Opine oxidoreductases in brachiopods, bryozoans, phoronids and molluscs

BiochemicalSystematics and Ecology,Vol. 19, No. 4, pp. 263-269, 1991. Printed in Great Britain.

0305-1978/91 $3.00+0.00 © 1991PergamonPressplc.

Opine Oxidoreductases in Brachiopods, Bryozoans, Phoronids and Molluscs CARL S. HAMMEN* and ROBERT C. BULLOCK Department of Zoology, University of Rhode Island, Kingston, RI 02881, U.S.A.

Key Word Index--Lophophorates; molluscs; opine dehydrogenase; lactate; tauropine; octopine; phylogenetics.

Abstract--The opine dehydrogenases catalyse the reductive condensation of pyruvate with various amino acids to form products resembling octopine (N-carboxyethyl arginine). They are widely distributed among marine invertebrates and function in metabolism in the place of lactate dehydrogenase, which often has very low activity, especially in molluscs. The reaction of pyruvate, both alone and with either alanine, arginine, I~-alanine, or taurine, catalysed by tissue extracts of various species, was measured by decrease in absorbance of NADH. The 27 species examined were brachiopods, bryozoans, phoronids and a wide variety of molluscs, including a scaphopod, two chitons and species representing the five subclasses of bivalves and three orders of prosobranch gastropods. Activities were coded 0-5, with 5 assigned to rates greater than 5 ~mol min 1 g-1. These numbers were entered by species into a program that performed average linkage cluster analysis. Results of this analysis were transformed into a dendrogram. The plesiomorphic state was defined as the presence of the total array of opine enzymes and the loss of one or more during evolution was taken as apomorphic. The resulting dendrogram has some features in common with standard phylogenetic trees based on morphology and development.

Introduction The terminal reaction of anaerobic glycolysis in animal cells is generally stated to be the reduction of pyruvate by means of reduced NAD, thereby forming L-lactate and regenerating oxidized NAD, so that glycolysis can continue. Among euryoxic invertebrates, however, several products other than lactic acid are formed, and an array of terminal oxidoreductases serving the same function as lactate dehydrogenase (LDH) has been identified. Especially among molluscs, newly discovered enzymes called opine dehydrogenases often take the place of LDH. These enzymes catalyse the reductive condensation of pyruvate with an amino acid to form compounds like octopine (N-carboxyethyl arginine) and alanopine (iminodipropanoate). Octopine dehydrogenase (EC 1.5.1.11)occurs not only in octopus and other cephalopods but also in many bivalves and gastropods, and when present its activity was found to be much greater than that of LDH [1]. The possible advantages of alternate end-products were discussed by Fields [2], and included: more favorable redox balance, smaller intracellular pH changes, interaction with osmotic adjustment and sometimes increased yield of ATP. The physiological role of the various pyruvate reductases was reviewed by G~de and Grieshaber [3], who suggested that some species may have two or more enzymes in order to cope with both functional and environmental hypoxia. The distribution of opine dehydrogenases among various species of invertebrates was examined closely by Livingstone et al. [4]. Activities of these enzymes were found in 52 of 73 species of marine invertebrates, representing five phyla in addition to the molluscs, but were lacking in all echinoderms, arthropods and chordates tested. Since this survey was published, two additional opine enzymes have been described. Tauropine dehydrogenase (TDH) was found in adductor muscle of several species of *Author to whom correspondence should be addressed. (Received 4 January 1991)

263

264

C.S. HAMMEN AND R. C. BULLOCK

Haliotis (ormer, abalone) by Sato and G~ide [5], and in the pedicle of Glottidia (inarticulate brachiopod) by D o u m e n and Ellington [6]. Prior to these discoveries, taurine was k n o w n only as an osmotic effector in marine invertebrates and was t h o u g h t to be metabolically inert. 13-Alanopine d e h y d r o g e n a s e (BDH) activity was f o u n d in all tissues of Scapharca broughtonfi (blood shell) by Sato et al., [7] and was the major e n z y m e a m o n g an array of d e h y d r o g e n a s e s in the f o o t muscle. The diversity of the opine enzymes and the limited distribution of s o m e of t h e m are difficult to explain on physiological grounds alone. The "reverse reaction" of LDH is stereospecific, and animals with a requirement for D-lactate are distinct along lines of p h y l u m and class from those producing and oxidizing L-lactate [8]. These considerations and the speculations of Livingstone et al. [4] suggest a phylogenetic interpretation. We have e x a m i n e d representative species of brachiopods, bryozoans, phoronids and species from 11 orders of molluscs w i t h respect to activities of LDH and four of the opine oxidoreductases. Materials and Methods Animals were collected from intertidal areas of Narragansett Bay, Rhode Island, or purchased from Pacific Bio-

Marine Laboratories,Venice, California,or Gulf Specimen Co., Panacea,Florida. Enzyme preparationsconsisted of the supernatant fractions of muscle tissue that had been homogenized with HEPESbuffer and centrifuged for 10 min at 9000 g. When animals were too small to allow isolation of muscle, the entire soft tissue, including muscle, was homogenized. Dehydrogenaseactivities were determined by a method similar to that described by G~de [9]. Absorbance at 340 nrn of reduced NAD was measuredwith a Zeiss M4QIIIspectrophotometerand PMI digital indicator.All chemicals were from Sigma Chemical Co., St. Louis, MO. The reaction mixture contained 100 mM HEPES buffer, pH 7.3, tissue extract adjustedto obtain measurableratesand NADHat a final concentrationof 0.15mM, in a total volume of 3.0 ml. The reactionwas started by the addition of sodium pyruvate.The final concentration of pyruvate in the reaction mixture was 2.0 mM. The temperature of the mixture was 19+1°C. Readingsof absorbance were taken every 30 s for 4 min. Usually, absorbance decreasedslowly in a linear manner and amino acids were then added as additional substrate at a final concentration of 40 raM. When a particular opine dehydrogenasewas present,there was a marked accelerationof the rate of reaction and the increasein rate is reported here. When the rate did not increase,that particular enzymewas presumedto be lacking. Extracts of muscle from 10 species were tested for activity of LDH in the direction of lactate oxidation. Oxidized NAD was supplied at a final concentration of 2.0 mM, and D-lactateor L-lactateat 100-300 mM. The buffer was HEPES,pH 8.0, or Tris, pH 8.2. Absorbance at 340 nm was measuredfor 4-6 rain, and the rate of increase was used to calculateactivity. The enzymeactivities reported here were given rating numbers of 0-5, as follows: 0, no activity detected; 1, up to 0.1 pmol rain 1 g ~; 2, 0.1 to 0.5; 3, 0.5 to 1.0; 4, 1.0 to 5.0; 5, >5.0 pmol rain ~g ~. These data were used to perform averagelinkagecluster analysiswith a SAS program at the URI Academic Computer Center.The results of this analysiswere transformed into the dendrogram of Fig. 1 by means of a program developed by Dan Jacobs, Maryland Sea Grant CollegeProgram.

Results Tissues of all 27 species e x a m i n e d had demonstrable lactate d e h y d r o g e n a s e (LDH) activity (Tables 1 and 2). In the majority, 21 species, the rate was between 0.007 and 1.2 p m o l min 1 g ~ tissue. Higher LDH activities were found in three gastropods, t w o chitons and one bivalve. Most species (21) contained one or more of the opine d e h y d r o g e n a s e s in addition to LDH. The exceptions w e r e t w o species of Phoronis, two chitons and t w o bivalves, Mya and Lyonsia, in which no evidence of activities other than LDH was found. A l a n o p i n e d e h y d r o g e n a s e occurred in 18 species, and octopine d e h y d r o g e n a s e in seven. Tauropine d e h y d r o g e n a s e was found to be the major c o m p o n e n t in seven species, four gastropods and three brachiopods and a m i n o r c o m p o n e n t in one bivalve and one gastropod. During the latter phase of this study, 19 species were e x a m i n e d for the presence of the enzyme catalysing f o r m a t i o n of l~-alanopine [7]. Activity was found in five species, the brachiopod Glottidia, two bivalves, Anadara and Crassostrea, the oyster drill

OPINE OXIDOREDUCTASES IN LOPHOPHORATES AND MOLLUSCS A~ AG2 AG4 A~5 BI~ BI7 CH1 CH2 AG~ SC~ BR1 BR3 BR2 EC1 EC3 EC2 PH1 PH2 ~la BN N~ ~15

~

265

-

I ~ } ~ ~

~H

~

~12

~

I

N ~

MG1 I MG2 [~,~,,,,,[,,~ 0 1

Illlr

IIIIIllll[ 2

Illlllllllll 3

4

r~lllllllffl] 5

I1[ I I ~ t l f 6

IIl[ll~lllll~[ 7

8

Distance FIG. 1. DENDROGRAM DEPICTING RESULTS OF AVERAGE LINKAGE CLUSTER ANALYSIS, BASED ON ACTIVITIES OF OPINE OXlDOREDUCTASES IN 27 SPECIES OF MOLLUSCS AND LOPHOPHORATES. See text for mode of construction.

TABLE 1. ACTIVITIES OF DEHYDROGENASES (DH) IN TISSUE EXTRACTS OF "LOPHOPHORATES"

Brachiopoda BR1 Glottidiapyramidata BR2 Laqueuscalifornianus BR3 Terebratalia transversa Phoronida PH1 Phoronis architecta PH2 R vancouverensis

L

A

O

T

B

0.163 0.007

0.431 0.020

0 0

7.984 0.121

0.260

0.118

0.051

0

3.115

0

0.014

0

0

0

0.018

0

0

0

Bryozoa EC1 Bugula neritina

0.007

0.003

0

0

EC2 Mernbranipora tenuis

0.050

0.114

0

0

EC3 Scht~oporella floridana

0.018

0.009

0

0

0

See text for conditions of assay. L = lactate DH, A = alanopine DH, O = octopine DH, T = tauropine DH, 13= ~]-alanopine DH (~mol rain -~ g ~). Symbols used in cluster analysis are in far left column.

Urosalpinx and the limpet Tectura testudinalis. Experiments to determine the substrate specificity of lactate oxidation indicated that Chaetopleura, three archaeogastropods, Nucella and Lyonsia had LDH specific for D-lactate, and two brachiopods, Glottidia and Terebratalia, had LDH specific for L-lactate. In each case, only one isomer was oxidized. Activities ranged from 0.007 to 0.755 ~mol min -1 g-l. Activity in the direction of lactate oxidation was very weak in the bryozoan Membranipora, but suggested specificity for L-lactate. The dendrogram shows three main clusters, one consisting of Iophophorates and the other two of molluscs. One molluscan group contains Scaphopoda, Polyplacophora, Archaeogastropoda and two Bivalvia. The other group contains all other gastropods and bivalves representative of four of the five subclasses. Discussion Tauropine dehydrogenase was confirmed as the major terminal oxidoreductase in the abalone Haliotis and in the lingulid brachiopod Glottidia, as reported by others. Activity of TDH was also high in two articulate brachiopods and in three additional species of

266

C. S, HAMMEN AND R, C. BULLOCK

TABLE 2. ACTIVITIES OF DEHYDROGENASESIN TISSUE EXTRACTS OF VARIOUS SPECIES OF MARINE MOLLUSCS

C1. Scaphopoda SC1 Dentalium pilsbryi C1. Polyplacophora (Amphineura) CH1 Chaetopleura apiculata CH2 Mopah~ muscosa C1. Bivalvia Subclass Palaeotaxodonta BI1 Nucula proxima Subclass Cryptodonta BI2 Solemya velum Subclass Pteriomorpha Order Arcoida BI3 Anadara ovalis Order Pterioida BI4 Crassostrea virginica Subclass Heterodonta BI5 Spatula soh~h~sima BI6 Mya arenaria Subclass Anomalodesmata 817 Lyonsia hyalina C1 Gastropoda Subctass Prosobranchia Order Archaeogastropoda AG1 Haliob~ rufescensfoot AG1 Haliot~ rufescens adductor AG2 Diodora cayenensis AG3 Tectura testudinalis AG4 Turbo castanea AG5 Teguta funebrah~ Order Mesogastropoda MG1 Littorina #ttorea MG2 Crepiduta fornicata Order Neogastropoda NG1 Urosalpinx c/nerea NG2 Nuce#a lap#/us

L

A

0

T

B

0.231

0

3.570

0

0

7.060 8.879

0 0

0 0

0 0

0

0.671

10.056

18.814

0

0

1.261

16.952

56.617

0

0

0.522

7.870

0.871

0.104

4.483

0.208

6.366

0

0

1.288

5.112 0.645

3.825 0

7.646 0

0 0

0 0

0.217

0

0

0

0

2.184 1.048 0.213 0.318 0.443 0.355

0 0 0.040 0 0.041 0.188

2.026 0 0 0 0 0

0 1.703 1.568 0.244 7.686 5.354

0

9.69 5.45

7.12 6.88

0 0

0 0

0 0

0.99 0.81

9.50 4.18

0 1.91

0 0

1.158 0

0 5.710

Assay conditions are described in the text. Abbreviations same as Table 1. (#mol min ~ g ~).

archaeogastropods. It was present as a minor component in one bivalve and in Tectura, the Atlantic plate limpet. Thus TDH is not rare, but at present seems important only in Brachiopoda and one order of Gastropoda. In Ha#otis rufescens the enzyme was found in adductor but not in foot muscle. This is in general agreement with the results of G&de [9] who observed that TDH activity in the adductor muscle of Ha#otis larnellosa was more than six times greater than in the foot muscle, and was practically absent from other tissues. The taurine concentration was 79.4 Bmol g-1 in this animal. In the pedicle of Glottidia the taurine concentration was an order of magnitude lower, 10.9 pmol g-l, but nevertheless accounted for 60% of the free amino acids in the tissue [6]. At a pyruvate concentration of 5 mM, the apparent Kr~ for taurine was 65 mM in Ha#otis and 13.9 mM in Glottidia. Thus, tissue concentrations and Kr~ were in the same range in each species. A decrease in taurine concentration and an increase in tauropine in the adductor of H. lamellosa during experimental anoxia were shown by G~de [10]. It is likely that TDH plays a significant role in anaerobic glycolysis in species that display high activity. Prior to these experiments, an enzyme specific for i~-alanine was known as a major component of the collection of dehydrogenases in the foot muscle of Scapharca broughton#[7]. It was not surprising, therefore, to find high activity in another species of the order Arcoida, Anadara ovalis (Table 2). J]-Alanine had been shown to be an

OPINE OXIDOREDUCTASES IN LOPHOPHORATESAND MOLLUSCS

267

alternate substrate of the TDH in Glottidia [6] and we also found activity in this species. The strombine dehydrogenase of Modiolus squamosus displayed limited activity with 16-alanine [11]. In this study, similar low activity was found in another bivalve, Crassostrea virginica, a member of the order Pterioida. The most striking new finding was the high activity of the I]-alanine enzyme in the limpet Tectura testudinalis, classified with the Archaeogastropoda. Two other species of this group had no activity with this substrate. This is particularly interesting because of recent proposals placing limpets well apart from other gastropods [12]. The choice of species used in this study depended partly on availability, but also on the goal of a broad representation within selected taxonomic groups. That biochemical data are sometimes relevant to phylogeny, in spite of questionable use in the past, was made clear by Mangum [13]. Two large questions of phylogeny that have been asked repeatedly are the validity of the concept of Iophophorates and the relation of this group to other phyla. Hyman [14] wrote that "the Iophophorates constitute a connecting link between the Protostomia and the Deuterostomia, but the details of the connection cannot be stated". The concept of Iophophorates is far from certain. Rowell and Grant [15] stated that the Phoronida, Bryozoa and Brachiopoda "may have shared a common evolutionary history not shared by other phyla," and they called for additional evidence from development, morphology and protein chemistry. The fossil record provides little guide to the origin of phyla and the relationship of the Iophophorates to other invertebrates remains obscure. In recent years, molecular resemblances of nucleic acids and proteins have been used more and more to supplement the classic morphological criteria of evolutionary descent. An attempt to construct a "molecular phylogeny" was carried out by Field et al. [16], based on comparison of nucleotide sequences in ribosomal RNA from representative species in 10 phyla. The tree resulting from this analysis shows Lingula reevivery near the polychaete Chaetopterus and the chiton Cryptochiton stelleriand more distant from two bivalves and a nudibranch. No archaeogastropods were examined and Lingula was the sole representative of the Iophophorate group. The same data were used by Lake [17] in an alternate statistical procedure to produce a tree in which Lingula and the molluscs are associated on a branch distinct from other invertebrates. Commenting on both papers, Patterson [18] stated that in dealing with relations between phyla, "we are certainly asking about events in the Precambrian, because arthropods, molluscs and brachiopods are all known in early Cambrian rocks". Since enzymes are specific products of the genes that direct their synthesis, phylogenetic meaning may be expected to reside in patterns of enzyme distribution. Similar levels of activity of the various dehydrogenases are expected among animals believed to be closely related on ordinary taxonomic grounds. A phylogenetic distribution of lactate and opine pathways was postulated by Livingstone eta/. [4] in their survey of dehydrogenases in 103 species, including 44 species of molluscs and two brachiopods. They found the opine pathways characteristic of lower and middle phyla and absent from the higher phyla. Their tree showed Brachiopoda more closely allied to the Deuterostomia than to the Mollusca. The assumption used in our analysis is that evolution ordinarily has proceeded through gradual loss of enzymes. In other words, the presence of all enzymes is the ancestral or plesiomorphic state and the absence of each is apomorphic or a "derived character state" [19]. This idea was expressed by Florkin [20], who gave the "disappearance" of uricolytic enzymes as his main example. Primitive crustaceans have the complete pathway of uric acid degradation, while "advanced" insects do not. The principle was also invoked by Baldwin [21] in his discussion of nutritional requirements of animals, which "have come to lose so large a part of the synthetic ability that their ancestors must at one time have possessed". The conclusion of Livingstone eta/. [4] is

268

C.S. HAMMEN AND R. C. BULLOCK

consistent with this idea: "..o all the terminal dehydrogenases were probably present in the early stages of metazoan evolution and that, subsequently, a selection occurred involving the loss of some or all opine dehydrogenase activities from certain phyla . . . . " The earliest shelled invertebrates probably had a complete array of opine dehydrogenases, or possibly fewer enzymes with broad specificity. In this work, the species nearest the presumed ancestral condition is Anadara ovalis, which has some activity of all five enzymes measured. The same abundance of enzymes had been found in the blood clam Scapharca, also in the family Arcidae [7]. Cluster analysis (Fig. 1) groups the phoronids and bryozoans with the articulate brachiopod Laqueus, supporting the concept of Iophophorates. The other two brachiopods are more distant, but nevertheless allied to this group. This result also favors the coherence of articulate and inarticulate brachiopods in a single phylum. The archaeogastropods cluster with a scaphopod, two chitons and two bivalves that contain only LDH. The other major branch of the molluscs consists of the other five bivalves and the higher gastropods that have the greatest array of dehydrogenases. One factor that contributed to the distance of molluscs from brachiopods in this analysis was the scoring of L-lactate-specific LDH as 5 and the D-lactate-specific enzyme as 0. Long [8] made the assumption that L-LDH is the basic, original, ancestral form and the stereospecificity for D-lactate is the result of very ancient mutations. This was based on postulating the minimum number of changes to account for the present distribution, namely eight phyla exclusively L, the Mollusca only g and two phyla, Annelida and Arthropoda, with g in a few classes. The data of Hammen [22] and Livingstone et al., [4] and the new data on Terebratalia, indicate that brachiopods in five of the existing seven superfamilies all have LDH specific for L-lactate. The tree resulting from this study has many features in accord with conservative trees based on morphology and development. Its greatest departure is the incomplete separation of bivalves from gastropods. One interesting feature is the distance of the limpet Tectura from the base of the diagram, suggesting that it has preserved the primitive condition for a long time and with respect to these physiological traits, it may represent best the ancestral state. A sample of muscle from Neopilina would have been very useful in this study, but in the last analysis no tree based on only a few traits can be assigned much validity. The essence of quantitative phylogenetic analysis is the cumulative weight of a large number of measurable traits. Thus, a combination of nucleotide sequences, opine dehydrogenase activities, shell morphology, gill structure, etc. should be superior to any one type of data in establishing plausible phylogenies. Acknowledgements--Wewish

to thank S. Woodbury and A. Gerardi for performing several important experiments and I. Thukral and C. Vye for computer program assistance and the production of the dendrogram.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Regnouf, F. and Thoai, N. V. (1970) Comp. Biochem. Physiol. 32, 411. Fields, J. H. A. (1983) J. Exp. ZooL 228, 445. G~de, G. and Grieshaber, M. K. (1986) Comp. Biochern. Physiol. 83B, 255. Livingstone, D. R., deZwaan, A., Leopold, M. and Marteijn, E. (1983) Biochem. Syst. Ecol. 11, 415. Sato, M. and G~de, G. (1986) Naturwissenschaften 73, 207. Doumen, C. and Ellington, W. R. (1987) J. Exp. ZooL 243, 25. Sato, M., Takahara, M., Kanno, N., Sato, S. and Ellington, W. R. (1987) Comp. Biochem. Physt~L 88B, 803. Long, G. L. (1976) Comp. Biochem. Physiol. 551], 77. G~de, G. (1986) Eur. J. Biochem. 160, 311. G~de, G. (1988) Biol. Bull 175, 122. Nicchitta, C. A. and Ellington, W. R (1984) Comp. Biochem. Physiol. 77B, 233. Haszprunar, G. (1988) J. Moll. Stud. 54, 367. Mangum, C. P. (1990) Proc. Biol. Soc. Wash. 103, 235.

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14. Hyman, L. H. (1959) The InvertebratesVol. 5, p. 604. McGraw-Hill, New York. 15. Rowell, A. J. and Grant, R. E. (1987) in Fossillnvertebrates(Boardman, R. S., Cheetham, A. H. and Rowell, A. J., eds), p. 461. Blackwell, Palo Alto. 16. Field, K. G., Olsen, G. J., Lane, D. J., Giovannoni, S. J., Ghiselin, M. T., Raft, E. C., Pace, N. R. and Raft, R. A. (1988) Science 239, 748. 17. Lake, J. A. (1990) Proc. Natn. Acad. Sci., USA 87, 763. 18. Patterson, C. (1990) Nature 344, 199. 19. Ridley, M. (1986) Evolution and Classification: The Reformation ofCladism, p. 55. Longman, London. 20. FIorkin, M. (1960) Unity and Diversity in Biochemistry, p. 343. Pergamon Press, New York. 21. Baldwin, E. (1964) An Introduction to Comparative Biochemistr~, 4th edn, p. 135. Cambridge University Press, Cambridge. 22. Hammen, C. S. (1977) Am. Zoo/. 17, 141.