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The phase relationship of glycogen and free fatty acids in tissues of aestivating giant African snails (Achatina achatina)
Comp. Biochem. Physiol. Vol. 78B, No. 2, pp. 461-466, 1984 Printed in Great Britain
0305-0491/84 $3.00 + 0.00 (~' 1984 Pergamon Press Ltd
THE PHASE R E L A T I O N S H I P OF G L Y C O G E N A N D FREE F A T T Y ACIDS IN TISSUES OF A E S T I V A T I N G G I A N T A F R I C A N SNAILS (ACHA TINA A CHA TINA ) *GABRIEL M. UMEZURIKE and EUGENE N. |HEANACHO Department of Biochemistry, University of Nigeria, Nsukka, Nigeria
(Received 29 September 1983) Abstract--1. The phase relationships of glycogen and free fatty acids (FFA) in the digestive (midgut)
gland, foot muscle and heart muscle of starved and aestivating giant African snails (Achatina achatina) were analysed by means of phase plane plots. 2. Phase plane plots of glycogen against FFA content indicated that these metabolites were out of phase in the digestive gland but in phase in the foot muscle, whereas the plot for the heart muscle had a long axis parallel to the abscissa. 3. Digestive gland glycogen was out of phase with foot muscle FFA and with heart muscle FFA. 4. Phase plane plots of foot muscle glycogen against digestive gland FFA had its long axis parallel to the abscissa, whereas the plot of foot muscle glycogen against heart muscle FFA showed that these metabolites were in phase. 5. Heart muscle FFA was in phase with either foot muscle FFA or digestive gland FFA. 6. Pyruvate kinase extracted from the foot muscle of starved and aestivating snails was very labile and behaved like the allosteric type by exhibiting co-operative interaction with respect to phosphoenolpyruvate, allosteric activation by fructose 1,6-diphosphate and inhibition by ATP. 7. The oscillatory motions of glycogen and FFA are discussed in terms of anaerobic metabolism in aestivating snails.
INTRODUCTION Oscillations of metabolic intermediates have been shown to occur during glycolysis, and these oscillations have been attributed to the regulatory properties of phosphofructokinase in yeast cells and beef heart extracts (Higgins, 1964; Betz and Chance, 1965; Frenkel, 1968). These studies have also indicated that a number of effector molecules (ATP, N A D H , etc) are involved in the regulation of oscillatory motions of glycolytic intermediates (Betz and Chance, 1965; Frenkel, 1968). It has been observed that in molluscan tissues alanine, succinate and fatty acids are accumulated as major end-products and lactate as a minor endproduct under anaerobic conditions, and that the pathway of anaerobic glucose breakdown involves a cytoplasmic reduction of oxaloacetate formed by carboxylation of phosphoenolpyruvate (obtained by the normal glycolytic sequence) and mitochondrial fumarate reductase system as in parasitic helminths (Simpson and Awapara, 1966; Stokes and Awapara, 1968; Chen and Awapara, 1969; de Zwaan and Van Marrewijk, 1973; Oudejans and Van der Horst, 1974; Van der Horst, 1974; M c M a n u s and James, 1975, Umezurike and Iheanacho, 1983). A metabolic scheme showing the mechanism for fatty acid synthesis in aestivating Achatina achatina has been proposed (Umezurike and Iheanacho, 1983). In view of the involvement of the glycolytic sequence in the proposed scheme, and of the observed changes in the contents of glycogen and free fatty *Present address: School of Biological Sciences, Imo State University, P. M. B. 2000, Etiti, Imo State, Nigeria. 461
acids ( F F A ) during aestivation in the tissues of A. achatina (Umezurike and Iheanacho, 1983), the oscillatory characteristics of glycogen and F F A in aestivating snails were investigated by examining the phase relationship of these metabolites in different tissues. To the best of our knowledge, no similar reports are available in the literature. MATERIALS AND METHODS
Materials A number of snails (Achatina achatina), bought from the local market, were starved and induced to aestivate as described previously (Umezurike and Iheanacho, 1983). All chemicals used were obtained from Sigma (London) Chemical Company Ltd., Kingston-upon-Thames, Surrey, UK. Glycogen and free fatty acid estimation The estimation of tissue glycogen and free fatty acids was carried out as described previously (Umezurike and Anya, 1978; Anya and Umezurike, 1978; Iwuala et al., 1981). The standard deviation of 2-3 replicates was not more than 10% of the mean values. Pyruvate kinase extraction About 150g of foot muscle from starved (18 days) and aestivating snails were chilled at 0°C and then minced and homogenized at 4°C in 50 mM Tris-HC1 (pH 7.5) containing 0.5mM EDTA (ethylenediaminetetra-acetate). The homogenate was squeezed through layers of cheese-cloth and then centrifuged at 10,000g for 30rain at 4°C. The supernatant solution was then subjected to (NH4)2SO 4 fractionation. The 30-60% saturated (NH4)2SO 4 fraction was dissolved in the extraction buffer and used for the studies reported here as attempts to further purify the enzyme by dialysis and gel-filtration on a column of Sephadex G-200 led to loss of enzymic activity.
462
GABRIEL M.
UMEZURIKE and
EU(IENE N, IHEANA('HO
parallel to the abscissa. Only slight changes in glyAssay (?['pvruvale kinase Pyruvate kinase activity was determined spec- cogen content accompanied changes in FFA content, trophotometrically at 30C in a Pye-Unicam SP.500 spec- indicating that the heart muscle was probably utiliztrophotometer fitted with a Unicam AR25 linear recorder in ing metabolites supplied by other tissues. The phase a system coupled to lactate dehydrogenase as described by plane plot for the digestive gland (Fig. 3b) consists of Stewart and Moore (1971) except that 50mM Tris HC1 a clockwise spiral, the long axis of which is inclined at buffer (pH 7.8) was used. Enzyme activity was expressed as 135", indicating that glycogen and FFA were out ot" nmol/min/ml sample. phase. The results presented in Figs 2 and 3a show that RESULTS during starvation and aestivation (up to 24 days) the foot muscle contained about 15 times more glycogen Changes in glycogen and ji'ee jcttty acid content than the heart muscle (450 600 rag/100 g foot muscle The results of studies on the effect of starvation as against 3~ 44 mg/100 g heart muscle), whereas lhe and aestivation on the glycogen and free fatty acid heart muscle contained about 10 limes more FFA (FFA) content of the foot muscle, digestive gland than the toot muscle (1.7 2.5mol 100g foot mus(midgut gland) and heart muscle of A. achatina are cle as against 10.0 18.4mol/100g heart muscle). presented in Fig. 1 as plots of the ratio of FFA The corresponding values for the digestive gland content to glycogen content against the duration of were 59.,V154.4mg glycogen/100g tissue and starvation. There are obvious oscillations in the ratio 16.6 28.2 tool FFA/100g tissue. In view of the lo~ for the foot muscle, digestive gland and heart muscle. glycogen content of the heart muscle of starved and The phase relationships of glycogen and FFA content aestivating snails and the possibility that some in the various tissues were determined by preparing metabolites (e.g. glucose, lactate, succinate and alaphase plane plots of glycogen content against FFA nine) derived from the digestive gland could be content, as it has been established that the phase plane plot gives the clearest graphical representation of the nature of oscillating metabolites (Betz and "~ 7(x Chance, 1965). The phase plane plot of glycogen content against 60( FFA content for the foot muscle (Fig. 2) has two g o sections (0-10 days and 13-24 days) each ot" which ~ 5o( 7 consists of a clockwise spiral, the long axis of which E is inclined at 45 '~, indicating that glycogen and FFA ~L.0£ o contents were in phase in each section. However, the ' ' >, 1!8 22 26 J.o second section (13 24 days) was operating at a higher Free Fatty Acids{mol/100g tissuel glycogen range than the first section. On the contrary, the phase plane plot for the heart muscle (Fig. 3a) Fig. 2. Phase plane plot of glycogen content against free gave an anti-clockwise spiral, the long axis of which is fatty acid (FFA) content in the loot muscle of starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation.
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Fig. I. Plots of the ratio of free fatty acid content to glycogen content against duration of starvation and aestivation in Achatina achatina heart muscle (©), digestive gland (@) and foot muscle (A).
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Fig. 3. Phase plane plots of glycogen content against free fatty acid (FFA) content in (a) the heart muscle and (b) the digestive gland of starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation.
Phase relationship of metabolites in Achatina transported in the haemolymph to the heart muscle and foot muscle for further metabolism to generate F F A , phase plane plots of digestive gland glycogen content against foot muscle F F A (Fig. 4a) or against heart muscle F F A (Fig. 4b) and foot muscle glycogen content against digestive gland F F A (Fig. 5a) or against heart muscle F F A content (Fig. 5b) were prepared. The plot of digestive gland glycogen content against foot muscle F F A content (Fig. 4a) or against heart muscle F F A (Fig. 4b) indicated that the digestive gland glycogen was out of phase with foot muscle F F A or with heart muscle FFA. A plot of digestive gland glycogen against foot muscle glycogen gave a similar result, indicating that digestive gland glycogen was out of phase with foot muscle glycogen. The phase plane plot of foot muscle glycogen content against digestive gland F F A content (Fig. 5a) consists of two sections of clockwise spirals the long axes of which are parallel to the abscissa, whereas the plot of foot muscle glycogen content against heart muscle F F A content (Fig. 5b) consists of two clockwise spirals inclined at 45 °. Fig. 5b indicates that the foot muscle glycogen content was increasing as the heart muscle F F A content was also increasing, and vice versa. However, Fig. 5a indicates that digestive gland F F A content was changing even when foot muscle glycogen content had reached its minima or maxima. Put together, these results suggest that the digestive gland was breaking down glycogen and providing metabolites (e.g. succinate, alanine or glucose) to the heart and foot muscles for F F A or glycogen synthesis. F F A may then be transported to the digestive gland for lipid synthesis. Thus a plot of heart muscle F F A against foot muscle F F A or against digestive
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Fig. 4. Phase plane plots of digestive gland glycogen content against (a) foot muscle free fatty acid (FFA) content or (b) heart muscle free fatty acid (FFA) content in starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation. C.B.P. 78/2B K
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Fig. 5. Phase plane plots of foot muscle glycogen content against (a) digestive gland free fatty acid (FFA) content or (b) heart muscle free fatty acid (FFA) content in starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation.
gland F F A (not shown) gave anti-clockwise spirals inclined at 45 °, indicating that F F A in the heart muscle was in phase with F F A in the foot muscle or digestive gland. Properties of pyruvate kinase
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Both pyruvate kinase and PEP carboxykinase activities were detected in the cytosol fraction derived from the foot muscle of starved and aestivating A. achatina (Umezurike and Iheanacho, 1983). It is well established that pyruvate kinase is an important regulatory enzyme in some organisms (cf. Hardie, 1981; Lehninger, 1982). The properties of pyruvate kinase extracted from the foot muscle of starved and aestivating snails were therefore studied. The 30-60~ saturated (NH4)2SO4 fraction obtained from the crude extract of starved and aestivating foot muscle was used in these studies as exhaustive dialysis at 4°C or gel-filtration of this fraction on Sephadex G-200 resulted in complete loss of pyruvate kinase activity. The relationship between initial velocity and the concentration of PEP in the absence and presence of fructose 1,6-diphosphate (FDP) or of ATP (adenosine triphosphate) are shown in Fig. 6. In the absence of FDP the curve was markedly sigmoidal. The sigmoidicity was increased in the presence of 0.5 mM ATP but was completely abolished in the presence of 5.0 mM FDP. Double-reciprocal plots were curved (concave-up) in the absence of F D P and in the presence of ATP but became linear in the presence of 5.0 mM FDP. Figure 7 shows the Hill plots of log (V/Vma x - - V ) against log [PEP]. The Hill cofficient (n) was calculated to be 1.5, 1.8 and 1.0 in the absence of FDP, presence of 5.0 mM ATP and presence of 5.0mM FDP respectively. Similar changes in substrate cooperativity have been oh-
464
GABRIEL M. UMEZURIKE a n d EUGENE N. IHEANACH{)
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Fig. 6. Effect of phosphoenolpyruvate (PEP) concentration on the activity of pyruvate kinase from the foot muscle of starved and aestivating Achatina achatina in the absence of fructose 1,6-diphosphate (O), presence of 5.0 mM fructose 1,6-diphosphate (O) and presence of 5.0 mM ATP (A).
°/~
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Fig. 8. Scheme of anaerobic carbohydrate metabolism in Achatina achatina. F6P, fructose &phosphate; FDP, fruc-
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-io-o'.s-o16-o!~-o!2 o!o o'.2 o'~ log[PEP]
Fig. 7. Hill plots of the activity of pyruvate kinase from the foot muscle of starved and aestivating Achatina achatina in the presence of various concentrations of PEP, (O) without fructose 1,6-diphosphate (FDP); ((D) with 5.0mM FDP and (A) with 5.0 mM ATP.
served with pyruvate kinase from a number of sources (Hess et al., 1966; Bailey et al., 1968; Stewart and Moore, 1971). The apparent Km for PEP was 0.75mM in the absence of FDP, 0.19mM in the presence of 5.0 mM F D P and 1.15 mM in the presence of 5.0 mM ATP. The inhibitory effect of 2 mM ATP on the activity of pyruvate kinase in the presence of increasing concentrations of PEP was virtually abolished in the presence of 5.0 mM FDP.
DISCUSSION
Studies on the steady-state content of some metabolites and the intracellular distribution and levels of activity of some major enzymes of anaerobic metabolism in the foot muscle of starved and aestivating A. achatina have indicated that this snail is a facultative anaerobe. Succinate, lactate, alanine and free fatty
tose 1,6-diphosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; ALA, alanine; LAC, lactate; MAL, malate; OAA. oxaloacetate; FFA, free fatty acids: Ac-CoA. acetyl-CoA: CIT, citrate; FUM, fumarate: SUC, succinate. (A) and (B) denote routes A and B for entry of metabolites into the mitochondria respectively. The numbered reactions are catalysed by the enzymes: 1, pyruvate kinase: 2, PEP carboxykinase; 3, malate dehydrogenase; 4, malic enzyme: 5, transaminase; 6, lactate dehydrogenase; 7, fumarase: 8, fumarate reductase; 9, malate dehydrogenasc: 10, malic enzyme; l I, pyruvate carboxylase: 12, pyruvale dehydrogenase complex; 13, citrate synthase: 14, ATP citrate lyase.
acids were found to be present in the foot muscle as end-products of anaerobic metabolism (Umezurike and Iheanacho, 1983), and the lactate dehydrogenase from the foot muscle was more active in the direction of lactate oxidation than in that of pyruvate reduction (Umezurike and Eke, 1983). The metabolic cycle proposed as a working scheme for anaerobic fatty acid synthesis (Umezurike and Iheanacho, 1983) is shown in a simplified form in Fig. 8. Each metabolic cycle involves enzymecatalysed reactions occurring in both the cytosol and mitochondria. Malate and pyruvate derived from carbohydrates (or pyruvate derived from lactate and alanine) enter the mitochondria by the action of dicarboxylate carriers. Succinate is then formed as end-product from malate via fumarate. The metabolites eventually give rise to citrate which is then transported out of the mitochondria by a tricarboxylate carrier. The acetyl-CoA formed from citrate by the action of ATP-citrate lyase is used for
465
Phase relationship of metabolites in Achatina synthesis of free fatty acids as end-products, and oxaloacetate reduction completes the cycle. Oxaloacetate may also be removed for glycogen synthesis. The results presented in Figs 1-5 indicate that only two damped oscillatory motions of glycogen and free fatty acids have been completed in 24 days of starvation and aestivation by A. achatina. Under conditions of aestivation, the foot becomes dormant whereas the heart continues beating, albeit at a reduced rate (Borradaile et al., 1963). More ATP would thus be needed to sustain the higher activity of the heart muscle relative to that of the inactive foot muscle. In starved and aestivating snails ATP is generated at the fumarate reductase step (reaction 8 in Fig. 8) if pyruvate kinase is inhibited, as indicated by studies on the succinate-DCPIP and NADH-fumarate oxidoreductases associated with the membrane of submitochondrial particles derived from the foot muscle of A. achatina (Umezurike and Chilaka, 1982). The pyruvate kinase from the foot muscle of aestivating A. achatina behaved like the regulatory or allosteric type in showing co-operative interaction with respect to PEP, specific allosteric activation by FDP and inhibition by ATP as in some other organisms (cf. Hess et al., 1966; Tanako et al., 1967a,b; Pogson, 1968; Stewart and Moore, 1971). Pyruvate kinase from gluconeogenic tissues are known to be strongly and specifically activated by FDP (Gancedo et al., 1967). Pyruvate kinase from some sources is inhibited by acetyl-CoA, long chain fatty acids, alanine and ATP (cf. Lehninger, 1982). Furthermore, the enzyme from some animal tissues is also subject to regulation by the hormone glucagon which increases the level of cyclic AMP, leading to reversible phosphorylation of pyruvate kinase by cyclic AMPdependent protein kinase (cf. Hardie, 1981). Pyruvate kinase is thus one of the candidates for the regulation of the flux and oscillatory motions of metabolites in the tissues of aestivating snails. Furthermore, the mechanism for fatty acid synthesis would be turned off if as in some animal tissues acetyl-CoA carboxylase, the enzyme which catalyses the rate-limiting step for fatty acid synthesis, is subject to inactivation by reversible phosphorylation or to allosteric inhibition by long-chain fatty acylCoA (cf. Hardie, 1981). The positive effector (citrate) and the negative effector (long-chain fatty acyl-CoA) of this enzyme are competitive with each other (cf. Hardie, 1981). The anaerobic metabolic cycle depicted in Fig. 8 thus explains satisfactorily the oscillatory characteristics of glycogen and free fatty acids in starved and aestivating A. achatina, as the mechanism for glycogen or fatty acid synthesis can be switched on and off depending on the levels of control intermediates (ATP, NADH, NADPH, and the effectors of regulatory enzymes) in the various tissues. Differences in the levels of these control intermediates would determine the metabolic routes (cf. Fig. 8) that predominate in these tissues. This would also account for the predominance of glycogen in the foot muscle and of free fatty acids in the heart muscle, and for the apparent prominence of both glycogen and free fatty acids in the digestive gland.
REFERENCES
Anya A. O. and Umezurike G. M. (1978) Respiration and carbohydrate energy metabolism of the lung-dwelling parasite Rhabdias bufonis (Nematoda: Rhabdiasoidea). Parasitology 76, 21-27. Bailey E., Stirpe F. and Taylor C. B. (1968) Regulation of rat liver pyruvate kinase. Biochem. J. 108, 427-436. Betz A. and Chance B. (1965) Phase relationship of glycolytic intermediates in yeast ceils with oscillatory metabolic control. Archs Biochem. Biophys. 109, 585-594. Borradaile L. A., Potts F. A., Eastham L. E. S., Saunders J. T. and Kerkut G. A. (1963) The Invertebrata, 4th edn., p. 610. Cambridge University Press, Cambridge. Chen C. and Awapara J. (1969) Intracellular distribution of enzymes catalysing succinate production from glucose in Rangia mantle. Comp. Biochem. Physiol. 30, 727-737. Frenkel R. (1968) Control of reduced diphosphopyridine nucleotide oscillations in beef heart extracts III. Purification and kinetics of beef heart phosphofructokinase. Archs Biochem. Biophys. 125, 166-174. Gancedo J. M., Gancedo C. and Sols A. (1967) Regulation of the concentration and activity of pyruvate kinase in yeasts and its relationship to gluconeogenesis.Biochem. J. 102, 23C. Hardie G. (1981) Fat and phosphorylation--the role of covalent enzyme modification in lipid synthesis. Trends Biochem. Sci. 6, 75-77. Hess B., Haeckell R. and Brand K. (1966) FDP-activation of yeast pyruvate kinase. Biochem. biophys. Res. Commun. 24, 824-831. Higgins J. (1964) A chemical mechanism for oscillation of glycolytic intermediates in yeast cells. Proc. Natn. Acad. Sci. U.S.A. 51, 989-994. Iwuala M. O. E., Umezurike G. M. and Nwadiogbu N. (1981) Quantitative studies on the proteins, free fatty acids and glycogen contents of eggs of Amblyomma variegatum and Boophilus decoratus (Koch) ticks, Ixodoidea: Ixodidae). Acarologia 22, 133-143. Lehninger A. L. (1982) Principles of Biochemistry, pp. 424-425. Worth Publishers, Inc., New York. McManus D. P. and James B. L. (1975) Anaerobic glucose metabolism in the digestive gland of Littorina saxatilis rudis (Maton) and in the daughter sporocysts of Microphallus similis (Jag) (Digenea: Microphallidae). Comp. Biochem. Physiol. 51B, 293-297. Oudejans R. and Van der Horst D. (1974) Biosynthesis of fatty acids in the pulmonate land snail, Cepaea nemoralis. Comp. Biochem. Physiol. 47B, 139-147. Pogson C. I. (1968) Adipose tissue pyruvate kinase. Biochem. J. 110, 67-77. Simpson J. W. and Awapara J. (1966) The pathway of glucose degradation in some invertebrates. Comp. Biochem. Physiol. 18, 537-548. Stewart G. R. and Moore D. (1971) Factors affecting the level and activity of pyruvate kinase from Coprinus lagopus sensu Buller. J. gen. Microbiol. 66, 361-370. Stokes T. M. and Awapara J. (1968) Alanine and succinate as end-products of glucose degradation in the clam Rangia cuneata. Comp. Biochem. Physiol. 25, 883892. Tanako T., Harano Y., Sue F. and Morimura H. (1967a) Crystallization, characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J. Biochem., Tokyo 62, 71-91. Tanako T., Harano Y., Sue F. and Morimura H. (1967b) Evidence for the presence of two types of pyruvate kinase in rat liver. Biochem. biophys. Res. Commun. 21, 5560. Umezurike G. M. and Anya A. O. (1978) Studies on energy metabolism in the endoparasite Strongyluris brevicaudata (Nematoda). Comp. Biochem. Physiol. 5OB, 147-151. Umezurike G. M. and Chilaka F. C. (1982)
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GABRIEL M. UMEZURIKEand EUGENE N. IHEANA('HO
Succinate-DCPIP and NADH-fumarate oxidoreductases in submitochondrial particles of the giant African snail (Achatina achatina) foot muscle. Comp. Biochem. Physiol. 71B, 181-185. Umezurike G. M. and Eke L. O. (1983) Some properties of lactate dehydrogenase from the foot muscle of the giant African snail (Achatina achatina ). Comp. Biochem. Physiol. 7513, 263-268. Umezurike G. M. and Iheanacho E. N. (1983) Metabolic
adaptations in aestivating giant African snail (Achatina achatma). Comp. Biochem. Physiol. 74B, 493-498. Van der Horst D. (1974) In citro biosynthesis of fatty acids in the pulmonate land snail ('epaca m'moralis under anoxic conditions. ('omp. Biochem. Physiol. 47B, 181 187. Zwaan A. de and Van Marrewijk W. J. A. (1973) Anaerobic glucose degradation m the sea mussel Ml'tilus edu/is k. Comp. Biochem. Physiol. 44B, 429 439.
0305-0491/84 $3.00 + 0.00 (~' 1984 Pergamon Press Ltd
THE PHASE R E L A T I O N S H I P OF G L Y C O G E N A N D FREE F A T T Y ACIDS IN TISSUES OF A E S T I V A T I N G G I A N T A F R I C A N SNAILS (ACHA TINA A CHA TINA ) *GABRIEL M. UMEZURIKE and EUGENE N. |HEANACHO Department of Biochemistry, University of Nigeria, Nsukka, Nigeria
(Received 29 September 1983) Abstract--1. The phase relationships of glycogen and free fatty acids (FFA) in the digestive (midgut)
gland, foot muscle and heart muscle of starved and aestivating giant African snails (Achatina achatina) were analysed by means of phase plane plots. 2. Phase plane plots of glycogen against FFA content indicated that these metabolites were out of phase in the digestive gland but in phase in the foot muscle, whereas the plot for the heart muscle had a long axis parallel to the abscissa. 3. Digestive gland glycogen was out of phase with foot muscle FFA and with heart muscle FFA. 4. Phase plane plots of foot muscle glycogen against digestive gland FFA had its long axis parallel to the abscissa, whereas the plot of foot muscle glycogen against heart muscle FFA showed that these metabolites were in phase. 5. Heart muscle FFA was in phase with either foot muscle FFA or digestive gland FFA. 6. Pyruvate kinase extracted from the foot muscle of starved and aestivating snails was very labile and behaved like the allosteric type by exhibiting co-operative interaction with respect to phosphoenolpyruvate, allosteric activation by fructose 1,6-diphosphate and inhibition by ATP. 7. The oscillatory motions of glycogen and FFA are discussed in terms of anaerobic metabolism in aestivating snails.
INTRODUCTION Oscillations of metabolic intermediates have been shown to occur during glycolysis, and these oscillations have been attributed to the regulatory properties of phosphofructokinase in yeast cells and beef heart extracts (Higgins, 1964; Betz and Chance, 1965; Frenkel, 1968). These studies have also indicated that a number of effector molecules (ATP, N A D H , etc) are involved in the regulation of oscillatory motions of glycolytic intermediates (Betz and Chance, 1965; Frenkel, 1968). It has been observed that in molluscan tissues alanine, succinate and fatty acids are accumulated as major end-products and lactate as a minor endproduct under anaerobic conditions, and that the pathway of anaerobic glucose breakdown involves a cytoplasmic reduction of oxaloacetate formed by carboxylation of phosphoenolpyruvate (obtained by the normal glycolytic sequence) and mitochondrial fumarate reductase system as in parasitic helminths (Simpson and Awapara, 1966; Stokes and Awapara, 1968; Chen and Awapara, 1969; de Zwaan and Van Marrewijk, 1973; Oudejans and Van der Horst, 1974; Van der Horst, 1974; M c M a n u s and James, 1975, Umezurike and Iheanacho, 1983). A metabolic scheme showing the mechanism for fatty acid synthesis in aestivating Achatina achatina has been proposed (Umezurike and Iheanacho, 1983). In view of the involvement of the glycolytic sequence in the proposed scheme, and of the observed changes in the contents of glycogen and free fatty *Present address: School of Biological Sciences, Imo State University, P. M. B. 2000, Etiti, Imo State, Nigeria. 461
acids ( F F A ) during aestivation in the tissues of A. achatina (Umezurike and Iheanacho, 1983), the oscillatory characteristics of glycogen and F F A in aestivating snails were investigated by examining the phase relationship of these metabolites in different tissues. To the best of our knowledge, no similar reports are available in the literature. MATERIALS AND METHODS
Materials A number of snails (Achatina achatina), bought from the local market, were starved and induced to aestivate as described previously (Umezurike and Iheanacho, 1983). All chemicals used were obtained from Sigma (London) Chemical Company Ltd., Kingston-upon-Thames, Surrey, UK. Glycogen and free fatty acid estimation The estimation of tissue glycogen and free fatty acids was carried out as described previously (Umezurike and Anya, 1978; Anya and Umezurike, 1978; Iwuala et al., 1981). The standard deviation of 2-3 replicates was not more than 10% of the mean values. Pyruvate kinase extraction About 150g of foot muscle from starved (18 days) and aestivating snails were chilled at 0°C and then minced and homogenized at 4°C in 50 mM Tris-HC1 (pH 7.5) containing 0.5mM EDTA (ethylenediaminetetra-acetate). The homogenate was squeezed through layers of cheese-cloth and then centrifuged at 10,000g for 30rain at 4°C. The supernatant solution was then subjected to (NH4)2SO 4 fractionation. The 30-60% saturated (NH4)2SO 4 fraction was dissolved in the extraction buffer and used for the studies reported here as attempts to further purify the enzyme by dialysis and gel-filtration on a column of Sephadex G-200 led to loss of enzymic activity.
462
GABRIEL M.
UMEZURIKE and
EU(IENE N, IHEANA('HO
parallel to the abscissa. Only slight changes in glyAssay (?['pvruvale kinase Pyruvate kinase activity was determined spec- cogen content accompanied changes in FFA content, trophotometrically at 30C in a Pye-Unicam SP.500 spec- indicating that the heart muscle was probably utiliztrophotometer fitted with a Unicam AR25 linear recorder in ing metabolites supplied by other tissues. The phase a system coupled to lactate dehydrogenase as described by plane plot for the digestive gland (Fig. 3b) consists of Stewart and Moore (1971) except that 50mM Tris HC1 a clockwise spiral, the long axis of which is inclined at buffer (pH 7.8) was used. Enzyme activity was expressed as 135", indicating that glycogen and FFA were out ot" nmol/min/ml sample. phase. The results presented in Figs 2 and 3a show that RESULTS during starvation and aestivation (up to 24 days) the foot muscle contained about 15 times more glycogen Changes in glycogen and ji'ee jcttty acid content than the heart muscle (450 600 rag/100 g foot muscle The results of studies on the effect of starvation as against 3~ 44 mg/100 g heart muscle), whereas lhe and aestivation on the glycogen and free fatty acid heart muscle contained about 10 limes more FFA (FFA) content of the foot muscle, digestive gland than the toot muscle (1.7 2.5mol 100g foot mus(midgut gland) and heart muscle of A. achatina are cle as against 10.0 18.4mol/100g heart muscle). presented in Fig. 1 as plots of the ratio of FFA The corresponding values for the digestive gland content to glycogen content against the duration of were 59.,V154.4mg glycogen/100g tissue and starvation. There are obvious oscillations in the ratio 16.6 28.2 tool FFA/100g tissue. In view of the lo~ for the foot muscle, digestive gland and heart muscle. glycogen content of the heart muscle of starved and The phase relationships of glycogen and FFA content aestivating snails and the possibility that some in the various tissues were determined by preparing metabolites (e.g. glucose, lactate, succinate and alaphase plane plots of glycogen content against FFA nine) derived from the digestive gland could be content, as it has been established that the phase plane plot gives the clearest graphical representation of the nature of oscillating metabolites (Betz and "~ 7(x Chance, 1965). The phase plane plot of glycogen content against 60( FFA content for the foot muscle (Fig. 2) has two g o sections (0-10 days and 13-24 days) each ot" which ~ 5o( 7 consists of a clockwise spiral, the long axis of which E is inclined at 45 '~, indicating that glycogen and FFA ~L.0£ o contents were in phase in each section. However, the ' ' >, 1!8 22 26 J.o second section (13 24 days) was operating at a higher Free Fatty Acids{mol/100g tissuel glycogen range than the first section. On the contrary, the phase plane plot for the heart muscle (Fig. 3a) Fig. 2. Phase plane plot of glycogen content against free gave an anti-clockwise spiral, the long axis of which is fatty acid (FFA) content in the loot muscle of starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation.
~
0.5
80,
,/"°"~o ~
O
°
[a]
6C 0
C
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7
~ 2o O
0.3
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Free Fatty Acids (mol/100g tissue] 18C
(b)
~ t~C ~100,
I.l-
r 60 o° 20 )1
0
6
12
18
24
DAYS
Fig. I. Plots of the ratio of free fatty acid content to glycogen content against duration of starvation and aestivation in Achatina achatina heart muscle (©), digestive gland (@) and foot muscle (A).
16
1~8
21O
212 24
2~6
I
28
I
30
Free Fatty Acids (mol/lO0g tissue)
Fig. 3. Phase plane plots of glycogen content against free fatty acid (FFA) content in (a) the heart muscle and (b) the digestive gland of starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation.
Phase relationship of metabolites in Achatina transported in the haemolymph to the heart muscle and foot muscle for further metabolism to generate F F A , phase plane plots of digestive gland glycogen content against foot muscle F F A (Fig. 4a) or against heart muscle F F A (Fig. 4b) and foot muscle glycogen content against digestive gland F F A (Fig. 5a) or against heart muscle F F A content (Fig. 5b) were prepared. The plot of digestive gland glycogen content against foot muscle F F A content (Fig. 4a) or against heart muscle F F A (Fig. 4b) indicated that the digestive gland glycogen was out of phase with foot muscle F F A or with heart muscle FFA. A plot of digestive gland glycogen against foot muscle glycogen gave a similar result, indicating that digestive gland glycogen was out of phase with foot muscle glycogen. The phase plane plot of foot muscle glycogen content against digestive gland F F A content (Fig. 5a) consists of two sections of clockwise spirals the long axes of which are parallel to the abscissa, whereas the plot of foot muscle glycogen content against heart muscle F F A content (Fig. 5b) consists of two clockwise spirals inclined at 45 °. Fig. 5b indicates that the foot muscle glycogen content was increasing as the heart muscle F F A content was also increasing, and vice versa. However, Fig. 5a indicates that digestive gland F F A content was changing even when foot muscle glycogen content had reached its minima or maxima. Put together, these results suggest that the digestive gland was breaking down glycogen and providing metabolites (e.g. succinate, alanine or glucose) to the heart and foot muscles for F F A or glycogen synthesis. F F A may then be transported to the digestive gland for lipid synthesis. Thus a plot of heart muscle F F A against foot muscle F F A or against digestive
~
{a)
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160
.~ 40 Jl
~ 2oo
I 1.7
I I i I i 1.9 2'1 23 2"5 2.7 Foot muscle FFAlmol/100g tissue)
219
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o 80 ~
4o
o
0
'~
~ Heort
1~
~'2 1~
1'6
t'8 2'0
muscle FFA(moW100g tissuel
Fig. 4. Phase plane plots of digestive gland glycogen content against (a) foot muscle free fatty acid (FFA) content or (b) heart muscle free fatty acid (FFA) content in starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation. C.B.P. 78/2B K
~
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2'2 2'4
Oigeshve gland
6O(
16 2'~
FFA (mol/100g tissue)
Ib)~ ~
13 17
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>, ol
'E
~
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e'~
~'0 Heart
1'2 ~'~ 1~
muscle
FFA(mol/100g
~
2'0
2'2
tissue)
Fig. 5. Phase plane plots of foot muscle glycogen content against (a) digestive gland free fatty acid (FFA) content or (b) heart muscle free fatty acid (FFA) content in starved and aestivating Achatina achatina. The numbers indicate the duration (in days) of starvation.
gland F F A (not shown) gave anti-clockwise spirals inclined at 45 °, indicating that F F A in the heart muscle was in phase with F F A in the foot muscle or digestive gland. Properties of pyruvate kinase
0
1-5
4c0~j ~
463
Both pyruvate kinase and PEP carboxykinase activities were detected in the cytosol fraction derived from the foot muscle of starved and aestivating A. achatina (Umezurike and Iheanacho, 1983). It is well established that pyruvate kinase is an important regulatory enzyme in some organisms (cf. Hardie, 1981; Lehninger, 1982). The properties of pyruvate kinase extracted from the foot muscle of starved and aestivating snails were therefore studied. The 30-60~ saturated (NH4)2SO4 fraction obtained from the crude extract of starved and aestivating foot muscle was used in these studies as exhaustive dialysis at 4°C or gel-filtration of this fraction on Sephadex G-200 resulted in complete loss of pyruvate kinase activity. The relationship between initial velocity and the concentration of PEP in the absence and presence of fructose 1,6-diphosphate (FDP) or of ATP (adenosine triphosphate) are shown in Fig. 6. In the absence of FDP the curve was markedly sigmoidal. The sigmoidicity was increased in the presence of 0.5 mM ATP but was completely abolished in the presence of 5.0 mM FDP. Double-reciprocal plots were curved (concave-up) in the absence of F D P and in the presence of ATP but became linear in the presence of 5.0 mM FDP. Figure 7 shows the Hill plots of log (V/Vma x - - V ) against log [PEP]. The Hill cofficient (n) was calculated to be 1.5, 1.8 and 1.0 in the absence of FDP, presence of 5.0 mM ATP and presence of 5.0mM FDP respectively. Similar changes in substrate cooperativity have been oh-
464
GABRIEL M. UMEZURIKE a n d EUGENE N. IHEANACH{)
GLYCOGEN
600 o
f
qF'6"P",-) FDP""
o
.c_
LIPIDS
E c > ~ 20[
w
,
/
O
•
0.5
1.0 115 21,0 [PEP](raM)
/PEP
/
CIT
Fig. 6. Effect of phosphoenolpyruvate (PEP) concentration on the activity of pyruvate kinase from the foot muscle of starved and aestivating Achatina achatina in the absence of fructose 1,6-diphosphate (O), presence of 5.0 mM fructose 1,6-diphosphate (O) and presence of 5.0 mM ATP (A).
°/~
Z~
ALA
\
UM~ MAL
I~
OAA~'j"
oo
,jo >e
U /
MITOCHONDRION
Fig. 8. Scheme of anaerobic carbohydrate metabolism in Achatina achatina. F6P, fructose &phosphate; FDP, fruc-
-I.C
-2-0
-io-o'.s-o16-o!~-o!2 o!o o'.2 o'~ log[PEP]
Fig. 7. Hill plots of the activity of pyruvate kinase from the foot muscle of starved and aestivating Achatina achatina in the presence of various concentrations of PEP, (O) without fructose 1,6-diphosphate (FDP); ((D) with 5.0mM FDP and (A) with 5.0 mM ATP.
served with pyruvate kinase from a number of sources (Hess et al., 1966; Bailey et al., 1968; Stewart and Moore, 1971). The apparent Km for PEP was 0.75mM in the absence of FDP, 0.19mM in the presence of 5.0 mM F D P and 1.15 mM in the presence of 5.0 mM ATP. The inhibitory effect of 2 mM ATP on the activity of pyruvate kinase in the presence of increasing concentrations of PEP was virtually abolished in the presence of 5.0 mM FDP.
DISCUSSION
Studies on the steady-state content of some metabolites and the intracellular distribution and levels of activity of some major enzymes of anaerobic metabolism in the foot muscle of starved and aestivating A. achatina have indicated that this snail is a facultative anaerobe. Succinate, lactate, alanine and free fatty
tose 1,6-diphosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; ALA, alanine; LAC, lactate; MAL, malate; OAA. oxaloacetate; FFA, free fatty acids: Ac-CoA. acetyl-CoA: CIT, citrate; FUM, fumarate: SUC, succinate. (A) and (B) denote routes A and B for entry of metabolites into the mitochondria respectively. The numbered reactions are catalysed by the enzymes: 1, pyruvate kinase: 2, PEP carboxykinase; 3, malate dehydrogenase; 4, malic enzyme: 5, transaminase; 6, lactate dehydrogenase; 7, fumarase: 8, fumarate reductase; 9, malate dehydrogenasc: 10, malic enzyme; l I, pyruvate carboxylase: 12, pyruvale dehydrogenase complex; 13, citrate synthase: 14, ATP citrate lyase.
acids were found to be present in the foot muscle as end-products of anaerobic metabolism (Umezurike and Iheanacho, 1983), and the lactate dehydrogenase from the foot muscle was more active in the direction of lactate oxidation than in that of pyruvate reduction (Umezurike and Eke, 1983). The metabolic cycle proposed as a working scheme for anaerobic fatty acid synthesis (Umezurike and Iheanacho, 1983) is shown in a simplified form in Fig. 8. Each metabolic cycle involves enzymecatalysed reactions occurring in both the cytosol and mitochondria. Malate and pyruvate derived from carbohydrates (or pyruvate derived from lactate and alanine) enter the mitochondria by the action of dicarboxylate carriers. Succinate is then formed as end-product from malate via fumarate. The metabolites eventually give rise to citrate which is then transported out of the mitochondria by a tricarboxylate carrier. The acetyl-CoA formed from citrate by the action of ATP-citrate lyase is used for
465
Phase relationship of metabolites in Achatina synthesis of free fatty acids as end-products, and oxaloacetate reduction completes the cycle. Oxaloacetate may also be removed for glycogen synthesis. The results presented in Figs 1-5 indicate that only two damped oscillatory motions of glycogen and free fatty acids have been completed in 24 days of starvation and aestivation by A. achatina. Under conditions of aestivation, the foot becomes dormant whereas the heart continues beating, albeit at a reduced rate (Borradaile et al., 1963). More ATP would thus be needed to sustain the higher activity of the heart muscle relative to that of the inactive foot muscle. In starved and aestivating snails ATP is generated at the fumarate reductase step (reaction 8 in Fig. 8) if pyruvate kinase is inhibited, as indicated by studies on the succinate-DCPIP and NADH-fumarate oxidoreductases associated with the membrane of submitochondrial particles derived from the foot muscle of A. achatina (Umezurike and Chilaka, 1982). The pyruvate kinase from the foot muscle of aestivating A. achatina behaved like the regulatory or allosteric type in showing co-operative interaction with respect to PEP, specific allosteric activation by FDP and inhibition by ATP as in some other organisms (cf. Hess et al., 1966; Tanako et al., 1967a,b; Pogson, 1968; Stewart and Moore, 1971). Pyruvate kinase from gluconeogenic tissues are known to be strongly and specifically activated by FDP (Gancedo et al., 1967). Pyruvate kinase from some sources is inhibited by acetyl-CoA, long chain fatty acids, alanine and ATP (cf. Lehninger, 1982). Furthermore, the enzyme from some animal tissues is also subject to regulation by the hormone glucagon which increases the level of cyclic AMP, leading to reversible phosphorylation of pyruvate kinase by cyclic AMPdependent protein kinase (cf. Hardie, 1981). Pyruvate kinase is thus one of the candidates for the regulation of the flux and oscillatory motions of metabolites in the tissues of aestivating snails. Furthermore, the mechanism for fatty acid synthesis would be turned off if as in some animal tissues acetyl-CoA carboxylase, the enzyme which catalyses the rate-limiting step for fatty acid synthesis, is subject to inactivation by reversible phosphorylation or to allosteric inhibition by long-chain fatty acylCoA (cf. Hardie, 1981). The positive effector (citrate) and the negative effector (long-chain fatty acyl-CoA) of this enzyme are competitive with each other (cf. Hardie, 1981). The anaerobic metabolic cycle depicted in Fig. 8 thus explains satisfactorily the oscillatory characteristics of glycogen and free fatty acids in starved and aestivating A. achatina, as the mechanism for glycogen or fatty acid synthesis can be switched on and off depending on the levels of control intermediates (ATP, NADH, NADPH, and the effectors of regulatory enzymes) in the various tissues. Differences in the levels of these control intermediates would determine the metabolic routes (cf. Fig. 8) that predominate in these tissues. This would also account for the predominance of glycogen in the foot muscle and of free fatty acids in the heart muscle, and for the apparent prominence of both glycogen and free fatty acids in the digestive gland.
REFERENCES
Anya A. O. and Umezurike G. M. (1978) Respiration and carbohydrate energy metabolism of the lung-dwelling parasite Rhabdias bufonis (Nematoda: Rhabdiasoidea). Parasitology 76, 21-27. Bailey E., Stirpe F. and Taylor C. B. (1968) Regulation of rat liver pyruvate kinase. Biochem. J. 108, 427-436. Betz A. and Chance B. (1965) Phase relationship of glycolytic intermediates in yeast ceils with oscillatory metabolic control. Archs Biochem. Biophys. 109, 585-594. Borradaile L. A., Potts F. A., Eastham L. E. S., Saunders J. T. and Kerkut G. A. (1963) The Invertebrata, 4th edn., p. 610. Cambridge University Press, Cambridge. Chen C. and Awapara J. (1969) Intracellular distribution of enzymes catalysing succinate production from glucose in Rangia mantle. Comp. Biochem. Physiol. 30, 727-737. Frenkel R. (1968) Control of reduced diphosphopyridine nucleotide oscillations in beef heart extracts III. Purification and kinetics of beef heart phosphofructokinase. Archs Biochem. Biophys. 125, 166-174. Gancedo J. M., Gancedo C. and Sols A. (1967) Regulation of the concentration and activity of pyruvate kinase in yeasts and its relationship to gluconeogenesis.Biochem. J. 102, 23C. Hardie G. (1981) Fat and phosphorylation--the role of covalent enzyme modification in lipid synthesis. Trends Biochem. Sci. 6, 75-77. Hess B., Haeckell R. and Brand K. (1966) FDP-activation of yeast pyruvate kinase. Biochem. biophys. Res. Commun. 24, 824-831. Higgins J. (1964) A chemical mechanism for oscillation of glycolytic intermediates in yeast cells. Proc. Natn. Acad. Sci. U.S.A. 51, 989-994. Iwuala M. O. E., Umezurike G. M. and Nwadiogbu N. (1981) Quantitative studies on the proteins, free fatty acids and glycogen contents of eggs of Amblyomma variegatum and Boophilus decoratus (Koch) ticks, Ixodoidea: Ixodidae). Acarologia 22, 133-143. Lehninger A. L. (1982) Principles of Biochemistry, pp. 424-425. Worth Publishers, Inc., New York. McManus D. P. and James B. L. (1975) Anaerobic glucose metabolism in the digestive gland of Littorina saxatilis rudis (Maton) and in the daughter sporocysts of Microphallus similis (Jag) (Digenea: Microphallidae). Comp. Biochem. Physiol. 51B, 293-297. Oudejans R. and Van der Horst D. (1974) Biosynthesis of fatty acids in the pulmonate land snail, Cepaea nemoralis. Comp. Biochem. Physiol. 47B, 139-147. Pogson C. I. (1968) Adipose tissue pyruvate kinase. Biochem. J. 110, 67-77. Simpson J. W. and Awapara J. (1966) The pathway of glucose degradation in some invertebrates. Comp. Biochem. Physiol. 18, 537-548. Stewart G. R. and Moore D. (1971) Factors affecting the level and activity of pyruvate kinase from Coprinus lagopus sensu Buller. J. gen. Microbiol. 66, 361-370. Stokes T. M. and Awapara J. (1968) Alanine and succinate as end-products of glucose degradation in the clam Rangia cuneata. Comp. Biochem. Physiol. 25, 883892. Tanako T., Harano Y., Sue F. and Morimura H. (1967a) Crystallization, characterization and metabolic regulation of two types of pyruvate kinase isolated from rat tissues. J. Biochem., Tokyo 62, 71-91. Tanako T., Harano Y., Sue F. and Morimura H. (1967b) Evidence for the presence of two types of pyruvate kinase in rat liver. Biochem. biophys. Res. Commun. 21, 5560. Umezurike G. M. and Anya A. O. (1978) Studies on energy metabolism in the endoparasite Strongyluris brevicaudata (Nematoda). Comp. Biochem. Physiol. 5OB, 147-151. Umezurike G. M. and Chilaka F. C. (1982)
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GABRIEL M. UMEZURIKEand EUGENE N. IHEANA('HO
Succinate-DCPIP and NADH-fumarate oxidoreductases in submitochondrial particles of the giant African snail (Achatina achatina) foot muscle. Comp. Biochem. Physiol. 71B, 181-185. Umezurike G. M. and Eke L. O. (1983) Some properties of lactate dehydrogenase from the foot muscle of the giant African snail (Achatina achatina ). Comp. Biochem. Physiol. 7513, 263-268. Umezurike G. M. and Iheanacho E. N. (1983) Metabolic
adaptations in aestivating giant African snail (Achatina achatma). Comp. Biochem. Physiol. 74B, 493-498. Van der Horst D. (1974) In citro biosynthesis of fatty acids in the pulmonate land snail ('epaca m'moralis under anoxic conditions. ('omp. Biochem. Physiol. 47B, 181 187. Zwaan A. de and Van Marrewijk W. J. A. (1973) Anaerobic glucose degradation m the sea mussel Ml'tilus edu/is k. Comp. Biochem. Physiol. 44B, 429 439.