Biochemical Systematics and Ecology, Vol. 20, No. 6, pp. 535-539, 1992. 0305-1978/92 $5.00÷0.00 Printed in Great Britain. © 1992 Pergamon Press Ltd.

Echinoderm (Holothuria atra) Lactate Dehydrogenase: Affinities with the Putative Ancestral Chordate Enzyme

JOHN BALDWIN,* ANNETTE PATAKt and KEITH MORTIMER:~ *Department of Ecology and Evolutionary Biology, Monash University, Clayton, Victoria 3168, Australia; tZoology Department and 1:Centre for Protein and Enzyme Technology, La Trobe University, Bundoora,

Victoria 3083, Australia

Key Word Index--Holothuria atra; Holothuriidae; Echinodermata; lower chordates; lactate dehydrogenase; amino acid; immunochemistry; evolutionary relationships.

Abstract--Lactate dehydrogenase (LDH) was purified from the echinoderm Holothuria atra. The enzyme occurs as a single L-specific homotetramer with a subunit molecular weight of 39,000, and kinetic properties that differ from those of the A 4, B 4 and C 4 enzymes of lower vertebrates and lower chordates. Amino acid compositional analysis and immunochemical titration indicate considerable structural affinity (=70% sequence similarity) with the single LDH subunit of the ascidian Pyura stolonifera. This supports the proposition that echinoderms lie close to the stemline leading from invertebrates to lower chordates, and that the ascidian LDH subunit represents the ancestral subunit type of vertebrate LDH.

Introduction Vertebrate lactate dehydrogenase [L-LDH; (S)-Iactate: NAD + oxidoreductase, EC 1.1.1.27] isoenzymes are tetramers composed of various combinations of at least three structurally and genetically distinct subunits, termed A, B and C. It is con- sidered that these subunits are products of gene duplication, having diverged from a single LDH subunit type present in lower chordates, or possibly prechordate ancestors (Markert et al., 1975; Li et aL, 1983).

Recently we demonstrated that the ascidian Pyura stolonifera (phylum Chordata, subphylum Urochordata) contains a single LDH subunit showing greater structural similarity to the C than to the A and B subunits of teleost fish (Baldwin et aL, 1988). Such a result was consistent with amino acid sequence data implying that the C subunit resembles most closely the ancestral subunit type of vertebrate LDH (Rehse and Davidson, 1986; Li et aL, 1983).

An obvious extension of this study was to determine the degree to which the ascidian LDH subunit shared affinities with the LDH subunits of modern inverteb- rates lying close to the stemline from which chordates arose. Evidence that the echinoderms occupy such a position comes from developmental and anatomical similarities with the Hemichordata (Ubaghs, 1969; Godeaux, 1974; Barnes, 1980), and their possible affinities with the Stylophora, a group of Palaeozoic fossils that have been interpreted as chordates ancestral to the urochordates, cephalochordates and vertebrates [Jefferies (1967), but also see Jollie (1982)].

We report that the single LDH of the echinoderm Holothuria atra displays unusual functional properties, but shares significant structural similarity with ascidian LDH.

Materials and Methods Sea cucumbers (Holothuria atra, phylum Echinodermata, class Holothuroidea, family Holothuriidae) were collected on the reef flat at Heron Island, Queensland, Australia. Ascidians (t~/ura sto/onifera, phylum Chordata, subphylum Urochordata, class Ascidiacea) came from the intertidal zone of rocky shores in South Eastern Victoria, Australia. Southern bastard red cod (Pseudophyc/s barbara, order Gadiformes, family Moridae) were purchased from a local Melbourne fish market.

(Received 9 March 1991)

535

536 J. BALDWIN ETAL.

The LDH pyruvate reductase reaction was assayed at 340 nm with a recording spectrophotometer in which the cuvette temperature was maintained at 25°C. Routine assays contained 5 mM pyruvate, 0.15 mM NADH and 100 mM phosphate buffer, pH 7.5.

The tissue distribution of LDH in H. atra was examined by cellulose acetate gel electrophoresis using a 75 mM Tris buffer in which the pH was adjusted to 6.9 with citric acid. Enzyme activity was detected by the method of Markert and Ursprung (1962). Fresh tissues were homogenized in ice-cold 100 mM potassium phosphate buffer, pH 7.5 (1 : 2 W/V), centrifuged at 13,000 g for 10 min at 4°C, and supernatants were used immediately.

LDH was purified from longitudinal muscle strips of H. atra by oxamate-Sepharose affinity chromato- graphy using the method described for lamprey LDH (Baldwin et al., 1987). Purification of the ascidian and teleost LDH enzymes is described in Baldwin et al. (1988). The purity of LDH preparations was assessed by protein staining following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Specific activities were calculated using protein concentrations determined by the method of Lowry et al. (1951) with bovine serum albumin standards.

The subunit molecular weight of purified echinoderm LDH was determined by SDS electrophoresis using marker proteins (LMW calibration kit, Pharmacia). The molecular weight of the native enzyme was obtained by gel filtration (FPLC, Sepharose 12 HR 16/10, Pharmacia), with catalyase, aldolase, pig LDHA 4 and bovine serum albumin run as standards.

Methods used for determining amino acid composition and for immunochemical comparisons are described in Baldwin et al. (1987) and Baldwin et al. (1988).

Results The LDH activities in tissues of H. atra are shown in Table 1. Electrophoresis of these tissue extracts gave single coincident anodal bands of LDH activity, with the exception of the body wall where activity was too low to detect. On the basis of these results the longitudinal muscle was selected for LDH purification. The purifica- tion procedure gave approximately 90% recovery of the activity present in the initial tissue homogenate. The final LDH preparation had a specific activity of 310 #mol NADH min -1 mg -1 protein at 25°C. It gave single coincident bands when stained for LDH activity and for protein following electrophoresis on cellulose acetate gel, and a single protein band of 39,000 M r after acrylamide geI-SDS electrophoresis. Gel filtration gave a molecular weight of 137,300 for the native enzyme. The amino acid composition of the echinoderm LDH subunit is given in Table 2.

Antisera produced against the echinoderm and ascidian LDH enzymes were tested for the ability to inhibit pyruvate reductase activity of purified echinoderm, ascidian and teleost LDH homopolymers (Table 3). Ascidian LDH and teleost LDHC 4 showed similar sensitivity to inhibition with anti-echinoderm LDH antiserum. However, the antiserum : protein ratio giving 50% inhibition of these enzymes gave only 39 and 33% inhibition, respectively, of teleost LDHB 4 and LDHA 4, and ratios as high as 1000 #1 antiserum : #g LDH protein failed to achieve 50% inhibition. Anti- ascidian LDH antiserum was most effective at inhibiting teleost LDHC 4, followed by echinoderm LDH and teleost LDHB 4. It failed to show any inhibition of teleost LDHA 4 at ratios up to 1000 #1 antiserum : #g LDH protein.

Echinoderm LDH oxidized L-, but not D-lactate. Kinetic properties of the enzyme are compared with published data for the ascidian and teleost LDH enzymes in Table 4.

TABLE 1. LDH ACTIVITY IN TISSUES OF HOLOTHURIA ATRA

Activity Tissue (Mmol NADH min 1 g ~ wet wt, 25°C)

Longitudinal muscle 33 Radial muscle 3.4 Respiratory tree 4.8 Gonad < 0.5 Intestine < 0.5 Body wall < 0.5

ECHINODERM LACTATE DEHYDROGENASE 537

TABLE 2. AMINO ACID COMPOSITION OF HOLOTHURIA ATRA

LDH SUBUNIT

Amino acid Number Amino acid Number

Cys 3 Met 10 Asx* 18 lie 24 Thr 14 Leu 34 Ser 24 Tyr 9 Glxl" 33 Phe 8 Pro 11 His 12 Gly 28 Lys 29 Ala 24 Trp 5 Val 39 Arg 12

Calculated on the assumption that amino acid residues.

*Aspartata + asparagine. 1"Glutamate + glutamine.

the subunit contains ca 337

TABLE 3. IMMUNOCHEMICAL TITRATION OF LDH PYRUVATE REDUCTASE ACTIVITY

Inhibition values* Anti-echinoderm LDH Anti-ascidian LDH

LDH antiserum antiserum

Echinoderm 1 126 Ascidian 214 1 Teleost A 4 > 314 N.l. Teleost B4 > 267 > 154 Teleost C 4 212 42

*Inhibition values are calculated from the number of microtitres of antiserum required to give 50% inhibition of pyruvate reductase activity of 1 i~g of LDH protein. Values are expressed relative to the inhibition value obtained for the LDH used to produce the antiserum.

N.l.=no inhibition detected at a ratio of 1000 I~1 antiserum: 1 ~g LDH.

TABLE 4. KINETIC PROPERTIES OF LDH ENZYMES

Specific K m pyruvatel" APAD Activity at 10 mM pyruvate Enzyme activity* (mM) ratios as % of maximalt

Echinoderm LDH 310 0.20 ± 0.03 2.6 _+ 0.2 92 + 5 Ascidian LDH 1000 0.41 1.6 92 Teleost LDHA 4 1300 0.68 1.6 87 Teleost LDHB 4 400 0.10 7.3 55 Teleost LDHC 4 410 0.13 4.9 66

*The assay contained the optimal pyruvate concentration, 0.15 mM NADH, and 100 mM potassium phosphate buffer, pH 7.5. Assay temperature 25°C. Units are pmol substrate min 1 mg 1 protein.

1"The assay was as for the above footnote, but with pyruvate concentrations varied between 0.01 and 10 raM.

SEnzyme (0.05 IU) was incubated for 10 min at 25°C with 0.33 or 10 mM pyruvata and 1.43x10 6 M 3-acetylpyridine adenine dinucleotide (APAD) in 100 mM potassium phosphate buffer, pH 7.5. Enzyme activity was then determined after the addition of 0.15 mM NADH. Activity at 0.33 mM pyruvate was divided by the activity at 10 mM pyruvate to give the APAD ratio (Kaplan et al., 1968).

Values for echinoderm LDH are means with S.D., for three determinations. Values for ascidian LDH and teleost LDH are from Baldwin et al. (1988).

538 J. BALDWIN ETAL.

Discussion Only one electrophoretic form of LDH was detected in tissues of H. atra. This enzyme has a subunit molecular weight and tetrameric structure similar to the LDH enzymes of chordates (Pesce et al., 1967; Markert et al., 1975), and is assumed to be a homopolymer. Attempts to confirm the presence of a single subunit type by identifying N-terminal amino acids were unsuccessful as the N-terminus, like that of ascidian LDH (Baldwin et aL, 1988), was blocked.

A number of properties of the echinoderm LDH distinguish this enzyme from the LDH homopolymers of lower chordates and lower vertebrates. While the low specific activity and low K m pyruvate value resemble the LDHB 4 lactate oxidases of elasmobranchs and teteosts and LDHC 4 of teleosts, the low APAD ratio, limited pyruvate inhibition and high histidine content are properties usually associated with the pyruvate reductase activities of the gnathostome and hagfish LDHA 4 isozymes and ascidian LDH. The single LDH enzymes of lampreys show higher specific activities, Km pyruvate values and APAD ratios, but similar histidine contents in comparison to echinoderm LDH (Baldwin et aL, 1987; Baldwin, 1988; Baldwin et aL, 1988, 1989). This unusual combination of properties makes it difficult to assess the physiological role of the echinoderm enzyme.

Both immunochemical and amino acid compositional data provide evidence that the ascidian LDH subunit shares greater structural similarity with the teleost C than with the teleost A and B subunits (Baldwin et al., 1988). In the present study, titration with both anti-echinoderm LDH antisera and anti-ascidian LDH antisera demonstrates greater immunochemical similarity between the echinoderm, ascidian and teleost C subunits than between the echinoderm or ascidian subunits and the teleost A and B subunits.

Using the method of Metzger et al. (1968) to compare amino acid compositions of echinoderm and ascidian LDH subunits (Table 5) gives a Metzger difference index of 6.46. As this value is below the critical value for proteins of this size (=8.0, Cornish-Bowden, 1983), it can be taken as strong evidence of homology, an interpretation supported by the immunochemical data. The degree of sequence similarity between these two subunits can be estimated from the relationship between Metzger difference indices and amino acid sequence differences established for LDH subunits of known amino acid sequence (Rehse and Davidson, 1986; Baldwin et aL, 1988). This extrapolation provides an estimate of 30.38_+4.32 (mean+95% confidence limits) for the percentage difference in amino acid sequence. The difference is equivalent to that between vertebrate LDHA and LDHB subunits, and between the mammalian LDHC subunit and the LDHA subunits of mammals and birds (Rehse and Davidson, 1986). To our knowledge this is the first estimate to be made of sequence similarity between invertebrate and chordate LDH enzymes. The results support the proposition that echinoderms lie close to the stemline leading from invertebrates to the lower chordates, and also that the ascidian LDH subunit represents the ancestral subunit type of vertebrate LDH.

However, while both the amino acid composition and immunochemical data indicate considerable structural affinity between the echinoderm and ascidian LDH

TABLE 5. AMINO ACID COMPOSITION RELATEDNESS OF ECHINODERM, ASCIDIAN AND TELEOST LDH SUBUNITS (Metzger difference indices)

Teleost

Ascidian A B C

Echinoderm 6.46 12.51 8.63 12,56 Ascidian 13.67 12,35 8.76 Teleost A 10.39 16.67 Teleost B 11.97

ECHINODERM LACTATE DEHYDROGENASE 539

subunits, relationships with the teleost subunits are less clear. For example, although the echinoderm subunit immunochemically is closer to the teleost C than to the teleost A or B subunits, Metzger difference indices imply closer relationships with the teleost B subunit. Similarly, the Metzger difference index groups the ascidian subunit more closely with the echinoderm than with the teleost C subunit, but the immunochemical data suggest the reverse. These discrepancies, which arise at Metzger difference indices greater than 8.0, imply that the structural differences between the echinoderm and teleost LDH subunits lie beyond the range for which amino acid composition can be expected to accurately reflect sequence similarity.

Acknowledgements--Amino acid analysis was carried out by Mr P. M. Strike, C.S.I.R.O. Division of Protein Chemistry. We thank the Heron Island Research Station for providing facilities used during this study.

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