Fisheries Science 63(3), 414-420 (1997)

Purification and Characterization of Tauropine Dehydrogenase from the Marine Sponge Halichondria japonica Kadota (Demospongia)*1

Nobuhiro Kan-no,*2,•õ Minoru Sato,*3 Eizoh Nagahisa,*2

and Yoshikazu Sato*2

*2School of Fisheries Sciences , Kitasato University, Sanriku, Iwate 022-01, Japan *3Faculty of Agriculture , Tohoku University, Sendai, Miyagi 981, Japan (Received July 8, 1996)

Tauropine dehydrogenase (tauropine: NAD ) was purified to homogeneity from the sponge Halichondria japonica Kadota (colony). Relative molecular masses of this in its native form and in its denatured form were 36,500 and 37,000, respectively, indicating a monomeric structure. The maximum rate in the tauropine-biosynthetic reaction was observed at pH 6.8, and that in the tauro pine-catabolic reaction at pH 9.0. Pyruvate and taurine were the preferred substrates. The enzyme showed significant activity for oxalacetate as a substitute for pyruvate but much lower activities for other keto acids and amino acids. The tauropine-biosynthetic reaction was strongly inhibited by the pyruvate. The optimal concentration of pyruvate was 0.25-0.35 mm and the inhibitory concen tration giving half-maximal rate was 3.2 mm. The tauropine-catabolic reaction was inhibited by the sub strate tauropine: the optimal concentration was 2.5-5.0 mm. Apparent K,,, values determined using con stant cosubstrate concentrations were 37.0 mm for taurine, 0.068 mm for pyruvate, and 0.036 mm for NADH in the tauropine-biosynthetic reaction; and 0.39 mm for tauropine and 0.16 mm for NAD+ in the tauropine-catabolic reaction. Key words: opine dehydrogenase, tauropine, tauropine dehydrogenase, Halichondria, sponge, purification, anaerobic glycolysis

To date, five unique imino acids called `opines', i.e., oc with emphasis on the from muscular tissues of topine, alanopine, strombine, tauropine and /3-alanopine, molluscs.8) Those characterization studies have shown the have been isolated from marine invertebrates.1-7) These similarities of the molecular structures and catalytic prop opines are biosynthesized by pyruvate reductases of a fami erties of OpDHs, and thus led to the hypothesis that all ly of opine dehydrogenases (imino acid: NAD oxidoreduc OpDHs are homologous.12) Recently, Sato et al.") deter tase; OpDH) which catalyze the reversible reductive con mined the distribution of OpDHs including TaDH and ƒÀ densation of pyruvate and amino acids with NADH as the - AlDH, and pointed out the importance of the study on coenzyme. Opine dehydrogenases (or activities) corre TaDH; the observation that TaDH represented a major sponding to the five opines have been discovered: i.e., octo OpDHs activity of the species belonging to the lower phy pine dehydrogenase (D-octopine: NAD oxidoreductase; la, Porifera and Coelenterata, led to a hypothesis that EC 1.5.1.11; OcDH), alanopine dehydrogenase (meso TaDH was the most ancient OpDH. Therefore, the charac alanopine: NAD oxidoreductase; EC 1.5.1.17; AlDH), terization of TaDH from the animals belonging to the low strombine dehydrogenase (D-strombine: NAD oxidoreduc er phyla is expected to provide valuable information on tase; EC 1.5.1.22; StDH), tauropine dehydrogenase (tauro the origin and evolution of OpDH molecules and on the pine: NAD oxidoreductase; TaDH), and ƒÀ-alanopine de physiological role of OpDHs. So far, TaDHs of the aba hydrogenase (ƒÀ-alanopine: NAD oxidoreductase; EC lone (ormer) Haliotis lamellosa18) and Haliotis discus han 1.5.1.26; ƒÀ-AlDH). It is widely accepted that the physiolog nai19) (Mollusca, Gastrapoda), and of the brachiopod Glot ical roles of OpDH in invertebrates are analogous to those tidea pyramidata20) (Tentaculata) have received significant of lactate dehydrogenase (EC 1.1.1.27, 28; LDH), i.e., attention. Thus we initiated a study on the TaDH from the balancing cytoplasmic redox potential and maintaining marine sponge Halichondria japonica, the phylogenetical rates of energy production during hypoxic conditions.8-12) ly `lowest' animal, in which TaDH activity had been The three main OpDHs, i.e., OcDH, AlDH, and StDH, demonstrated.17) Furthermore, it is interesting to compare have been extensively studied on their phylogenetic distri the properties of TaDH from the sponge which has no mus bution13-17) and on the catalytic and molecular properties cular tissue with the known TaDHs of muscular tis-

*1 A preliminary report was presented at the 4th International Congress of Comparative Physiology and Biochemistry (1995; Birmingham, UK)

with a brief abstract in Physiological Zoology 68. •õ To whom correspondence should be addressed. Abbreviations: AlDH, alanopine dehydrogenase; ƒÀ-AlDH, ƒÀ-alanopine dehydrogenase; IEF-TLPAG, isoelectric focusing in thin-layer polyacrylamide gel; LDH, lactate dehydrogenase; OcDH, octopine dehydrogenase; OpDH, opine dehydrogenase; StDH, strom bine dehydrogenase; TaDH, tauropine dehydrogenase. Tauropine Dehydrogenase from Halichondria 415 sues.18-20) In this paper, the purification and some properties enzyme solution was loaded onto a column (10 x 230 mm) of the sponge TaDH are described. of Macro-Prep ceramic hydroxyapatite (20 ƒÊm particle size; Bio-Rad Laboratories, Richmond, Ca., USA) Materials and Methods equilibrated with 1 mM KH2PO4/KOH (pH 7.2) contain ing 10 mM 2-mercaptoethanol. The column was eluted by a Materials linear gradient of KH2PO4/KOH (1 to 150 mm, pH 7.2, Specimens of the sponge Halichondria japonica (colo 200 ml in total volume) at a flow rate of 0.25 ml/ min. The ny) were collected from the seashore of Sanriku, Iwate, TaDH activity was eluted at about 50 mm region. The enzy Japan, in May 1995 and were immediately subjected to en matically active fractions were pooled for further studies. zyme extraction. Four opine compounds, i.e., meso-alano pine, D-strombine, tauropine, and ƒÀ-alanopine, were pre Analytical Isoelectric Focusing in Thin-Layer Poly pared according to the methods described by Sato et al.4-7) acrylamide Gels (IEF-TLPAG) D-Octopine was purchased from Sigma Chemical (St. Analytical IEF-TLPAG was carried out using a slab gel Louis, Mo., USA). of 90 mm length, 200 mm width, and 0.5 mm thickness containing Pharmalyte 3-10 (1:16 dilution; Pharmacia Purification of Tauropine Dehydrogenase Biotech) (acrylamide concentration was 5%T and 3%C) All enzyme extraction and purification steps were per by the method described in our previous paper.211 Focusing formed at 4•Ž unless otherwise indicated. The fresh was carried out, using 0.04 M aspartic acid (anode) and 1 M sponges were dissected into pieces, washed well with NaOH (cathode) as electrode solutions, for 4000 volt-hour filtered seawater to remove all encrusting organisms, sand, at a constant power of 6 W (with a maximum voltage of and other foreign materials (we sometimes scraped off the 1500 V) in a flat-bed IEF apparatus (Atto, Tokyo, Japan) surface part of sppponnn and blotted with filter paper to re with cooling water (10•Ž) circulation. After the focusing, move seawater. The cleaned sponges (100-200 g wet wt) the gel was immersed at room temperature for 5-20 min in were homogenized for 5-10 min with 3 volumes of ice-cold a mixture of 1.0 mm NAD+, 3.0 mm tauropine (adjusted 20mM KH2PO4/KOH (pH 7.2) containing 1 mm EDTA pH 9.0 with NaOH), 0.1 mm phenazine methosulfate, and and 10 mm 2-mercaptoethanol by using a disperser. Then 1 mm nitroblue tetrazolium in 100 mm Tris/HCl buffer the homogenate was centrifuged for 20 min at 10,000 x g (pH 9.0) for the localization of TaDH activity. The gel was and 4•Ž. The supernatant was used as crude enzyme. stained for protein with Coomassie brilliant blue G250.22) Solid (NH4)2SO4 was slowly added to the crude enzyme The isoelectric point of the enzyme was estimated using pI to 45% saturation. After stirring for 1 hr, the solution was - marker proteins (broad pI range; Pharmacia Biotech). centrifuged for 20 min at 10,000 x g and 4°C. Then addi tional (NH4)2SO4 was added to the supernatant to 75% Determination of Relative Molecular Mass saturation, and the solution was stirred and centrifuged as The relative molecular mass of the enzyme in its native above. The protein precipitated was dissolved in a minimal form was estimated on a TSK G3000SW rapid gel-filtra volume of 2 mm KH2PO4/KOH (pH 7.2) containing 1 mm tion column (7.5 x 600 mm; Tosoh) attached to a PLC-10 EDTA and 10 mm 2-mercaptoethanol (referred to as HPLC system (Eyela, Tokyo, Japan), using 20 mm Buffer A). The enzyme solution was loaded onto a column KH2PO4/KOH (pH 7.2) containing 1 mm EDTA, 10mM (44 x 900 mm) of Sephadex G75 (Pharmacia Biotech, Up 2-mercaptoethanol, and 0.2 M NaCl as a running buffer. psala, Sweden) and eluted with Buffer A at a flow rate of 1 The standard proteins and the purified TaDH were run ml/min. The column eluate was monitored for absor through the column at a flow rate of 0.5 ml/ min. The stan bance at 280 nm and TaDH activity. The fractions show dard proteins (Pharmacia Biotech) were: aldolase, 158 ing TaDH activity were pooled, and loaded onto a column kDa; bovine albumin, 67 kDa; ovalbumin, 43 kDa; (32 x 200 mm) of Blue-Sepharose CL6B (Pharmacia chymotrypsinogen A, 25 kDa; and ribonuclease, 13 kDa. Biotech). The column was washed with Buffer A, and then The relative molecular mass of the enzyme in its dena eluted by a linear gradient of NaCl in Buffer A (0 to 1 M, tured form was determined by SDS-PAGE carried out un 500 ml in total volume) at a flow rate of 1 ml/ min. TaDH der reducing conditions. A homogeneous-pore slab gel of was eluted between 0.2-0.4 M NaCl concentration. The 12.5%T (80 mm length, 90 mm width, 1 mm thickness) fractions showing TaDH activity were pooled, concentrat was prepared and run according to the method of Laem ed by ultrafiltration on a Diaflo YM3 membrane (3-kDa mli.23)The gel was stained for protein with Coomassie bril molecular mass cut-off; Amicon, Beverly, Ma., USA), and liant blue R250. Bio-Rad low range SDS-PAGE standards desalted by passing through a small column (20 x 100 mm) (97.4 kDa-14.4 kDa) were used for the calibration. of Sephadex G25 (Pharmacia Biotech) using Buffer A as a running buffer. The enzyme solution was then loaded onto Enzyme Assays a column (10 x 230 mm) of Super Q-Toyopearl 650S The activities of OpDHs and LDH were measured by (Tosoh, Tokyo, Japan) equilibrated with Buffer A. The monitoring the rate of enzymatic conversion of NADH column was initially washed with Buffer A, and eluted by a into NAD+ (opine-biosynthetic reaction) or of NAD+ into linear gradient of NaCl in Buffer A (0 to 50 mM, 200 ml in NADH (opine-catabolic reaction) at 340 nm using an total volume) at a flow rate of 0.3 ml/min. The fractions Ubest V-560 UV/VIS spectrophotometer equipped with showing TaDH activity were pooled, concentrated by the an on-line data processing computer system and a ther ultrafiltration, and desalted by passing through the Sepha mostated cell holder (Jasco Co., Tokyo, Japan). The com dex G25 column using 1 mm KH2PO4/KOH (pH 7.2) con plete assay mixture for the opine-biosynthetic reaction con taining 10 mm 2-mercaptoethanol as a running buffer. The tained 100ƒÊmol of Pipes/NaOH (pH 6.8), 100,ƒÊmol of 416 Kan-no et al.

amino acid (neutralized), 0.3 ƒÊmol of sodium pyruvate, The crude enzymes from 5 individuals of the sponge 0.3 ƒÊmol of NADH, and an aliquot of enzyme preparation were examined by IEF-TLPAG (data not shown). Of the in a final volume of 1.0 ml. The amino acid substrate was five OpDHs and LDH, only TaDH was detected as a single varied depending on the target enzyme: L-arginine for activity band; StDH activity band was not demonstrated. OcDH (20 mm in the reaction mixture limited to this ami We could not confirm any multiple form of TaDH or any no acid), L-alanine for AlDH, glycine for StDH, taurine genetic variations. for TaDH, ƒÀ-alanine for ƒÀ-AlDH, and none for LDH. The assay mixture for the opine-catabolic reaction contained Purification of TaDH 100 ƒÊmol of Tris/HCl (pH 9.0), 1.0 ƒÊmol of NAD+, 3.0 The course of a typical purification from the sponge ƒÊ mol of opine (adjusted to pH 9.0 with NaOH), and an (200 g wet wt) is summarized in Table 1. A 430-fold aliquot of enzyme preparation in a final volume of 1.0 ml. purification with 26% recovery was achieved relative to the The substrate opine was varied depending on the target en crude enzyme step. The final TaDH preparation, having a zyme (D or L-lactate was used for LDH). For both assays, specific activity of 780.9 units/mg protein (the standard the reaction mixture without enzyme was preincubated at tauropine-biosynthetic reaction), was homogeneous based 30•Ž for 2 min, and the reaction at 30•Žwas started by on SDS-PAGE and IEF-TLPAG (Figs. 1 and 2). adding enzyme. The reaction was monitored for at least 3 The activity and catalytic properties of the purified min, and reaction rate was calculated using a time-scan TaDH were maintained at 4•Žin Buffer A for at least two ning computer program. Real OpDH activity in the opine weeks. The enzyme was unstable to freezing and thawing, - biosynthetic reaction was calculated by subtracting LDH and easily lost its activity. activity from the apparent OpDH activity. One enzyme unit was defined as the amount of enzyme oxidizing or producing 1ƒÊmole of NADH per min under the specified conditions. Kinetic data were analyzed by double-reciprocal plots. Calculations were performed by a least-squares linear regression analysis. Results were ex

pessed as means•} S.E.M. of three independent determina tions.

Protein Assay Protein concentration was determined by the Coomassie brilliant blue G250 binding method,24) with bovine serum a lbumin as the standard.

Results

Activities of OpDHs and LDH in Crude Extracts Activities of the five OpDHs and LDH in the crude ex tracts of the sponge H. japonica were determined (the opine or lactate-biosynthetic reaction). TaDH activity was dominant in this sponge (14.2•}2.3 units/g wet wt, n=5). Additionally, minor StDH activity (0.92±0.11 units/g wet wt, n = 5) was also observed, while the activi ties of the other OpDHs and LDH were not detected at all. The TaDH activity observed in this study is higher than that reported in our previous paper.17) This discrepancy Fig. 1. SDS-PAGE analysis of the purified TaDH from H. japonica. can be accounted for by the use of an inadequate concen Lane 1, the purified TaDH (1 ƒÊg). Lane 2, molecular-mass stan tration of pyruvate as the substrate (pyruvate concentra dard markers: phosphorylase b, 97.4 kDa; bovine serum albumin, tion of inhibitory range; see below for details) in the previ 66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; soybean ous enzyme assay. trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa.

Table 1. Purification of TaDH from the sponge H. japonica (200 g wet wt)

* TaDH activity was determined using the standard conditions for the tauropine-biosynthetic reaction . See Materials and Methods for 'unit' definition. Tauropine Dehydrogenase from Halichondria 417

Table 2. Substrate specificity of TaDH purified from the sponge H. japonica

Fig. 2. IEF-TLPAG analysis of the purified TaDH from H. japonica. IEF was carried out on 5% polyacrylamide gel containing Pharma lyte 3-10 (dilution 1:16) for 4000 volt-hour at 10•Ž. Lane 1, the crude enzyme stained for TaDH activity; lane 2, the purified enzyme stained for the activity; lane 3, the purified enzyme stained for pro tein with Coomassie brilliant blue G250; lane 4, pI marker proteins: trypsinogen, pI 9.3; lentil lectin, pIs 8.65, 8.45 and 8.15; myoglobin, *1 The tauropine-biosynthetic reactions conducted at pH 6.8. pI 7.35; human carbonic anhydrase, pI 6.55; bovine carbonic anhy *2 The tauropine-catabolic reactions conducted at pH 9.0. drase, pI 5.85; ƒÀ-lactoglobulin, pI 5.2; soybean trypsin inhibitor, pI 4.55. C and A denote inner margin of cathode and anode, respec tively. reaction to the tauropine-catabolic reaction was approxi mately 21 under the standard assay conditions.

Relative Molecular Mass The molecular mass of the purified TaDH in its native Substrate Specificity form was determined to be 36,500 ± 500 (n = 3) by gel filtra The sponge TaDH showed a specific requirement for tion on a TSK G3000SW column (data not shown). The NAD(H), which was not substituted by NADP(H) (Table molecular mass of the denatured form was estimated to be 2). This property is common to all OpDHs characterized 37,000•}400 (n=3) by SDS-PAGE (Fig. 1). These data are so far.8) consistent with a monomeric structure for TaDH from H. The amino acid substrate specificity of the sponge TaDH is summarized in Table 2. The enzyme was highly japonica. The isoelectric point of the sponge TaDH was estimated specific to taurine. Glycine was also utilized, while the ac to be 4.8•}0.1 (n=3) by IEF-TLPAG (Fig. 2). tivity was much lower. Taurine analogues tested were uti lized at rates less than 36% of the activity for taurine, in pH Optimum the decreasing order of hypotaurine, aminomethanesulfon The effect of pH on the activity of TaDH was examined ic acid, and homotaurine. However, ,6-alanine, which is a using five overlapping buffers systems. The buffers used structural analogue of taurine, exhibited no detectable ac were all at 100 mm (final concentration): Mes/NaOH, pH tivity. It seems that all of an amino group in the alpha posi 5.5-6.8; Pipes/NaOH, pH 6.3-7.5; NaH2PO4/NaOH, tion, a carbon chain length, and a sulfonic acid group in pH 6.0-7.8; Tris/HCl, pH 7.2-9.0; and Ches/NaOH, pH the beta position were critical factors for amino acid sub 8.8-10.0. Other conditions were the same as for the stan strate-binding or catalytic function of this enzyme. dard assays. The activity of the sponge TaDH was max As presented in Table 2, the low activities (less than 13% imal at pH 6.8 (half-maximal at pH 5.5 and 7.4) in the of the activity for pyruvate) were observed when glyoxy tauropine-biosynthetic direction, and at pH 9.0 (half-max late, ƒÀ-hydroxypyruvate, 2-ketobutyrate, or 2-ketovaler imal at pH 8.0) in the tauropine-catabolic direction. The ate were used as a substitute for pyruvate. On the other ratio of the reaction velocity of the tauropine-biosynthetic hand, oxalacetate was utilized with 86% of the activity for 418 Kan-no et al .

[keto acid] (mM) [tauropine] (mM)

Fig. 3. Pyruvate saturation kinetics for the purified sponge TaDH. Fig. 4. Tauropine saturation kinetics for the purified sponge TaDH. The enzyme activity was determined using the standard conditions The enzyme activity was determined using the standard conditions for the tauropine-biosynthetic reaction, except that the concentra for the tauropine-catabolic reaction, except that the concentration tion of pyruvate was varied from 0 to 10 mM under fixed concentra of tauropine was varied from 0 to 50 mm. The inset shows the double tions of cosubstrates. Saturation profiles for oxalacetate (-•¡-) and reciprocal plot of the data from the saturation profile. 2-ketobutyrate (-•›-) obtained in a similar manner to that for pyru vate (-•œ-) are also shown. The inset shows the double reciprocal plots of the data from the saturation profiles for pyruvate (-•œ-) and oxalacetate (-•¡-). mM tauropine and 1 mm NAD+ at pH 9.0).

Discussion pyruvate. In the tauropine-catabolic reaction, tauropine was not The sponge Halichondria japonica contains a dominant substituted at all by octopine, alanopine, strombine, or, TaDH activity and a minor StDH activity, while activities ƒÀ- alanopine (Table 2). This result is consistent with IEF of the other three OpDHs and LDH are not detected. The - TLPAG data described above. StDH activity detected in the crude enzyme seems to be long to TaDH, because the ratio of StDH activity to Apparent Michaelis Constants TaDH activity in the crude enzyme coincided with the The tauropine-biosynthetic reaction was markedly in result of the amino acid substrate specificity obtained with hibited by the substrate pyruvate (Fig. 3). Its optimum con the highly purified TaDH (Table 2). Therefore, TaDH may centration was 0.25-0.35 mm and the inhibitory concentra be the sole terminal enzyme of the anaerobic glycolysis in tion yielding the half-maximal reaction rate was 3.2 mm. this sponge. On the other hand, taurine and NADH exhibited no inhibi The StDH activity and the formation of strombine dur tory effect on the reaction up to concentrations of 200 mM ing environmental hypoxia have been demonstrated in the and 1 mM, respectively. Apparent Km values determined us sponge Halichondria panicea,25,26)while the TaDH activity ing constant cosubstrate concentrations (0.3 mM pyruvate, and the formation of tauropine have not been tested. Both 100 mM taurine, and 0.3 mM NADH at pH 6.8) were H. japonica and H. panicea inhabit shallow sea, and may 37.0•}1.5 mM for taurine, 0.068•}0.004 mM for pyruvate, face a similar environmental hypoxia. Thus it is ques and 0.036•}0.002 mM for NADH. The kinetic pattern ob tioned why the two species of sponges select different types tained using oxalacetate as a substitute for pyruvate (Fig. of OpDH. It has been noted that the opine de 3) was strikingly similar to that for pyruvate. The concen hydrogenases that had evolved were those that would tration of oxalacetate giving the maximal reaction rate was make use of the most abundant free amino acids in the spe 0.25-0.4 mM, and the inhibitory concentration yielding the cies.27)This is true for H. panicea.25,26) However, the most half-maximal rate was 2.0 mM. The apparent Km for ox abundant amino acid in the free amino acid pools of H. alacetate was estimated to be 0.11 •}0.006 mM using 100 japonica is glycine, and the subordinate one is taurine mM taurine and 0.3 mM NADH. On the contrary, 2 (Kan-no, unpublished observation). It is unclear at present - ketobutyrate showed no inhibitory effect up to 10 mm (Fig. why TaDH, not StDH, turns up in H. japonica. 3). The results of the characterization studies indicate that In the tauropine-catabolic reaction, the substrate tauro the properties of the sponge TaDH resemble those of other pine inhibited the reaction. The optimum concentration of OpDHs from various organisms in many aspects such as tauropine was 2.5-5.0 mM and the inhibitory concentra monomeric structure, molecular mass, coenzyme specifici tion of tauropine yielding half-maximal rate was 22.1 mM ty, and pH optima.8) The strict amino acid substrate (Fig. 4). Apparent Km values for tauropine and NAD+ specificity against taurine is a characteristic of the sponge were estimated to be 0.39•}0.02 mM and 0.16•}0.01 mM, TaDH. A similar extent of strictness of the amino acid sub respectively, using constant cosubstrate concentrations (3 strate specificity has been demonstrated for TaDHs from Tauropine Dehydrogenase from Halichondria 419 the abalone (ormer), H. lamellosa18) and H. discuss han tions in both the forward and the reverse reactions. This nai.19)Judging from the activities for taurine analogues as limitation of tauropine accumulation during anaerobic a substitute for taurine, the substrate specificity of TaDH conditions seems necessary for sponges, which have no cir from H. discuss hannai seems to be more strict than that culatory system. We are now investigating the catalytic of the sponge TaDH: the taurine analogues (homotaurine, properties of the sponge TaDH in further detail and the hypotaurine, and aminomethanesulfonic acid) are not uti tauropine formation during experimental hypoxia to clari lized by the abalone TaDH.19) The narrow amino acid sub fy the physiological role of TaDH in the sponge. strate specificity seems to be a common property of TaDH, but the partially purified G. pyramidata enzyme has been Acknowledgments The authors wish to thank Mr. G. Nishiyama, Mr. noted to use alanine as an alternative substrate to some ex T. Yamamoto, and Mr. D. Moriyama, Laboratory of Marine Food Che tent.20)Storey and Dando28) have postulated, on the basis mistry, School of Fisheries Sciences, Kitasato University, for their techni cal assistance in this study. of amino acid substrate specificity of OcDHs from various animals, that the main evolutionary modification of References OpDHs was in the narrowing of amino acid specificity. However, it is not possible to recognize the such relation ship in TaDHs characterized so far. With respect to the keto acid substrate specificity, the sponge TaDH slightly differs from the abalone enzymes and the brachiopod enzyme which exhibit rather narrow keto acid specificity and, surprisingly, do not utilize ox alacetate at all.18-20)The utilization of several keto acids other than pyruvate has been reported in most of OpDHs.8)However, in general, keto acids other than pyruvate are taken as having no physiological meaning because of their relatively high (non-physiological) Km values. In the case of the sponge TaDH, however, we can not entirely neglect a physiological significance of the reaction involv ing oxalacetate, since the activity observed for oxalacetate is exceedingly high and the apparent Km for this keto acid is low. Furthermore, kinetic patterns for pyruvate and ox alacetate are similar (Fig. 3). Thus, it would be useful to ex amine whether oxalacetate serves as the substrate in vivo. The most striking properties of the sponge TaDHHHHare low Km values for pyruvate and tauropine. These Km values are the lowest level compared to those obtained not only for TaDHs18-20)but also for other OpDHs from vari ous invertebrates8) The catalytic properties of the sponge TaDH resemble those of a heart-type (H-type) opine de hydrogenase, i.e., the high affinity for pyruvate, the potent substrate inhibition by high concentrate pyruvate, the high affinity for opine, and significant substrate inhibition by opine substrate.29) The H-type opine dehydrogenase has been found as a tissue-specific isoenzyme in non-muscular tissues such as brain and functions as opine oxidase utiliz ing opine as an aerobic fuel in contrast to the muscle-type (M-type) opine dehydrogenase playing a major role in the redox regulation. While the inhibitory pyruvate concentra tion yielding half-maximal reaction rate is about 10 mm for the typical H-type LDH and H-type OcDH,29) that ob served for the sponge TaDH was lower than 10 mm. The sponge TaDH was detected as a single form. Hence, if the sponge TaDH is of H-type, the source of e tauropine can not be accounted for. So, it is difficult to regard the sponge TaDH as the H-type enzyme. We expect that the character istics in the catalytic properties of the sponge TaDH are related to the lower capacity of anaerobic energy produc tion. This is explicable by no requirement of high energy production during anaerobic conditions for sponges, which are sessile animals having no muscular tissue. The ki netic data indicates that the concentration of tauropine, the end of the anaerobic glycolysis, in this animal is maintained at a lower level by the potent substrate inhibi- 420 Kan-no et al.

18) G. Gade: Purification and properties of tauropine dehydrogenase response to different proteins of the coomassie blue G dye-binding from the shell adductor muscle of the ormer, Haliotis lamellosa. assay for protein. Anal, Biochem., 116, 53-64 (1981). Eur. J. Biochem., 160, 311-318 (1986). 25) J. Barrett and P. E. Butterworth: A novel amino acid linked de 19) M. Sato, M. Takeuchi, N. Kanno, E. Nagahisa, and Y. Sato: hydrogenase in the sponge Halichondria panicea (Pallas). Comp. Characterization and physiological role of tauropine de Biochem. Physiol., 70B, 141-146 (1981). hydrogenase and lactate dehydrogenase from muscle of abalone, 26) U. Kreutzer, B. R. Siegmund, and M. K. Grieshaber: Parameters Haliotis discus hannai. Tohoku J. Agr. Res., 41, 83-95 (1991). controlling opine formation during muscular activity and environ 20) C. Doumen and W. R. Ellington: Isolation and characterization of mental hypoxia. J. Comp. Physiol. B, 159, 617-628 (1989). a taurine-specific opine dehydrogenase from the pedicles of the 27) G. Gade: Energy metabolism during anoxia and recovery in shell ad brachiopod, Glottidea pyramidata. J. Exp. Zool., 243, 25-31 ductor and foot muscle of the gastropod mollusc Haliotis lamello (1987). sa: formation of the novel anaerobic end product tauropine. Biol. 21) N. Kanno, K. Kamimura, E. Nagahisa, M. Sato, and Y. Sato: Ap Bull., 175, 122-131 (1988). plication of isoelectric focusing in thin layer polyacrylamide gels to 28) B. S. Storey and P. R. Dando: Substrate specificities of octopine de the study of opine dehydrogenases in marine invertebrates. Fish hydrogenases from marine invertebrates. Comp. Biochem. Phys eries Sci., 62, 122-125 (1996). i ol., 73B, 521-528 (1982). 22) R. W. Blakeslay and J. A. Boezi: A new staining technique for pro 29) K. B. Storey and J. M. Storey: Kinetic characterization of tissue teins in polyacrylamide gels using Coomassie brilliant blue G250. specific isozymes of octopine dehydrogenase from mantle muscle Anal. Biochem., 82, 580-582 (1977). and brain of Sepia officinalis: functional similarities to the M4 and 23) U. K. Laemmli: Cleavage of structural proteins during the assembly H4 isozymes of lactate dehydrogenase. Eur. J. Biochem., 93, 545 of the head of bacteriophage T4. Nature, 227: 680-685 (1970). -552 (1979). 24) S. M. Read and D. H. Northcote: Minimization of variation in the