the free guanidine and polyamine pools of bivalve mollusks in relation to their ecology

*Corresponding author. Fax: #33-5-56-83-51-04.E-mail address: [email protected] (C. Audit)

Biochemical Systematics and Ecology 28 (2000) 209}218

The free guanidine and polyamine pools of bivalvemollusks in relation to their ecology

SteH phane Gasparini, Christine Audit*Laboratoire d'Oce&anographie Biologique, Universite& Bordeaux I, CNRS UMR-EPOC 5805, 2 rue Professeur

Jolyet, 33120 Arcachon, France

Received 14 September 1998; accepted 29 March 1999

Abstract

The compositions of free guanidine and polyamine pools of three marine bivalves, the oysterCrassostrea gigas, the japanese clam Ruditapes philippinarum and the common cockle Ceras-toderma edule were investigated. The major arginine-related metabolites were 4-guanidinobutyric acid in the oyster C. gigas and octopine (N2-(1-D-carboxyethyl)-L-arginine) inthe clam R. philippinarum. The diamine and polyamine pattern of the cockle C. edule showed thepresence of unusual polyamines, norspermidine and norspermine. In the three bivalves studied,spermine appears to be the major polyamine and spermidine the second. The occurrence of theguanidine octopine and the presence of spermine as the major polyamines in bivalves isdiscussed in relation to the ecology of these organisms. ( 2000 Elsevier Science Ltd. All rightsreserved.

Keywords: Crassostrea gigas; Ruditapes philippinarum; Cerastoderma edule; Bivalve mollusks; Guanidino

compounds; Polyamines

1. Introduction

Until now most studies on the free pools of intracellular metabolites in marineinvertebrates have been directed towards the role of amino acids as osmotic regula-tors. In this intracellular pool, the presence of more basic compounds such aspolyamines and guanidine derivatives can also be observed.

0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 0 5 9 - 9

A variety of guanidine derivatives have been identi"ed in marine invertebrates(Thoai and Robin, 1969). As the base moiety of various phosphagens, guanidinesparticipate in the energy-requiring cell processes of these organisms. Additionally, bytheir involvement in the "xation, transfer and excretion of nitrogen, and due to theirhigh nitrogen content, the guanidino compounds play a major role in the recycling ofnitrogen.

Although polyamines have attracted much less attention in marine invertebratesthan in other organisms, it is obvious that in these animals polyamines are alsoinvolved in the functions of prime importance previously described for these com-pounds. Polyamines are known to be required for normal cellular growth anddi!erentiation (Bachrach, 1973; Heby, 1981). In the processes of cell growth andembryonic development, polyamines (spermidine, spermine) play a speci"c function asorganizers of the cleavage organelle and inducers of the egg/cell division (Audit, 1996).Polyamines may also regulate DNA replication and stabilize the membrane lipids(Marton and Morris, 1987). Moreover, some recent papers have revealed modi"ca-tions of polyamine metabolism promoted by osmotic and nutritional stresses inmarine invertebrates (Lovett and Watts, 1995; Stuck et al., 1996; Watts et al., 1996).Thus, polyamines could play a role as biochemical indicators of the marine environ-ment.

The aim of this study in marine bivalves was to investigate whether guanidine orpolyamine metabolism could re#ect a strategy used by these animals to adapt to theirenvironment. With this goal, the arginine-related metabolites in the oyster Crassostreagigas and the short-necked clam Ruditapes philippinarum as well as the polyaminepattern in the common cockle Cerastoderma edule were analysed and compared tothose of the other marine bivalves.

2. Materials and methods

2.1. Animals

The oyster Crassostrea gigas, the cockle Cerastoderma edule and the short-neckedclam Ruditapes philippinarum were collected in Arcachon Bay, near Bordeaux, France,in April and May 1997. Fresh animals were immediately frozen following an estima-tion of their wet weight.

2.2. Free amine pool extraction

Guanidine derivatives and polyamines were extracted from the whole homogenatesof "ve individuals of each species. Tissues were homogenized in the presence ofperchloric acid ("nal concentration: 0.45 N) and incubated on ice for 30 min. Follow-ing centrifugation, the resulting pellets were washed twice in perchloric acid. Proteinconcentrations of the pellets were determined by the biuret method. The combinedsupernatants were analyzed by ion-exchange chromatography for guanidino com-pounds and high-performance liquid chromatography for polyamines.

210 S. Gasparini, C. Audit / Biochemical Systematics and Ecology 28 (2000) 209}218

2.3. Guanidino compound analysis

After neutralization with KOH and centrifugation, the supernatants of perchloricextracts were subjected to a Dowex-50X4-200 resin. The removal of taurine and mostamino acids was achieved by washing the resin, followed by 0.6 N HCl. Octopine waseluted with 1.5 N HCl and 4-guanidinobutyric acid with 2 N HCl. Arginine andpolyamines were obtained with 6 N HCl. The guanidino compounds were furtherpuri"ed onto Amberlite CG-50 pyridine form, equilibrated with pH 7.0 1 M-pyridineacetate bu!er. Octopine or 4-guanidinobutyric acid was obtained with this pH 7.0bu!er and arginine was eluted with pH 6.0 pyridine acetate bu!er. Standardguanidino compounds were subjected to the same procedure.

The identi"cation of guanidine derivatives was carried out by thin-layerchromatography using as solvents: 1-butanol, pyridine, acetic acid, water (4 : 1 : 1 : 2)and 1-propanol, acetic acid, water (4 : 1 : 1). Guanidines were identi"ed usingSakaguchi reagent and their concentrations were estimated by the 1-naphthol-dia-cetyl method (Robin, 1964). The data shown correspond to the mean values ($ S.D.)of triplicate measurements performed on the two pools of "ve individuals for eachspecies.

2.4. Polyamine analysis

After neutralization and centrifugation, the perchloric extracts were submitted toan ion-exchange clean-up procedure on Dowex-50 to remove most amino acids andguanidine derivatives. Polyamines were derivatized using the dansyl chloride methodaccording to Flores and Galston (1982). The separation of dansylated polyamines wasachieved by the reversed-phase HPLC using a Purosphere RP18 column(240]4 mm, 5 lm particles) from Merck. The solvent was a gradient of acetonitrileand water (tri#uoroacetic acid: 0.1%)

3. Results and discussion

3.1. Guanidino compound pools

The number of guanidino compounds is low in vertebrates but highly variableamong invertebrates (Thoai and Robin, 1969). The biosynthesis of these compounds isrelated to the metabolism of arginine. In all mollusks, arginine is a guanidinocompound of prime importance since it is the basis of their phosphagen, phospho-L-arginine (Thoai and Roche, 1960). Phosphagen formation is catalyzed by the enzymearginine kinase (EC 2.7.3.3).

When the free guanidine pool of the oyster Crassostrea gigas was analyzed, thepresence of 4-guanidinobutyric acid was revealed. The concentrations of arginine and4-guanidinobutyric acid were estimated to 20.9$1.8 and 2.1$0.1 nmol mg~1 pro-tein, respectively. 4-Guanidinobutyric acid is produced from arginine by an oxidativedeamination catalyzed by L-aminoacid oxidase (EC 1.4.3.2) with the intermediary

S. Gasparini, C. Audit / Biochemical Systematics and Ecology 28 (2000) 209}218 211

Table 1Main guanidines occurring in various marine bivalves

Subclass and species Arginine 4-Guanidino butyric acid Agmatine Arcaine Octopine

PteriomorphaCrassostrea gigas!," # # #

Mytilus edulis# # # #

Pecten maximus# # # #

Arca noae# # # #

HeterodontaCerastoderma edule# # #

Ruditapes philippinarum!," # # #

!This study."Hamana et al. (1991).#Florkin and Bricteux-GreH goire (1972).

formation of 2-ketoarginine (Roche and Robin, 1962). The immediate end product ofthis deamination reaction is ammonia.

Another guanidino compound, agmatine, has been detected in Crassostrea gigas aswell as in the short-necked clam Ruditapes philippinarum by Hamana et al. (1991)(Table 1). Agmatine results from the enzymatic degradation of the carbon chain ofarginine catalyzed by the enzyme arginine decarboxylase (EC 4.1.1.9). Agmatine anda diguanidino compound named arcaine have also been found in Arca noae (Kutscheret al., 1931 cited by Florkin and Bricteux-GreH goire, 1972) (Table 1). Investigations onthe biosynthesis of this diguanidino compound in worms have indicated that theformation of arcaine (diamidinoputrescine) results from the transfer of two amidinegroups of arginine to putrescine with the intermediary formation of agmatine (mono-amidinoputrescine) (Audit et al., 1967). The transamidination pathway leading toarcaine appears to occur in animals living in polluted water as it is the case for Arcanoae or the marine polychaete Audouinia tentaculata in which the biosynthesis ofarcaine has been investigated (Robin and Audit, 1966).

Arginine is also the precursor of the guanidine octopine (N2-(1-D-carboxyethyl)-L-arginine) whose biosynthesis results from the reductive condensation of the aminogroup of arginine with pyruvic acid (Thoai and Robin, 1959). This reaction isreversibly catalyzed by octopine dehydrogenase (EC 1.5.1.11). In marine bivalves, thepresence of octopine has been "rst identi"ed in the mussel Mytilus edulis, the scallopPecten maximus and the cockle Cerastoderma edule (Thoai and Robin, 1959) (Table 1).

The genetic variation at the octopine dehydrogenase locus in the adductor musclealso has been investigated in the three above-mentioned species as well as in the otherspecies belonging to the pteriomorpha subclass, the mussel Modiolius modiolius andvarious species of the genus Chlamys (C. varia, C. opercularis, C. distorta) (GaK de,1980a). The formation of octopine has also been observed in Pecten alba and Pectenjacobaeus (GaK de, 1980a). In oysters, the presence of octopine has not been detected inCrassostrea gigas during this study, or in Ostrea edulis (Regnouf and Thoai, 1970). In


the heterodonta subclass, besides the cockle Cerastoderma edule, octopine has beenidenti"ed in Cerastoderma oeculatum and Cerastoderma tuberculatum and in the threespecies of the genus Tapes (T. decussatus, T. rhomboideus, T. pullastra) (Regnouf andThoai, 1970). In this study, octopine was detected in the short-necked clam Ruditapesphilippinarum. The concentrations of arginine and octopine in the free guanidine poolof this species were estimated to 119.7$12.5 and 7.03$0.48 nmol mg~1 protein,respectively. The role of octopine formation has been analyzed in marine mollusks(Grieshaber and GaK de, 1976; GaK de, 1980a,b). In various species of Pectinidae capableof swimming activity (Pecten alba, Pecten jacobeus, Pecten maximus, Placopectenmagellanicus) energy is generated via the breakdown of arginine phosphate during theescape response with a smaller contribution from octopine production. In Cardiidae,experiments in vitro in the jumping cockle Cerastoderma tuberculatum have shownthat during contractile activity in the presence of an insu$cient supply of oxygen,energy is generated from arginine phosphate (82%) and glycolysis via the octopinepathway (18%). The contractile activity leads to an increased concentration ofarginine while octopine accumulates as the main anaerobic end product (GaK de,1980a). By trapping the product of glycogen breakdown, pyruvate, in octopine, thiscockle avoids dependence upon oxygen supply during periods of intense activity.Further comparative experiments in the cockles Cerastoderma edule and Ceras-toderma tuberculatum have indicated that octopine accumulates in the jumping cockleduring burst activity while this metabolite increases slightly in the two species duringexperimental anoxia (GaK de, 1980b; Meinardus and GaK de, 1981). The sudden andextremely high requirement for energy during jumping movements can only beful"lled by the depletion of the phosphagen pool. The higher level of octopinesynthesis during jumping movements may compensate for the increased phosphagendemand. Indeed, by synthesizing high levels of octopine, the jumping cockle canpreserve high amounts of arginine from degradation by its binding to pyruvate.Thereby, high levels of arginine are available as substrate for the arginine kinasereaction in order to replenish the phosphagen pool. Thus, the formation of high levelsof octopine in bivalves could appear as an adaptive response of the moving species totheir high-energy demand when they are subjected to hypoxia.

3.2. Polyamine pool

Polyamines are normal constituents of all cells and they are distributed ubiquitous-ly in vertebrates and invertebrates. The aliphatic diamine putrescine is formed bydirect decarboxylation of ornithine and this diamine is the biosynthetic precursor ofthe two polyamines, spermidine and spermine.

When HPLC analysis was performed on the free polyamine pool of the cockleCerastoderma edule, the polyamines spermidine (1, 8-diamino-4-azaoctane) and sper-mine (1, 12-diamino-4, 9-diazadodecane) as well as the diamines 1, 3-diaminopropane,putrescine (1, 4-diaminobutane) and cadaverine (1, 5-diaminopentane) were found(Fig. 1). Low amounts of the unusual polyamines, norspermidine (1, 7-diamino-4-azaheptane) and norspermine (1, 11-diamino-4, 8-diazaundecane) were also present(Fig. 1). Norspermidine and norspermine were "rst discovered in the extremely


Fig. 1. RP-HPLC elution pro"le of dansyl-derivatized polyamines extracted from whole homogenates ofthe cockle Cerastoderma edule. The compounds are identi"ed as follows: 1: 1, 3-diaminopropane, 2:putrescine, 3: cadaverine, 4: norspermidine, 5: spermidine, 6: tyramine, 7: norspermine, 8: spermine, X, Y:unknowns.

thermophilic bacterium, Thermus thermophilus by Oshima (1983) but have never beendetected in vertebrates. In marine invertebrates, they have been revealed in cnidarian,arthropods, echinoderms (Zappia et al., 1978) and in invertebrate chordates (Hamanaet al., 1991). In mollusks, Zappia et al., (1978) have detected norspermidine in thebivalves Mytilus galloprovincialis and Pinna nobilis (Table 2) and the gastropod Patellacoerula. These authors have also found norspermine in Pinna nobilis (Table 2) and inthe cephalopod Octopus vulgaris. In the oyster Crassostrea gigas and the Japaneseclam Ruditapes philippinarum, norspermidine and norspermine as well as anotherunusual polyamine, homospermidine (1, 9-diamino-5-azanonane) have been identi"edby Hamana et al. (1991) (Table 2). As the amounts of spermidine and spermine inoyster and clam were not estimated by these authors, an HPLC analysis on these twospecies was carried out in order to determine their polyamine content in comparisonto that of cockle's. Spermine appears as the main polyamine in the three bivalvesstudied (Fig. 2) and this result is in agreement with previous polyamine estimation inthe two other bivalves, Mytilus galloprovincialis and Pinna nobilis (Zappia et al., 1978).

The occurrence of the tetraamine spermine as the main polyamine in molluskscontrasts with the presence of the triamine spermidine as the major polyamine in mosttissues of vertebrates (Rosenthal and Tabor, 1956). The identity of the major poly-amine in organisms may be an important feature. Indeed, polyamines are present in


Table 2Main polyamines occurring in various marine bivalves

Subclass and species Spermine Spermidine Nor-spermine Nor-spermidine Homo-spermidine

PteriomorphaCrassostrea gigas!," # # # # #

Mytilus gallo-provincialis#

# # #

Penna nobilis# # # # #

HeterodontaCerastoderma edule! # # # # n.d.Ruditapes philip-pinarum!,"

# # # # #

!This study."Hamana et al. (1991).#Zappia et al. (1978).n.d. Not determined.

Fig. 2. Levels of spermine (white) and spermidine (black) in various marine bivalves. Data concerningMytilus galloprovincialis and Pinna nobilis are from Zappia et al. (1978).

their native form in the free intracellular pool and their chemical structure is the clueto a variety of interactions with anionic constituents such as phospholipids (Igarashiet al., 1982) and proteins (Oriol-Audit, 1978). The biophysical attributes of polyaminesinvolve electrostatic forces and ditopic e!ects due to their amino groups as well asa degree of basicity and hydrogen forces dependent on their aliphatic chains (Oriol-Audit et al., 1985). Spermine is the strongest natural polyamine in cells and atphysiological pH, the number of net charges is 4` for spermine and 3` for spermi-dine (Ganem, 1982). The ionic interactions between polyamines and other cellular


constituents are the basis of essential functions. The electrostatic interactions betweenthe amino groups of polyamines and the carboxyl groups of the protein actin inducethe formation of the cleavage organelle and promote the egg/cell division (Audit,1996). Both spermidine and spermine can induce egg/cell cytokinesis but experimentalstudies have demonstrated that spermine is the most e$cient polyamine (Oriol-Audit,1982; Grant et al., 1984). Owing to its strong biophysical attributes, the tetraaminespermine appears as the most appropriate polyamine to succeed in promoting eggdivisions during the course of embryonic development that takes place in seawater.

Other interesting functions lie in the interactions that polyamines establish with cellmembranes. These interactions are involved in the stabilization of membranes as wellas in the regulation of its #ow and ion channels (Marton and Morris, 1987; Williams,1997). The electrostatic interactions of the amino groups of spermine with acidicgroups of membrane phospholipids may stabilize cell membranes against lysis. Poly-amines may also regulate the intracellular membrane #ow by controlling the mem-brane fusion. Experiments in vitro have shown that polyamines promote the fusion ofmembrane vesicles (Hong et al., 1983) and in the presence of cellular conditions,spermine proved to be the most e$cient natural cation (Tilley et al., 1988). Theactivity of ion channels in membranes also appears to be modulated by polyaminesand in Aplysia california, spermine was found to reduce the outward currents throughK` channels in neurones of this mollusk (Williams, 1997). These experimental studiesshow the plurality of roles assumed by polyamines in cell membranes.

In marine environment, the membrane functions and the embryonic developmentas well as other physiological functions must be accomplished in the presence of#uctuating salinity concentrations. By selecting spermine as the major polyamine intheir tissues, bivalve mollusks have the advantage of the most e$cient polyamine ableto withstand environmental pressures.

Acknowledgements

We are most grateful to Pr. Michel Broquedis and Dr. Laurence Geny for theHPLC facilities and the helpful advice. We thank LomKc Denis for technical assistance.

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the free guanidine and polyamine pools of bivalve mollusks in relation to their ecology

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