Comp. Biochem. PhysioL, 1974, VoL 48B, pp. 513 to 517. Pergamon Press. Printed in Great Britain

OCTOPINE DEHYDROGENASE IN THE FRESH WATER BIVALVE, ANODONTA CYGNEA

GERD GADE

Zoologisches Institut der Westf'alischen-Wilhelms-Universit~t, Lehrstuhl fiir Tierphysiologie, D-44 Miinster, Hindenburgplatz 55, BRD

(Received 20 August 1973)

Abstract--1 . Activities of the enzymes GAPDH, LDH and ODH were assayed in the adductor muscle, foot, heart, mantle, gill and hepatopancreas of the fresh-water bivalve, Anodonta cygnea.

2. The presence of ODH was shown in this species. Therefore, ODH is not characteristic of marine invertebrates, as it appeared to be previously.

3. The enzyme apparently occurs only in muscle tissue. 4. The biological significance of ODH is discussed in relation to the function

of the slow (white) and fast (yellow) part of the adductor muscle.

INTRODUCTION So FAg octopine has been found only in many species of molluscs (Morizawa, 1927; Ackermann & Mohr, 1937; Moore & Wilson, 1937a, b; Irvin, 1938; Roche et al., 1952; Regnouf & Thoai, 1970) and in one species of Sipunculida (Thoai & Robin, 1959). It is the product of the reductive condensation of pyruvate and arginine (Thoai & Robin, 1959). This reaction is catalyzed by a specific octopine dehydro- genase (ODH), an enzyme which according to previous investigations is present in several marine molluscs (Regnouf & Thoai, 1970), but appears to be lacking in fresh-water organisms (Thoai & Robin, 1960).

Usually high activities of ODH correspond to low activities of lactate dehydro- genase (LDH) (Regnouf & Thoai, 1970). Therefore, the pathway via ODH might possibly represent a modification of classical glycolysis.

Many of the earlier studies were concerned with the anaerobic carbohydrate breakdown in marine intertidal bivalves; only a little information is available on the metabolism of fresh-water molluscs. A rather low LDH activity and/or little or no lactate production was found in the gills of Drdssena (Wernstedt, 1944), in the adductor muscle of Unio (Karpiak et al., 1962), in the heart of Anodonta cygnea (Bass et al., 1972) and in the whole animal, Pleurobema coccineum (Badman & Chin, 1973).

In this paper the LDH and ODH pathway of a fresh-water bivalve was investi- gated. The clam A. cygnea seems to be an especially favourable subject, since this species lives in the mud of slow-flowing rivers or ponds and, therefore, should be adapted to conditions of poor oxygen supply.

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514 GERDG~DE

The activities of three key enzymes were compared in several tissues: (1) muscle tissue (adductor, foot, heart) and non-muscular tissue (mantle, gill, hepato- pancreas); and (2) the slow (white) part of the adductor muscle to the fast (yellow) part.

MATERIALS AND METHODS

Specimens of A. cygnea were collected in early winter at the M6hnetalsperre, Germany, kept in running tap water at 10°C and analyzed after 1 week of acclimation.

The dissected tissues were dried on towelling, weighed and homogenized in a tenfold volume of 0"1 M phosphate buffer, pH 7"3, in an Ultra Turrax at high speed for 30 sec. The homogenate was centrifuged at 15,000 g for 20 rain. The activities of the soluble enzymes were assayed spectrophotometrically at 25°C : (1) GAPDH--3 glyceraldehyde-3-phosphate- dehydrogenase--D-glyceraldehyde-3-phosphate : NAD-oxidoreductase (phosphorylating), E.C. 1.2.1.12; according to Bergmeyer (1970). (2) LDH and ODH--lactate dehydrogenase and octopine dehydrogenase--L-lactate : NAD-oxidoreductase, E.C. 1.1.1.27, and octopine : NAD-oxidoreductase (donor cleaving), E.C. 1.5.1.a; according to G~ide & Zebe (1973).

RESULTS AND DISCUSSION

The absolute activities measured in the different tissues are shown in Table 1. From a comparative point of view, relations between the activities of certain enzymes are especially interesting. Such relative activities, expressed as a percent- age of the G A P D H activity, are given in Table 2.

TABLE 1 - - A C T I V I T I E S OF ENZYMES IN DIFFERENT TISSUES OF A. cygnea (/zmole substrate/min and g wet wt., U/g wet wt.)

Adductor muscle Hepato-

Slow part Fast part Foot Heart Mantle Gill pancreas

GAPDH 21"0 43"2 37.7 101.2 25"7 26"5 25.2 LDH 3"2 6"3 6.0 4.1 2"0 3'3 1.5 ODH 7"3 11"0 5.2 0.97 0"67 0"0 0.0

TABLE 2 - - A C T I V I T I E S OF ENZYMES IN DIFFERENT TISSUES OF A. cygnea EXPRESSED AS A P E R -

C E N T A G E OF G A P D H ACTIVITY

Adductor muscle Hepato-

Slow part Fast part Foot Heart Mantle Gill pancreas

GAPDH 100 100 100 100 100 100 100 LDH 15 15 16 4.0 7"8 13 5.9 ODH 35 25 14 1.0 2.6 - - - -

OCTOPINE DEHYDROGENASE IN A N O D O N T A C Y G N E A 515

In all the tissues studied LDH is present, although without exception in rather low activities, which amounts maximally to 15 per cent of GAPDH activity in the adductor muscle and the foot.

ODH is distributed quite differently. Muscle tissue contains absolute activities five to ten times higher than in the heart and mantle. The relative activity is highest in the slow part of the adductor muscle (35 per cent). In the gills and the hepato- pancreas ODH is lacking completely.

The relative activity of LDH in a tissue can be taken as a measure of its capacity to produce lactate under anaerobic conditions. Therefore only small amounts of lactate will probably be formed in the various tissues of the clam A. cygnea during periods of short oxygen supply. In fact several instances are known in which little lactate or none at all could be found after experimental anaerobiosis (Wernstedt, 1944; Simpson & Awapara, 1966; de Zwaan & Zandee, 1972; Badman & Chin, 1973 ; G~ide, unpublished data). Instead, the accumulation of alanine and succinate as metabolic end-products was demonstrated in some cases (Stokes & Awapara, 1968; de Zwaan & van Marrewijk, 1973). Since ODH seems more or less to take the place of LDH in the muscles of certain bivalves, the formation and accumulation of octopine might be expected in these tissues in the absence of oxygen. However, at the present time evidence for the occurrence of "octopine fermentation" under physiological conditions is lacking, probably because of the difficulty in quanti- tatively estimating octopine.

The slow part of the adductor muscle, which has a tonic function, keeps the shell closed--sometimes for long periods--working against the ligament. The ability to produce energy anerobically is particularly important here and this apparently is done by "octopine fermentation". In the fast part, which is responsible for the quick closing movement of the shell, the relative activity of ODH is considerably lower. Different rates of 02 consumption indicating metabolic differences between both parts of the adductor muscle of A. celensis have already been reported (Brecht et al., 1955).

In the foot muscle LDH and ODH have about the same activity. A similar ratio (ODH/LDH: 0.87) was found in the foot muscle of the marine bivalve, Cardium edule (G~ide & Zebe, 1973).

There is a difference in the enzymatic pattern of muscular and non-muscular tissue. The mantle and gill contain very little LDH activity; the hepatopanereas, however, contains as much as the muscles. Traces of ODH were detected in the mantle but none at all in the gill and hepatopanereas. Probably the ODH in the mantle comes from contaminated paUial muscle. The sole presence of ODH in muscles is in agreement with previous studies (Regnouf & Thoai, 1970; Hiltz & Dyer, 1971).

While the glycolytie capacity of muscles is rather high and distinctly lower in non-muscular tissues, the heart is a special ease. Metabolically, it is the most active organ (GAPDH: 101.2 U/g wet wt.), but there is only little LDH activity (4.1 U/g wet wt.) and even less ODH-activity. The data of GAPDH and LDH are very close to those of Bass et al. (1972) for the heart muscle of A. cygnea (GAPDH: 117.0

516 G~I~ G~E

U/g wet wt. and L D H : 7.6 U/g wet wt.). In conclusion, evidence has been presented that O D H is not restricted to marine animals, as it has appeared hitherto, but is also common in fresh-water molluscs such as A. cygnea, which are adapted to an environment with a low oxygen content. I t appears as if octopine formation might be a modification of the classical pathway of glycolysis.

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Key Word Index--Octopine dehydrogenase; lactate dehydrogenase; glyceraldehyde-3- phosphate dehydrogenase; anaerobiosis; fresh-water bivalve; Anodonta cygnea.