Anaerobic glucose degradation in the sea mussel Mytilus edulis l.

Comp. Biochem. Physiol., 1973, Vol. 44B, pp. 429 to. 439 Pergamon Press. Printed in Great Britain

ANAEROBIC GLUCOSE D E G R A D A T I O N IN T H E SEA MUSSEL M Y T I L U S E D U L I S L. ALBERTUS DE ZWAAN and W I L L I B R O R D U S J. A. VAN MARREWIJK Laboratory of Chemical Animal Physiology, 40 Jan van Galenstraat, State University of Utrecht, The Netherlands

(Received 12 ffune 1972) Abstract--1. The distribution of radioactivity over different classes of chemical

compounds from glucose-uL-xiCinjected into the sea mussel Mytilus edulis was examined after 24 and 48 hr of anaerobiosis. 2. The amino- and organic acids together incorporated about 85 per cent of the total radioactivity of mussel homogenate both after 24 and 48 hr of anaerobiosis. 3. The incorporation of radioactivity by the amino acids was mainly confined to alanine and glutamic acid. The level of alanine remained much the same after 24 or 48 hr of incubation but the level of glutamic acid increased. 4. The incorporation of radioactivity by the organic acids was mainly confined to succinate.

INTRODUCTION BIVALWtissues are rich in glycogen. Amounts ranging from 10 to 35 per cent dry weight of soft parts were found in the sea mussel Mytilus edulis (De Zwaan & Zandee, 1971). It has been shown that during exposure there is consumption of glycogen from the muscle tissue and hepatopancreas, resulting in accumulation of succinate and alanine. Lactate production was very low (De Zwaan & Zandee, 1972). These results show that anaerobic glycogen degradation in the sea mussel differs from that in skeletal muscle where lactate is the only fermentation product. This is in agreement with findings for other intertidal bivalves such as the American oyster, Crassostrea virginica (Hammen, 1966) and the brackish water clam, Rangia cuneata (Stokes & Awapara, 1968) which also accumulate succinate as the major end product of anaerobic glycolysis. Anaerobic glycogen degradation in the sea mussel has been examined by determining succinate and alanine levels by means of specific enzymes (De Zwaan & Zandee, 1972) and from this study it was found that these acids only accounted for about 50 per cent of the total degraded glycogen. For this reason we reinvestigated fermentation in the sea mussel by a radio-isotope method using glucose-uLJ4C. 429

430

ALBERTU$ DE ZWAAN AND WILLIBRORDUS J. A. VAN MARREWIJK

MATERIALS AND METHODS*

Experimental procedure Mussels were collected from a natural bed in the Waddenzee (northern part of T h e Netherlands). Animals with a shell length of 5"5 + 0.2 cm were used. Eight mussels were injected with glucose-oL-z4C (New England Nuclear Corp., Boston, Massachusetts). Each animal received 0"36 mg glucose with an activity of 5 FCi by injection into the hepatopanereas via the ligament. Whilst withdrawing the needle the site of injection was screened with Kleenex tissue to absorb any injection fluid pressed out by the animal. T h e mussels were then placed in a desiccator containing 1"5 1. of previously boiled sea water. Nitrogen was bubbled through the sea water for 3 hr before closing the desiccator. Twenty-one hr later four mussels were removed from the desiccator for analysis (group I) and a sample of the sea water was taken (see water sample 1). T h e remaining four mussels were examined after a further 24 hr (group II) when another sample of sea water was taken (sea water sample 2).

Fractionization of the mussels T h e following procedures, describing how the mussels were separated into different classes of chemical compounds, are summarized in Fig. 1. Incubation Potter- Elvehjem homogenizer

Dried homogenate B

Homogenate A

Lyophilization

HCCL3 exiT,

I BUchnerfunnel

t

~

Filtrate

Precipitate

Vacuum C~ ~ evaporator

Ethanol extr. centrifugation

HCCL3 fraction C

i

Sediment

Supernantant I

[0-3 N HOt extr. TCA centrifugation

HCCL3 phase Lyophilisation dissolv,in Io'zN HCL HCCLa extraction

t

Supem!tant Proteins J Ethanol D cenfrifugation

Aqueous p hose

Supernofant 2 " Amber te IR - 120 (H +)

Glycogen E

t

(Ammonia eluant) Amino acids F

Water eluant

Oowex Ix2(OH-) (Water eluant) Neutral compounds G

t

(HCOOH eluant) Organic compounds H

FIo. 1. Flow diagram showing the fractionization of the sea mussels. *List of abbreviations: Pyr, pyruvate; Glu, glutamate; Ala, alanine; 2-Kg, 2ketoglutarate; N A D , nieotinamide adenine dinueleotide; FCi, 10-Ci e (222 x 104 dpm); T C A , triehloroaeetic acid.

ANAEROBIC GLUCOSE DEGRADATION I N THE SEA MUSSEL

431

After incubation the mussels were cooled in ice and removed from their shells. The soft parts were cut into small pieces with scmsors and homogenized at 0-2°C using a PotterElvehjem homogeniser. Mussels within each group were pooled and analysed together. The homogenate was filtered through cheese cloth (homogenate A) and then lyophillzed (becoming dried homo= genate B). One hundred mg of dried homogenate B were weighed into a centrifuge tube with a screw cap and shaken continuously with 25 ml of chloroform for 4 hr on a Vortex mixer. After filtering through a Biichner funnel the precipitate was washed with 50 ml of chloroform and dried in a vacuum desiccator. The filtrate and washing were pooled, the chloroform was removed by a rotating vacuum evaporator and the remaining residue was dried in a vacuum desiccator (chloroform fraction C1). T h e dried precipitate was extracted from the filter with 40 m170% ethanol in a centrifuge tube with a screw cap and shaken for 18 hr at 4°C on a Vortex mixer. T h e filter was removed and the extract was centrifuged in a cooled centrifuge. The sediment and residue on the filter were extracted twice more by shaking for 4 hr with 40 and 20 ml of 70% ethanol respectively. T h e supernatants were pooled (supernatant 1). T h e sediments and residue on the filter were dissolved in 10 -s N hydrochloric acid and an equal volume of 6% (w/v) trichloroacetic acid was added. T h e precipitated proteins were centrifuged and then dried over phosphorous pentoxide in a vacuum desiccator (crude protein extract D). T o the supernatant 0.5 ml of a saturated solution of sodium sulphate was added and 96% ethanol was added to three times its volume. After standing overnight at 0°C the precipitated glycogen was collected by centrifugation and dried in a vacuum desiccator (crude glycogen fraction E). The supernatant (supernatant 2) was added to supematant 1 and ethanol was removed using a rotating vacuum evaporator and the residue was freeze dried. The dry material was suspended in 25 ml 10 -z N hydrochloric acid in a separator and extracted twice with 25 ml of chloroform previously saturated with 10 -~ N hydrochloric acid. The chloroform extracts were washed with 10 ml of 10 -2 N hydrochloric acid, evaporated and dried in a vacuum desiccator (chloroform fraction C2). The dilute hydrochloric acid phase and the washing were combined and partially evaporated to remove any remaining chloroform. Separation of the residue into ampholytes, anions and non-polar substances was obtained by the use of ion-exchange resins using the principle according to Stokes & Awapara (1968). The residue was passed through a cation exchanger Amberlite IR-120 (H + form) (column height 4--6 cm, diam. 2 crn). The column was eluted with 150 ml bidistilled water. T h e eluant contained the anions, the neutral components and the strongly acidic taurine. The column was further eluted with 100 ml 4 N ammonium hydroxide and rinsed with 50 mi bidistilled water. Thus the amino acids were removed from the column. The ammonia was removed by evaporation and the fraction was freeze dried (amino acid fraction F). T h e water eluant was adjusted to p H 5"5-6"5 and passed through an anion exchanger Dowex 1 x 2 ( O H - form) (column height 4-6 cm, diam. 2 cm). The column was eluted with 150 ml bidistilled water which removed the neutral components including glucose. This fraction was freeze dried (fraction G). After measurement of the radioactivity no further study was made on the neutral components. The organic acids were eluted from the Dowex column with 150 ml of 4 N formic acid after which the column was washed with 20 ml bidistilled water. This fraction was also freeze dried (organic acid fraction H).

Separation of the amino acid fiaction The amino acids (fraction F) were dissolved in 0"2 N lithium citrate buffer p H 2"2 and separated by an amino acid analyser (Biocal BC-200). I n series with the amino acid analyser was a liquid scintillation counter (Packard 2002) fitted with a flow cell, filled with anthracene crystals, for direct measurement of the radioactivity of the eluted amino acids (Sehram & Lombaert, 1962).

432

ALBP.RXUS DE ZWAANANDWILLIBRORDUS J. A. VAN MARREWIJK

From the dry material fraction B of experimentalgroup I a second sample of 100 mg was prepared partially accordingto the above-describedprocedure, but the ethanol soluble components (supernatants I and 2) were not separated into ampho]ytes, anions and neutral components. They were dissolved in 0"2 N lithium citrate buffer and analysed by

an amino acid analyser immediately after freeze drying. In this way it was found that the use of Amberlite IR-120 isolates taurine from the other amino acids.

Separation of the organic acid fraction The organic acid fraction G from experimental group I was dissolved in a mixture of 5 ml 96"5% ethanol and 1 ml 10 -2 N hydrochloric acid and partially separated into individual acids by means of one-dimensional descending paper chromatography (Whatman No. 1). Two 50-/zl spots of the solution were made with a 10/A Haak pipette. Reference samples of pyruvate, lactate, malate, fumarate, succinate and citrate were also applied in the amount of ca 1/~mole. The moving liquid was ether-water-formic acid (organic phase) = 18 : 9 : 5. The chromatogram was developed at 4°C for 11 hr, whereby the solvent front had progressed a distance of 48 cm from the origin. After drying the chromatogram a strip was cut off lengthwise containing one spot of the unknown mixture of organic acids. This strip was cut across the width into small strips of 3"5 or 7 mm and the radioactivity of each strip was measured by placing it in a counting vial containing toluene-Omnifluor scintillation fluid. The remaining part of the chromatogram was sprayed with a bromocresol green-bromophenol blue-potassium permanganate reagent (Krebs et al., 1967).

Measurement of radioactivity Radioactivities were determined with a Nuclear-Chicago Liquid Scintillation Counter Model Mark II. The scintillation medium used for counting aqueous solutions consisted of 4 g Omnifluor (NEN Chemicals GmbH), 60 ml naphthalene, 100 ml methanol and p-dioxane to a total volume of 1 1. Samples of chloroform soluble compounds and dry materials (after dissolving in Soluene 100 from Packard) were counted in a scintillation medium consisting of 4 g Omnifluor/l. toluene.

RESULTS AND DISCUSSION

Distribution of the radioactivity in the different classes of compounds Table 1 shows that from the 40 ~Ci glucose-UL-14C administered 30.4/~Ci was recovered (15.1/~Ci from the sea water, 8.7 and 6.6/~Ci from extract A of groups I and I I respectively). T h e unrecovered 9.6/~Ci may be accounted for by the following two reasons: 1. Losses during experimental procedure. T h e radioactivity on the Kleenex tissue used to screen the site of injection was not measured. Also the insoluble fraction of the homogenate plus the adhering fluid which remained on the cheese cloth after filtration was not measured. 2. 1~CO~ production. It is possible that during the initial period, aerobic breakdown takes place due to oxygen stored in the tissues and enclosed sea water. D u r i n g lyophilization of the homogenate there is also a loss in radioactivity (16 and 11 per cent for groups I and I I respectively). This may be due to the formation from glucose of volatile compounds such as CO 2 and short chained fatty acids.

ANAEROBIC GLUCOSE DEGRADATION I N THE SEA MUSSEL

433

TABLE 1--THE DISTRIBUTIONOF RADIOACTIVITYIN THE SEAMUSSELBETWEEN THE DIFFERENT CLASSESOF COMPOUNDSAFTERINCUBATIONTIMESOF 24 hr (GROUP I) and 48 hr (GROUP II) Lr~DERANAEROBICCONDITIONSAFTERINJECTIONWITH 5 ~Ci GLUCOSE-UL-14C. Group I (24 hr anaerobiosis) Fraction Homogenate Dried homogenate t Chloroform fraction Proteins Glycogen Amino acids Neutral compounds Organic acids Not recovered,+ Sea water §

Code* A B C D E F G H

Radioactivity /zCi % 8"71 7"29 0'07 0"30 0"22 3"85 0"13 2'63 0"31 15"06

100 1"0 4"1 0"3 52-5 1"8 36"1 4'2

Group II (48 hr anaerobiosis) Radioactivity /zCi % 6"65 5"99 0"02 0"24 0"01 3"37 0"00 2"20 0"14 14"94

100 0"3 4"0 0"2 56"3 0"0 36-8 2"4

* For code see Fig. 1. t The dry weight of group I was 3"9 g and group II was 4"2 g. Not recovered radioactivity refers to the total activity in the dry material, fraction B (= 100 per cent). §Activity in sea water: after 24 hr the sea water contained 15"06/LCi (sea water sample 1) and after 48 hr 14"94/~Ci (sea water sample 2). Table 1 shows that the distribution of radioactivity over the different fractions after incubation times of 24 hr (group I) and 48 hr (group II) was almost the same. F r o m the radioactivity of glucose-vL-14C which was recovered after incubation approximately half was incorporated in amino acids (52.5 and 56.3 per cent for groups I and II respectively). T h e organic acids accounted for just over one-third of the radioactivity (36.1 and 36.8 per cent for groups I and II respectively). Together the amino and organic acids represented about 85 per cent of the total activity of the mussel homogenate. T h e resulting 15 per cent was distributed over four other fractions. T h e radioactivity of these fractions was small compared to the acid fractions and is therefore regarded as insignificant. T h e activity in the neutral fraction is probably due in part to sugars, whilst that in the protein fraction may be due to the incorporation of radioactive amino acids.

Distribution of radioactivity in the amino acid fraction Table 2 gives the breakdown of the amino acids in fraction B. T h e incorporation of x4C is confined to only four of these acids, namely alanine, glutamic acid, aspartie acid and glutamine. Alanine accounts for about three-quarters of the activity followed by glutamic acid (about 20 per cent) whilst the activity in aspartic acid and glutamine is low (altogether about 5 per cent).

434

ALBERTUS DE ZWAA.N AND WILLIBRORDU$

J.

A. vAN M A ~ I J K

TABL~ 2 m T I ~

DISTRIBUTION OF RADIOACTIVITY AMONG THE AMINO ACIDS IN THE SEA MUSSEL FROMDRYHOMOGENATESOF GROUPI (24 hr INCUBATION) AND GROUPII (48 hr INCUBATION) UNDER ANAEROBIC CONDITIONS AFTER INJECTION WITH 5 ~ C i GLUCOSE-L-14C

Group I (24 hr anaerobiosis) /~mole Radioactivity 100 mg dry homogenate Tau Asp Thr Ser Asn Gin Glu Pro Gly Ala Val Met Ile Leu Tyr Phe fl-Ala Orn Lys His Try Arg Not identified

15.56" 0.90 1.62 3.47 1.70 0.85 2.91 1.14 6"48 6"60 0"42 0"21 0"44 0"32 0.48 0'20 0.47 0"59" 0"85* 0.27* 0"27* 0"30"

/zCi

%

0.14

3.8

0"08 0"61

2.2 16.7

2"66

72"7

0.17

4.6

Group II (48 hr anaerobiosis) /zmole Radioactivity 100 mg dry homogenate 0.01 0"63 1.71 2.57 1-10 0.48 2.65 0.68 6.11 7"54 0"44 0"33 0-30 0"28 0.30 0.17 0"24

/zCi

%

0-12

3"9

0"06 0"77

1"9 24"2

2"12

66'1

0-12

3"9

*These values were determined from the dry homogenate B immediately after ethanol extraction and freeze drying (see Materials and Methods). Table 3 shows how much radioactive carbon is incorporated in these amino acids as a percentage of the total activity in the dry homogenate (B). The level of alanine remains much the same after 24 or 48 hr of incubation. The amount of 14C incorporated in glutamic acid increases during anaerobiosis from 24 to 48 hr. The amino acid composition of group I was determined by analysis of the ammonia eluant from the Amberlite IR-120 column together with direct analysis of the ethanol extract of the dry homogenate B. A good conformity was found between the chromatograms and radiograms of the amino acids from both samples. Use of the ion exchanger gave slightly lower results for asparagine and glutamine with subsequent slight increases in aspartic acid and glutamic acid respectively. Taurine did not remain attached to the resin but was eluted in the water phase. From the radiogram of the ethanol extract no radioactivity was found in taurine, so this had no influence on the results.

ANAEROBIC GLUCOSE DEGRADATION I N THE SEA MUSSEL

TABLE 3--THs

435

INCORPORATION OF RADIOACTIVITY I N THE AMINO ACIDS AS A PERCENTAGE OF THE TOTAL ACTIVITY I N THE DRY HOMOGENATE B

Aspartic acid Glutamine Glutamic acid Alanine Not identified Total

Group I

Group II

2.0 1"1 8"8 38"2 2"4 52"5

2-2 1"1 13"6 37"2 2-2 56"3

Taurine is present in a very high concentration in the mussel (Table 2) as in many other marine invertebrates. However taurine is undetectable in fresh water and land snails (Simpson et al., 1959). The synthesis of taurine in M. edulis has been shown (Allen & Garrett, 1971). In contrast to the brackish water bivalve R. cuneata which can also synthesize taurine, M. edulis is capable of maintaining this amino acid in high concentrations. Taurine may have a function in osmoregulation (Lange, 1963) or maybe it is incorporated in compounds with a surface tension lowering activity which is important for the digestion of fat in many crustaceans (Van der Oord, 1965).

Distribution of radioactivity in the organic acid fraction The organic acid fraction of group I was separated by paper chromatography. Figure 2 shows the distribution of radioactivity over the chromatogram. In total, 1148 dpm were counted for the whole chromatogram. A sample of 50/zl was put on the chromatogram from a total volume of 6 ml. The organic acid fraction was made from 100 mg of dry homogenate B which in total weighed 3.9 g. Thus the total radioactivity of the organic acids were calculated: 1148 x ~

6

3.9 1 x ~7~ x ~ - ~

2-4~Ci.

By direct measurement of the radioactivity of this fraction 2.63/~Ci was found (fraction H of Table 1). The activity was distributed over the identified acids as follows (in percentages): succinate plus lactate, 52.3; malate, 14.7; citrate, 2.2; fumarate, 2.2; and pyruvate, 1.0. The remaining 27.6 per cent is mainly localized in the part with a low R! value (< 0-3). Presumably there are in this region strong polar intermediates, such as phospho-compounds. In a previous paper (De Zwaan & Zandee, 1972) it was reported that lactate accumulation in the sea mussel is negligible. The radioactivity detected in the spot on the chromatogram representing succinate and lactate is therefore almost entirely accounted for by succinate. In Table 4 the radioactivity in the different organic acids is given as a percentage of the dry extract B.

436

ALBERTUS DE Z W A A N AND WILLIBRORDUS

J. A.

VAN MARREWIJK

This study shows that the sea mussel, M. edulis and the brackish-water bivalve, R. cuneata (Stokes & Awapara, 1968) possess similar metabolic features under anaerobic conditions. Under these conditions both animals accumulate succinate I00ClT

MAL

PYR

SUC

FUM

LAC,

80-

= 60-

"~ 4020-

,,,,,4,

Ill T r I I I I

05

I!O

Rf

FIG. 2. The distribution of radioactivity over the chromatogram of the organic acid fraction of sea mussels kept for 24 hr under anaerobic conditions after injection with 5/~Ci glucose-gL-14C. The chromatogram was cut across the width into small strips of 3"5 or 7 mm and the radioactivity of each strip is given in the figure. in preference to lactate. Parasitic organisms living in environments with low oxygen tension as do many helminths (Saz & Lescure, 1969) and the liver fluke Fasciola hepatica (De Zoeten et al., 1969a, b) also ferment carbohydrates by producing succinate. For these animals it has been proved that energy-rich phosphate bonds can be generated by reduction of fumarate to succinate. In the case of F. hepatica a P/H ratio of 1 was found, resulting in a net yield of 3 moles ATP for every mole of glucose degraded. In the Embden-Meyerhof-Parnas pathway there is a net yield of only 2 moles ATP. For this reason succinate accumulation is of some advantage to organisms adapted to anaerobiosis. Excess succinate can be oxidized more rapidly than lactate through the Krebs cycle on the resumption of aerobic conditions. Simpson & Awapara (1966) propose for the clam R. cuneata that most of the phosphoenolpyruvate formed in glucose degradation is readily carboxylated to oxalacetate by the action of very active phosphoenolpyruvate carboxykinase, followed by reduction of oxalacetate and dehydration of malate. This part of the glycolysis is cytoplasmic and the re-oxidation of glycolytic formed NADH2, normally performed by lactate dehydrogenase, is taken over by malate dehydrogenase. Fumarate becomes reduced to succinate in the mitochondrion and this redox system also requires N A D H 2. For this reason Stokes & Awapara (1968) postulate that alanine is formed from pyruvate by transamination. For every mole of alanine formed an equimolar amount of NADH~ is delivered in the cytoplasm. This will migrate to the mitochondrion, thus feeding the fumarate-succinate redox system. This supposition implies that mitochondria of R. cuneata, unlike that of mammals, are permeable to N A D H 2. Chen & Awapara (1969) offer some experimental

437

ANAEROBIC GLUCOSE DEGRADATION I N THE SEA MUSSEL

evidence that this might be true. Because alanine and succinate constituted the major portion of end products and were formed in a ratio of 1 : 1, these acids can account for the balance of oxidations and reductions as well as for the balance of carbons. De Zwaan (1972) also found a very active phosphoenolpyruvate carboxykinase in M. edulis and we suppose that succinate production occurs according to the mechanism proposed by Stokes & Awapara (1968) for R. cuneata. However, it is clear from our results that succinate and alanine are not produced in equimolar amounts. After 24 hr of anaerobiosis alanine and succinate were formed in a ratio of about 2 : 1 (Tables 3 and 4). But after a successive period of 24 hr anaerobiosis there was no further formation of alanine (Table 3). T.~J3LE 4

THE

INCORPORATION OF RADIOACTIVITY I N THE ORGANIC ACIDS OF GROUP I AS A

PERCENTAGE OF THE TOTAL ACTIVITY I N THE DRY HOMOGENATE

Organic acid

B

Radioactivity in ~o of dry homogenate B

Succinate (+ lactate) Malate Citrate Fumarate Pyruvate Not identified

19"0 5"5 1-0 1"0 0"5 9"0

Total

36"0

In a previous study (De Zwaan & Zandee, 1972) it was found that alanine and succinate were formed in almost equimolar amounts after 48 hr exposure. These results suggest that alanine is the initial major end product of anaerobic glycolysis and that conversion of phosphoenolpyruvate to alanine is gradually prevented in favour of the further formation of succinate. This assumption is supported by studies on pyruvate kinase in the sea mussel which show that the activity of this enzyme is strongly inhibited by alanine accumulation and the gradual decrease of pH (De Zwaan, 1972; De Zwaan & Holwerda, 1972). Because anaerobic glycolysis is only maintained when no net oxidation occurs, the end products are formed by routes which involve an equal number of oxidative and reductive steps. It is therefore possible that alanine is formed in a reaction involving oxidoreduction. This might be by the direct action of an alanine dehydrogenase (reaction 3) or by an initial transamination, followed by reduction of 2-ketoglutarate by glutamate dehydrogenase (reactions 1 and 2). (1) (2) (3)

Pyr + Glu -~ Ala + 2-Kg 2-Kg + N H 3+ N A D H + H+ ~ Glu + NAD + + H~O Pyr + N H 3 + N A D H + H + ~ Ala + NAD + + H 2 0

438

ALBERTUSDE ZWAANAND WILLIBRORDUSJ. A. vAN MARREWlJK

In both cases for every mole of alanine formed an equimolar amount of N A D H 2 is re-oxidized. It has been proved in our laboratory that a cytoplasmic glutamate dehydrogenase is present in the sea mussel (Addink, unpublished results). According to this concept alanine and malate are formed from glucose in a route involving one oxidative and one reductive step. An advantage of this suggested alanine formation is that it involves ammonia fixation. This might be of physiological significance, because the sea mussel reacts to exposure by closing its shells. As protein catabolism will not stop immediately, this results in some production of ammonia. The formation of succinate occurs probably in the mitochondrion. We believe that malate migrates into the mitochondrion and becomes partly transformed into succinate via the reverse direction of Krebs cycle and partly into glutamate via 2-ketoglutarate in Krebs cycle. This route involves one reduction (fumarate-+ succinate) and three oxidations (malate-+oxalacetate, pyruvate-+ acetyl-CoA and isocitrate--+ 2-ketoglutarate). This implies that the ratio of succinate and glutamate formation would approximate 3 : 1. From Tables 3 and 4 it can be seen that the isotope distribution over succinate and glutamate is 19 : 9. By taking into account that succinate gains three and glutamate four radioactive carbons this ratio becomes 19 : 7. This means that the formation of glutamate as well as succinate is important for the balance of reduced and oxidized coenzymes in the mitochondrion. The increased formation of succinate after the first 24 hr together with the subsequent production of glutamate (Table 3) is in agreement with this suggestion. Acknowledgements--The authors are grateful to Professor Dr. D. I. Zandee for his guidance and advice throughout this study and also thank Mr. H. J. L. Ravestein for his technical assistance. REFERENCES

ALLENJ. A. & GARRETTM. R. (1971) Taurine in marine invertebrates. Adv. Mar. Biol. 9, 205-253. CHEN C. & AWAPARAJ. (1969) Intracellular distribution of enzymes catalyzing succinate production from glucose in Rangia mantle. Comp. Biochem. Physiol. 30, 727-737. HAMMENC. S. (1966) Carbon dioxide fixation in marine invertebrates--V. Rate and pathway in the oyster. Comp. Biochem. Physiol. 17, 289-296. KREBS K. G., I-I~ussER D. & WINNra~ H. (1967) Spriihreagenbien. In DunnschichtChromatographic (Edited by STAI-ILE.), pp. 813-861. Springer-Verlag, Berlin. LANGER. (1963) The osmotic function of amino acids and taurine in the mussel, Mytilus edulis. Comp. Biochem. Physiol. 10, 173-179. SAz J. H. & LESCUREO. L. (1969) The functions of phosphoenolpyruvate carboxykinase and malic enzyme in the anaerobic formation of succinate by Ascaris lumbricoides. Comp. Biochem. Physiol. 30, 49-60. SCHRAM E. & LOMBAERTR. (1962) Determination of tritium and carbon-14 in aqueous solution with anthracene powder. Analyt. Biochem. 3, 68-74. SIMPSONJ. W., ALLENK. & AWAPARAJ. (1959) Free amino acids in some aquatic invertebrates. Biol. Bull. 117, 371-381. SIMPSONJ. W. & AWAPARAJ. (1966) The pathway of glucose degradation in some invertebrates. Comp. Biochem. Physiol. 18, 537-548.

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STOKES T. & AWAeAP.AJ. (1968) Alanine and succinate as endproducts of glucose degradation in the clam Rangia cuneata. Comp. Biochem. Physiol. 25, 883-892. VAN DEN GORY A., DANmLSSON H. & RYHAGE R. (1965) On the structure of the emulsifiers in gastric juice from the crab Cancer pagurus L. 3. biol. Chem. 240, 2242-2247. DE ZOETZN L. W., POSTHUMA D. & Tn~mm J. (1969) Intermediary metabolism of the liver fluke Fasciola hepatica--I. Hoppe-Seyler's Z. physiol. Chem. 350, 683-690. DE ZO~ZN L. W. & T I P K ~ J. (1969) Intermediary metabolism of the liver fluke Fasciola hepatica--II. Hoppe-Seyler's Z. physiol. Chem. 350, 691-695. DE ZWAAN A. (1972) Pyruvate kinase in muscle extracts of the sea mussel Mytilus edulis L. Comp. Biochem. Physiol. 42B, 7-14. DE ZWAANA. & HOLWEaVA D. A. (1972) T h e effect of phosphoenolpyruvate, fructose-l,6diphosphate and p H on allosteric pyruvate kinase in muscle tissue of the bivalve Mytilus edulis L. Bioehim. biophys. Acta 276, 430-433. DE ZWAANA. & ZANDEED. I. (1972) Body distribution and seasonal changes in the glycogen content of the sea mussel Mytilus edulis. Comp. Biochem. Physiol. 43A, 53-58. DE ZWAANA. & ZANDEED. I. (1972) T h e utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L. Comp. Biochem. Physiol. 43B. 47-54.

Key Word Index---Glucose metabolism; Mytilus edulis; alanine; glutamic acid; succinate; anaerobic metabolism.