phosphate dehydrogenase Nucleoside diphosphate kinase PEP-carboxykinase Octopine dehydrogenase Alanopine dehydrogenase Strombine dehydrogenase Fructose- 1,6-bisphosphatase Glutamate-oxaloacetate transaminase Glutamate-pyruvate transaminase Myofibrillar ATPase
Activity 0.69 ± 0.03 (10) 0.24 ± 0.02 (10) 20.84 _+ 1.72 (9) N.D.* N.D. 1.10 + 0.10(10) 0.40 ± 0.05 (7) 42.86 _+ 3.53 (9) 0.13 + 0.02 (10) N.D. N.D. N.D. 0.08 ± 0.01 (8) 7.06 + 0.27 (10) 3.23 + 0.26 (10) 14.49 _+ 1.20 (6)
*Not detectable, The activities are expressed as #mol/min/g fresh wt at 25°C and are given as means _+ SEM. The number of assays performed for each enzyme is shown in parentheses.
496
BASILE MICHAELIDIS and
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Fig. I. Changes in the concentrations of glycolytic metabolites in the foot muscle of P. caerulea during exposure to air. (a) Glucose; (b) glucose-6-phosphate; (c) fructose-6-phosphate; (d) fructose-l,6-bisphosphate; (e) dihydroxyacetone phosphate; (f) glyceraldehyde-3-phosphate. Each point represents the mean of six determinations. remained almost constant. A D P and A M P concentrations followed similar but reciprocal patterns to that of ATP, showing an initial increase in the first two hours of anaerobiosis and a decrease at the fourth hour (Fig. 5b, c respectively). However, the sum of adenosine phosphates content remained fairly constant at all stages of anaerobiosis. DISCUSSION
From the results of the present study it appears that the foot muscle of P. caerulea has a complete
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sequence of glycolytic enzymes. The high activity of phosphorylase (3.48 _+ 0.23 #mol/min/g fresh wt) in comparison to hexokinase activity (0.47_+ 0.05 pmol/min/g fresh wt) and the considerable activity of P F K (7.93 + 0.65/~mol/min/g fresh wt) support the suggestion that glycogen is the main fuel oxidized. A similar pattern of activities was found by Zammit and Newsholme (1976) in a number of muscles from other marine invertebrates. The increased concentrations of glycolytic intermediates in the foot muscle of P. caerulea in the first four hours of anaerobiosis (Figs 1 and 2) indicate an increased rate of glycolysis.
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duration of anaerobiosis h Fig. 2. Changes in the concentrations of glycolytic metabolites in the foot muscle of P, caerulea during exposure to air. (a) 1,3-Diphosphoglycerate; (b) 3-phosphoglycerate; (c) 2-phosphoglycerate; (d) phosphoenolpyruvate; (e) pyruvat¢; (f) D-lactate. Each point represents the mean of six determinations.
Anaerobic metabolism in Patella muscle
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h
Fig. 3. Changes in the concentrations of Krebs cycle metabolites in the foot muscle of P. caerulea during exposure to air. (a) Citrate; (b) oxaloacetate; (c) malate; (d) ~-ketoglutarate; (e) succinate. Each point represents the mean of six determinations. However, the reduction of aspartate content in the same period of anaerobiosis (Fig. 4a), in combination with the accumulation of alanine (Fig. 4b) and the presence of considerable activities of GOT (7.06+0.27#mol/min/g fresh wt) and GPT
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4 8 lfi duration of anaerobiosis h Fig. 4. Changes in the concentrations of free amino acids in the foot muscle of P. caerulea during exposure to air. (a) Aspartate; (b) alanine; (c) glutamate. Each point represents the mean of six determinations. 2
(3.23 -I- 0.26 #mol/min/g fresh wt), indicate a coupled metabolism of glycogen and aspartate in the foot muscle of P. caerulea during exposure to air. Glycogen and aspartate are considered as the major sources for energy production in the tissues of marine gastropods (Livingstone and de Zwaan, 1983) and bivalves (de Zwaan, 1983) under anoxia leading to accumulation of various end-products including succinate, alanine, octopine, strombine, alanopine, D-lactate, acetate and propionate. Succinate, glutamate and to a lesser extent Dlactate are also accumulated in the foot muscle of P. caerulea during exposure to air. However, their accumulation depends on the stage of anaerobiosis. Specifically, in the first two hours, alanine, glutamate and o-lactate are accumulated (Figs 4b, c and 2f respectively), while in the next two hours succinate and alanine levels are increased (Figs 3e and 4b respectively). This indicates that, in the formation of these end-products, separate metabolic routes are involved after the step of PEP, possibly by the presence of the enzymes PEP-carboxykinase and PK as in the bivalves (de Zwaan, 1977). The slight increase of D-lactate and the accumulation of alanine in the first two hours of anaerobiosis show that a small amount of pyruvate is converted to o-lactate but most of it is transaminated to alanine with aspartate serving as the source of amino groups. The small accumulation of D-lactate is consistent with the low maximal activity of D-LDH measured in the foot muscle of P. caerulea (Table 1). It has been found that o-lactate is mainly accumulated in land molluscs during anaerobiosis (Wieser, 1980; Long et al., 1979), while the transamination of pyruvate to alanine at the expense of aspartate is the main metabolic fate of pyruvate in some marine gastropods (Livingstone and de Zwaan, 1983) and in most marine bivalves (de Zwaan, 1983).
498
BASILEM1CHAELIDISand ]SIDOROSBEIS (
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Fig. 5. Changes in the concentrations of adenosine phosphates and energy charge values (E.C.) in the foot muscle of P. caerulea during exposure to air. (a) ATP; (b) ADP; (c) AMP; (d) energy charge. Each point represents the mean of six determinations. In contrast to bivalves (de Zwaan, 1983), the absence of opine dehydrogenases in the foot muscle excludes the case of pyruvate conversion to octopine, strombine and alanopine in this muscle during anaerobiosis and it is in agreement with the data reported for P. vulgata as regards the existence of these dehydrogenases in the foot muscle (Livingstone et al., 1983). Opine dehydrogenases seem to be present especially in muscles with high energy demand, replacing LDH to reoxidize glycolytic N A D H (Gfide and Grieshaber, 1986). From the examined dehydrogenases in the foot muscle of P. caerulea only malate dehydrogenase measured in the direction of oxaloacetate reduction gave the highest maximal activity (Table 2), showing that reoxidation of glycolytic N A D H by the reaction catalysed by the malate dehydrogenase can supply the demanded N A D ÷ for the maximum glycolytic rate. The accumulation of glutamate in the foot muscle of P. caerulea in the first two hours of anaerobiosis (Fig. 4c) shows that an amount of pyruvate is also converted to glutamate. The presence of citrate synthetase, aconitase, NAD+(P) isocitrate dehydrogenase (Table 2) and glutamate dehydrogenase, which is distributed in both cytosolic and mitochondrial compartments in the foot muscle of P. caerulea (Michaelidis, 1984), in combination with the decrease of citrate concentration in the foot muscle in the same period which glutamate rises, indicates that glutamate is formed by the reactions catalysed by the enzymes of the first part of the Krebs cycle and glutamate dehydrogenase. This metabolic route for glutamate formation and a slight accumulation of glutamate had been observed in the tissues of some marine molluscs during anaerobiosis (de Zwaan and van Marrewijk, 1973; de Zwaan et al., 1981; Widdows et al., 1979). It has been suggested that the role of glutamate dehydrogenase is related to the reoxidation of cytosolic N A D H at the onset of anaerobiosis if insufficient substrates are available for other cytosolic dehydrogenases (de Zwaan, 1983).
No accumulation of o-lactate (Fig. 2f) and glutamate (Fig. 4c) is observed in the foot muscle in the period between the second and fourth hour of anaerobiosis. In addition, during the same period the concentration of pyruvate remains constant (Fig. 2e), while that of PEP and succinate increases (Figs 2d and 3e respectively). These observations indicate that PK activity is inhibited after the second hour of anaerobiosis. It has been demonstrated that inhibition of PK in the foot muscle is caused by the phosphorylation of enzyme (Michaelidis et al., 1988) and the inhibitors [H ÷] and alanine (Michaelidis et al., 1985). In particular, after the second hour of P. caerulea exposure to air, the degree of phosphorylation of PK from the foot muscle increased, resulting in a less active form as regards the substrate PEP and a more sensitive form with respect to inhibition by alanine (Michaelidis et al., 1988). Similar to the present results, Storey et al. (1989) have found that the glycolytic rate is reduced in the foot of the marine whelk Busycotypus canaliculatum in the first two hours of anoxia and that phosphorylation of PK plays a key role in depression of glycolytic rate. Although PK is inhibited after the second hour of anaerobiosis, the concentration of alanine continues to increase (Fig. 4b), while that of aspartate decreases (Fig. 4a). In addition, the concentration of succinate increases after this period of anaerobiosis (Fig. 3e). At this point the question of how alanine is produced at the expense of aspartate after PK has been inhibited arises, de Zwaan et al. (1983) postulated a modified reaction scheme of the classical aspartatemalate cycle in order to explain the accumulation of alanine and succinate at the expense of aspartate in the tissues of Mytilus edulis under anoxia. In this reaction scheme malate derived from the reduction of oxaloacetate can follow two different metabolic routes in mitochondria. Firstly, it can be decarboxylated to pyruvate by malic enzyme and then to alanine by GPT. Secondly, it can be converted to succinate by the first part of the Krebs cycle operating in the
Anaerobic metabolism in Patella muscle opposite direction. Both malic enzyme and GPT are located in the mitochondria of P. caerulea (Michaelidis, 1984), indicating that such a mechanism of alanine and succinate production at the expense of aspartate could operate in this muscle after the inhibition of PK. An alternative route of succinate production in the foot muscle of P. caerulea could be the conversion of PEP to oxaloacetate by PEP-carboxykinase and consequently to succinate via the first part of the Krebs cycle as has been reported above. The reaction catalysed by PEP-carboxykinase results in the production of an extra high energy nucleotide (possible ITP) which is converted to ATP by the nucleoside diphosphate kinase (de Zwaan, 1977). Both PEP-carboxykinase and nucleoside diphosphate kinase are present in the foot muscle of P. caerulea, showing the existence of the above described metabolic route of succinate production. PEP-carboxykinase displays optimum activity at low pH values (pH 6.9) (Michaelidis, 1984) compared to that of PK (pH 7.5) (Michaelidis et al., 1985). So, after the first two hours of anaerobiosis, phos° phorylation of PK and the increased concentrations of alanine and [H ÷] could result in inhibition of enzyme activity. This would enable PEP- carboxykinase to compete with PK for the substrate PEP, although the former enzyme has a lower maximal acitivity (Table 3) compared to the latter (Table 1). Prolonged anoxia results in conversion of succinate to propionate in bivalves (Schulz et al., 1982). In the present work the case of succinate conversion to propionate was not studied. As far as the rest of the enzymes of Table 3 are concerned, it is shown that gluconeogenesis does not take place in this muscle, since pyruvate carboxylase is not detectable and because of the very low activity of fructose-l,6-bisphosphatase. The presence of glucose-6-phosphate and gluconate-6-phosphate dehydrogenase indicate that the pentose phosphate pathway could operate in this muscle. However, the amount of phosphoexose residues which are metabolized through this pathway is unknown at present and is under investigation. The presence of arginine phosphokinase instead or creatine phosphokinase is in accordance with results obtained from other muscles of marine molluscs (Zammit and Newsholme, 1976), indicating that arginine phosphate reserves are oxidized during the first stages of muscle contraction for ATP supply. The low concentration of adenosine phosphates measured in P. caerulea are consistent with the low rate of energy expenditure by these muscles during muscular contraction (Riiegg, 1971; Baguet and Gilles, 1968). The increase of energy charge, however, in the period between the second and fourth hours of anaerobiosis (Fig. 5d) indicates that during this period an ATP producing metabolic process is activated. This, in combination with the increase of succinate concentration during the same period, indicates that the succinate pathway must be responsible for the observed increase of ATP concentration in the foot muscle of P. caerulea from the second to the fourth hour of anaerobiosis.
499 REFERENCES
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