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 oxidoreductase) was purified to homogeneity from the sponge Halichondria japonica Kadota (colony). Relative molecular masses of this enzyme 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 tauropine-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 substrate pyruvate. The optimal concentration of pyruvate was 0.25-0.35 mm and the inhibitory concentration giving half-maximal rate was 3.2 mm. The tauropine-catabolic reaction was inhibited by the substrate tauropine: the optimal concentration was 2.5-5.0 mm. Apparent K,,, values determined using constant 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

topine, alanopine, strombine, tauropine and /3-alanopine,

have been isolated from marine invertebrates.1-7) These

opines are biosynthesized by pyruvate reductases of a fami

ly of opine dehydrogenases (imino acid: NAD oxidoreduc

tase; OpDH) which catalyze the reversible reductive con

densation of pyruvate and amino acids with NADH as the

coenzyme. Opine dehydrogenases (or activities) corre

sponding to the five opines have been discovered: i.e., octo

pine dehydrogenase (D-octopine: NAD oxidoreductase;

EC 1.5.1.11; OcDH), alanopine dehydrogenase (meso

alanopine: NAD oxidoreductase; EC 1.5.1.17; AlDH),

strombine dehydrogenase (D-strombine: NAD oxidoreduc

tase; EC 1.5.1.22; StDH), tauropine dehydrogenase (tauro

pine: NAD oxidoreductase; TaDH), and ƒÀ-alanopine de

hydrogenase (ƒÀ-alanopine: NAD oxidoreductase; EC

1.5.1.26; ƒÀ-AlDH). It is widely accepted that the physiolog

ical roles of OpDH in invertebrates are analogous to those

of lactate dehydrogenase (EC 1.1.1.27, 28; LDH), i.e.,

balancing cytoplasmic redox potential and maintaining

rates of energy production during hypoxic conditions.8-12)

The three main OpDHs, i.e., OcDH, AlDH, and StDH,

have been extensively studied on their phylogenetic distri

bution13-17) and on the catalytic and molecular properties

with emphasis on the enzymes from muscular tissues of

molluscs.8) Those characterization studies have shown the

similarities of the molecular structures and catalytic prop

erties of OpDHs, and thus led to the hypothesis that all

OpDHs are homologous.12) Recently, Sato et al.") deter

mined the distribution of OpDHs including TaDH and ƒÀ

- AlDH, and pointed out the importance of the study on

TaDH; the observation that TaDH represented a major

OpDHs activity of the species belonging to the lower phy

la, Porifera and Coelenterata, led to a hypothesis that

TaDH was the most ancient OpDH. Therefore, the charac

terization of TaDH from the animals belonging to the low

er phyla is expected to provide valuable information on

the origin and evolution of OpDH molecules and on the

physiological role of OpDHs. So far, TaDHs of the aba

lone (ormer) Haliotis lamellosa18) and Haliotis discus han

nai19) (Mollusca, Gastrapoda), and of the brachiopod Glot

tidea pyramidata20) (Tentaculata) have received significant

attention. Thus we initiated a study on the TaDH from the

marine sponge Halichondria japonica, the phylogenetical

ly `lowest' animal, in which TaDH activity had been

demonstrated.17) Furthermore, it is interesting to compare

the properties of TaDH from the sponge which has no mus

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

of the sponge TaDH are described.

Materials and Methods

Materials

Specimens of the sponge Halichondria japonica (colo

ny) were collected from the seashore of Sanriku, Iwate,

Japan, in May 1995 and were immediately subjected to en

zyme extraction. Four opine compounds, i.e., meso-alano

pine, D-strombine, tauropine, and ƒÀ-alanopine, were pre

pared according to the methods described by Sato et al.4-7)

D-Octopine was purchased from Sigma Chemical (St.

Louis, Mo., USA).

Purification of Tauropine Dehydrogenase

All enzyme extraction and purification steps were per

formed at 4•Ž unless otherwise indicated. The fresh

sponges were dissected into pieces, washed well with

filtered seawater to remove all encrusting organisms, sand,

and other foreign materials (we sometimes scraped off the

surface part of sppponnn and blotted with filter paper to re

move seawater. The cleaned sponges (100-200 g wet wt)

were homogenized for 5-10 min with 3 volumes of ice-cold

20mM KH2PO4/KOH (pH 7.2) containing 1 mm EDTA

and 10 mm 2-mercaptoethanol by using a disperser. Then

the homogenate was centrifuged for 20 min at 10,000 x g

and 4•Ž. The supernatant was used as crude enzyme.

Solid (NH4)2SO4 was slowly added to the crude enzyme

to 45% saturation. After stirring for 1 hr, the solution was

centrifuged for 20 min at 10,000 x g and 4°C. Then addi

tional (NH4)2SO4 was added to the supernatant to 75%

saturation, and the solution was stirred and centrifuged as

above. The protein precipitated was dissolved in a minimal

volume of 2 mm KH2PO4/KOH (pH 7.2) containing 1 mm

EDTA and 10 mm 2-mercaptoethanol (referred to as

Buffer A). The enzyme solution was loaded onto a column

(44 x 900 mm) of Sephadex G75 (Pharmacia Biotech, Up

psala, Sweden) and eluted with Buffer A at a flow rate of 1

ml/min. The column eluate was monitored for absor

bance at 280 nm and TaDH activity. The fractions show

ing TaDH activity were pooled, and loaded onto a column

(32 x 200 mm) of Blue-Sepharose CL6B (Pharmacia

Biotech). The column was washed with Buffer A, and then

eluted by a linear gradient of NaCl in Buffer A (0 to 1 M,

500 ml in total volume) at a flow rate of 1 ml/ min. TaDH

was eluted between 0.2-0.4 M NaCl concentration. The

fractions showing TaDH activity were pooled, concentrat

ed by ultrafiltration on a Diaflo YM3 membrane (3-kDa

molecular mass cut-off; Amicon, Beverly, Ma., USA), and

desalted by passing through a small column (20 x 100 mm)

of Sephadex G25 (Pharmacia Biotech) using Buffer A as a

running buffer. The enzyme solution was then loaded onto

a column (10 x 230 mm) of Super Q-Toyopearl 650S

(Tosoh, Tokyo, Japan) equilibrated with Buffer A. The

column was initially washed with Buffer A, and eluted by a

linear gradient of NaCl in Buffer A (0 to 50 mM, 200 ml in

total volume) at a flow rate of 0.3 ml/min. The fractions

showing TaDH activity were pooled, concentrated by the

ultrafiltration, and desalted by passing through the Sepha

dex G25 column using 1 mm KH2PO4/KOH (pH 7.2) con

taining 10 mm 2-mercaptoethanol as a running buffer. The

enzyme solution was loaded onto a column (10 x 230 mm)

of Macro-Prep ceramic hydroxyapatite (20 ƒÊm particle

size; Bio-Rad Laboratories, Richmond, Ca., USA)

equilibrated with 1 mM KH2PO4/KOH (pH 7.2) contain

ing 10 mM 2-mercaptoethanol. The column was eluted by a

linear gradient of KH2PO4/KOH (1 to 150 mm, pH 7.2,

200 ml in total volume) at a flow rate of 0.25 ml/ min. The

TaDH activity was eluted at about 50 mm region. The enzy

matically active fractions were pooled for further studies.

Analytical Isoelectric Focusing in Thin-Layer Poly

acrylamide Gels (IEF-TLPAG)

Analytical IEF-TLPAG was carried out using a slab gel

of 90 mm length, 200 mm width, and 0.5 mm thickness

containing Pharmalyte 3-10 (1:16 dilution; Pharmacia

Biotech) (acrylamide concentration was 5%T and 3%C)

by the method described in our previous paper.211 Focusing

was carried out, using 0.04 M aspartic acid (anode) and 1 M

NaOH (cathode) as electrode solutions, for 4000 volt-hour

at a constant power of 6 W (with a maximum voltage of

1500 V) in a flat-bed IEF apparatus (Atto, Tokyo, Japan)

with cooling water (10•Ž) circulation. After the focusing,

the gel was immersed at room temperature for 5-20 min in

a mixture of 1.0 mm NAD+, 3.0 mm tauropine (adjusted

pH 9.0 with NaOH), 0.1 mm phenazine methosulfate, and

1 mm nitroblue tetrazolium in 100 mm Tris/HCl buffer

(pH 9.0) for the localization of TaDH activity. The gel was

stained for protein with Coomassie brilliant blue G250.22)

The isoelectric point of the enzyme was estimated using pI

- marker proteins (broad pI range; Pharmacia Biotech).

Determination of Relative Molecular MassThe relative molecular mass of the enzyme in its native

form was estimated on a TSK G3000SW rapid gel-filtration column (7.5 x 600 mm; Tosoh) attached to a PLC-10 HPLC system (Eyela, Tokyo, Japan), using 20 mm KH2PO4/KOH (pH 7.2) containing 1 mm EDTA, 10mM 2-mercaptoethanol, and 0.2 M NaCl as a running buffer. The standard proteins and the purified TaDH were run through the column at a flow rate of 0.5 ml/ min. The standard proteins (Pharmacia Biotech) were: aldolase, 158 kDa; bovine albumin, 67 kDa; ovalbumin, 43 kDa; chymotrypsinogen A, 25 kDa; and ribonuclease, 13 kDa.

The relative molecular mass of the enzyme in its denatured form was determined by SDS-PAGE carried out under reducing conditions. A homogeneous-pore slab gel of 12.5%T (80 mm length, 90 mm width, 1 mm thickness) was prepared and run according to the method of Laemmli.23) The gel was stained for protein with Coomassie brilliant blue R250. Bio-Rad low range SDS-PAGE standards (97.4 kDa-14.4 kDa) were used for the calibration.

Enzyme Assays

The activities of OpDHs and LDH were measured by

monitoring the rate of enzymatic conversion of NADH

into NAD+ (opine-biosynthetic reaction) or of NAD+ into

NADH (opine-catabolic reaction) at 340 nm using an

Ubest V-560 UV/VIS spectrophotometer equipped with

an on-line data processing computer system and a ther

mostated cell holder (Jasco Co., Tokyo, Japan). The com

plete assay mixture for the opine-biosynthetic reaction con

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,

0.3 ƒÊmol of NADH, and an aliquot of enzyme preparation

in a final volume of 1.0 ml. The amino acid substrate was

varied depending on the target enzyme: L-arginine for

OcDH (20 mm in the reaction mixture limited to this ami

no acid), L-alanine for AlDH, glycine for StDH, taurine

for TaDH, ƒÀ-alanine for ƒÀ-AlDH, and none for LDH. The

assay mixture for the opine-catabolic reaction contained

100 ƒÊmol of Tris/HCl (pH 9.0), 1.0 ƒÊmol of NAD+, 3.0

ƒÊ mol of opine (adjusted to pH 9.0 with NaOH), and an

aliquot of enzyme preparation in a final volume of 1.0 ml.

The substrate opine was varied depending on the target en

zyme (D or L-lactate was used for LDH). For both assays,

the reaction mixture without enzyme was preincubated at

30•Ž for 2 min, and the reaction at 30•Žwas started by

adding enzyme. The reaction was monitored for at least 3

min, and reaction rate was calculated using a time-scan

ning computer program. Real OpDH activity in the opine

- biosynthetic reaction was calculated by subtracting LDH

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 albumin 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

can be accounted for by the use of an inadequate concen

tration of pyruvate as the substrate (pyruvate concentra

tion of inhibitory range; see below for details) in the previ

ous enzyme assay.

The crude enzymes from 5 individuals of the sponge were examined by IEF-TLPAG (data not shown). Of the five OpDHs and LDH, only TaDH was detected as a single activity band; StDH activity band was not demonstrated. We could not confirm any multiple form of TaDH or any genetic variations.

Purification of TaDH

The course of a typical purification from the sponge

(200 g wet wt) is summarized in Table 1. A 430-fold

purification with 26% recovery was achieved relative to the crude enzyme step. The final TaDH preparation, having a

specific activity of 780.9 units/mg protein (the standard

tauropine-biosynthetic reaction), was homogeneous based

on SDS-PAGE and IEF-TLPAG (Figs. 1 and 2).

The activity and catalytic properties of the purified

TaDH were maintained at 4•Žin Buffer A for at least two

weeks. The enzyme was unstable to freezing and thawing,

and easily lost its activity.

Fig. 1. SDS-PAGE analysis of the purified TaDH from H. japonica.

Lane 1, the purified TaDH (1 ƒÊg). Lane 2, molecular-mass stan

dard markers: phosphorylase b, 97.4 kDa; bovine serum albumin,

66.2 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; soybean

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

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,

pI 7.35; human carbonic anhydrase, pI 6.55; bovine carbonic anhy

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.

Relative Molecular Mass

The molecular mass of the purified TaDH in its native

form was determined to be 36,500 ± 500 (n = 3) by gel filtra

tion on a TSK G3000SW column (data not shown). The

molecular mass of the denatured form was estimated to be

37,000•}400 (n=3) by SDS-PAGE (Fig. 1). These data are

consistent with a monomeric structure for TaDH from H.

japonica.

The isoelectric point of the sponge TaDH was estimated

to be 4.8•}0.1 (n=3) by IEF-TLPAG (Fig. 2).

pH OptimumThe effect of pH on the activity of TaDH was examined

using five overlapping buffers systems. The buffers used were all at 100 mm (final concentration): Mes/NaOH, pH 5.5-6.8; Pipes/NaOH, pH 6.3-7.5; NaH2PO4/NaOH, pH 6.0-7.8; Tris/HCl, pH 7.2-9.0; and Ches/NaOH, pH 8.8-10.0. Other conditions were the same as for the standard assays. The activity of the sponge TaDH was maximal at pH 6.8 (half-maximal at pH 5.5 and 7.4) in the tauropine-biosynthetic direction, and at pH 9.0 (half-maximal at pH 8.0) in the tauropine-catabolic direction. The ratio of the reaction velocity of the tauropine-biosynthetic

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

*1 The tauropine-biosynthetic reactions conducted at pH 6.8.*2 The tauropine-catabolic reactions conducted at pH 9.0.

reaction to the tauropine-catabolic reaction was approxi

mately 21 under the standard assay conditions.

Substrate Specificity

The sponge TaDH showed a specific requirement for

NAD(H), which was not substituted by NADP(H) (Table

2). This property is common to all OpDHs characterized

so far.8)

The amino acid substrate specificity of the sponge

TaDH is summarized in Table 2. The enzyme was highly

specific to taurine. Glycine was also utilized, while the ac

tivity was much lower. Taurine analogues tested were uti

lized at rates less than 36% of the activity for taurine, in

the decreasing order of hypotaurine, aminomethanesulfon

ic acid, and homotaurine. However, ,6-alanine, which is a

structural analogue of taurine, exhibited no detectable ac

tivity. It seems that all of an amino group in the alpha posi

tion, a carbon chain length, and a sulfonic acid group in

the beta position were critical factors for amino acid sub

strate-binding or catalytic function of this enzyme.

As presented in Table 2, the low activities (less than 13%

of the activity for pyruvate) were observed when glyoxy

late, ƒÀ-hydroxypyruvate, 2-ketobutyrate, or 2-ketovaler

ate were used as a substitute for pyruvate. On the other

hand, oxalacetate was utilized with 86% of the activity for

418 Kan-no et al .

[keto acid] (mM)

Fig. 3. Pyruvate saturation kinetics for the purified sponge TaDH.

The enzyme activity was determined using the standard conditions

for the tauropine-biosynthetic reaction, except that the concentra

tion of pyruvate was varied from 0 to 10 mM under fixed concentra

tions of cosubstrates. Saturation profiles for oxalacetate (-•¡-) and

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 (-•¡-).

pyruvate.

In the tauropine-catabolic reaction, tauropine was not

substituted at all by octopine, alanopine, strombine, or,

ƒÀ- alanopine (Table 2). This result is consistent with IEF

- TLPAG data described above.

Apparent Michaelis Constants

The tauropine-biosynthetic reaction was markedly in

hibited by the substrate pyruvate (Fig. 3). Its optimum con

centration was 0.25-0.35 mm and the inhibitory concentra

tion yielding the half-maximal reaction rate was 3.2 mm.

On the other hand, taurine and NADH exhibited no inhibi

tory effect on the reaction up to concentrations of 200 mM

and 1 mM, respectively. Apparent Km values determined us

ing constant cosubstrate concentrations (0.3 mM pyruvate,

100 mM taurine, and 0.3 mM NADH at pH 6.8) were

37.0•}1.5 mM for taurine, 0.068•}0.004 mM for pyruvate,

and 0.036•}0.002 mM for NADH. The kinetic pattern ob

tained using oxalacetate as a substitute for pyruvate (Fig.

3) was strikingly similar to that for pyruvate. The concen

tration of oxalacetate giving the maximal reaction rate was

0.25-0.4 mM, and the inhibitory concentration yielding the

half-maximal rate was 2.0 mM. The apparent Km for ox

alacetate was estimated to be 0.11 •}0.006 mM using 100

mM taurine and 0.3 mM NADH. On the contrary, 2

- ketobutyrate showed no inhibitory effect up to 10 mm (Fig.

3).

In the tauropine-catabolic reaction, the substrate tauro

pine inhibited the reaction. The optimum concentration of

tauropine was 2.5-5.0 mM and the inhibitory concentra

tion of tauropine yielding half-maximal rate was 22.1 mM

(Fig. 4). Apparent Km values for tauropine and NAD+

were estimated to be 0.39•}0.02 mM and 0.16•}0.01 mM,

respectively, using constant cosubstrate concentrations (3

[tauropine] (mM)

Fig. 4. Tauropine saturation kinetics for the purified sponge TaDH.

The enzyme activity was determined using the standard conditions

for the tauropine-catabolic reaction, except that the concentration

of tauropine was varied from 0 to 50 mm. The inset shows the double

reciprocal plot of the data from the saturation profile.

mM tauropine and 1 mm NAD+ at pH 9.0).

Discussion

The sponge Halichondria japonica contains a dominant TaDH activity and a minor StDH activity, while activities of the other three OpDHs and LDH are not detected. The StDH activity detected in the crude enzyme seems to belong to TaDH, because the ratio of StDH activity to TaDH activity in the crude enzyme coincided with the result of the amino acid substrate specificity obtained with the highly purified TaDH (Table 2). Therefore, TaDH may be the sole terminal enzyme of the anaerobic glycolysis in this sponge.

The StDH activity and the formation of strombine during environmental hypoxia have been demonstrated in the sponge Halichondria panicea,25,26) while the TaDH activity and the formation of tauropine have not been tested. Both H. japonica and H. panicea inhabit shallow sea, and may face a similar environmental hypoxia. Thus it is questioned why the two species of sponges select different types of OpDH. It has been noted that the opine dehydrogenases that had evolved were those that would make use of the most abundant free amino acids in the species.27) This is true for H. panicea.25,26) However, the most abundant amino acid in the free amino acid pools of H. japonica is glycine, and the subordinate one is taurine (Kan-no, unpublished observation). It is unclear at present why TaDH, not StDH, turns up in H. japonica.

The results of the characterization studies indicate that the properties of the sponge TaDH resemble those of other OpDHs from various organisms in many aspects such as monomeric structure, molecular mass, coenzyme specificity, and pH optima.8) The strict amino acid substrate specificity against taurine is a characteristic of the sponge TaDH. A similar extent of strictness of the amino acid substrate specificity has been demonstrated for TaDHs from

Tauropine Dehydrogenase from Halichondria 419

the abalone (ormer), H. lamellosa18) and H. discuss hannai.19) Judging from the activities for taurine analogues as a substitute for taurine, the substrate specificity of TaDH from H. discuss hannai seems to be more strict than that of the sponge TaDH: the taurine analogues (homotaurine, hypotaurine, and aminomethanesulfonic acid) are not utilized by the abalone TaDH.19) The narrow amino acid substrate specificity seems to be a common property of TaDH, but the partially purified G. pyramidata enzyme has been noted to use alanine as an alternative substrate to some extent.20) Storey and Dando28) have postulated, on the basis of amino acid substrate specificity of OcDHs from various animals, that the main evolutionary modification of OpDHs was in the narrowing of amino acid specificity. However, it is not possible to recognize the such relationship 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 oxalacetate 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 involving 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 oxalacetate are similar (Fig. 3). Thus, it would be useful to examine whether oxalacetate serves as the substrate in vivo.

The most striking properties of the sponge TaDHHHH are 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 various invertebrates8) The catalytic properties of the sponge TaDH resemble those of a heart-type (H-type) opine dehydrogenase, 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 utilizing 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 concentration yielding half-maximal reaction rate is about 10 mm for the typical H-type LDH and H-type OcDH,29) that observed 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 cannot be accounted for. So, it is difficult to regard the sponge TaDH as the H-type enzyme. We expect that the characteristics in the catalytic properties of the sponge TaDH are related to the lower capacity of anaerobic energy production. This is explicable by no requirement of high energy

production during anaerobic conditions for sponges, which are sessile animals having no muscular tissue. The kinetic data indicates that the concentration of tauropine, the end product of the anaerobic glycolysis, in this animal is maintained at a lower level by the potent substrate inhibi-

tions in both the forward and the reverse reactions. This

limitation of tauropine accumulation during anaerobic

conditions seems necessary for sponges, which have no cir

culatory system. We are now investigating the catalytic

properties of the sponge TaDH in further detail and the tauropine formation during experimental hypoxia to clari

fy the physiological role of TaDH in the sponge.

Acknowledgments The authors wish to thank Mr. G. Nishiyama, Mr.

T. Yamamoto, and Mr. D. Moriyama, Laboratory of Marine Food Che

mistry, School of Fisheries Sciences, Kitasato University, for their techni

cal assistance in this study.

References

420 Kan-no et al.

18) G. Gade: Purification and properties of tauropine dehydrogenase

from the shell adductor muscle of the ormer, Haliotis lamellosa. Eur. J. Biochem., 160, 311-318 (1986).19) M. Sato, M. Takeuchi, N. Kanno, E. Nagahisa, and Y. Sato:

Characterization and physiological role of tauropine dehydrogenase and lactate dehydrogenase from muscle of abalone, Haliotis discus hannai. Tohoku J. Agr. Res., 41, 83-95 (1991).

20) C. Doumen and W. R. Ellington: Isolation and characterization of

a taurine-specific opine dehydrogenase from the pedicles of the brachiopod, Glottidea pyramidata. J. Exp. Zool., 243, 25-31

(1987).21) N. Kanno, K. Kamimura, E. Nagahisa, M. Sato, and Y. Sato: Ap

plication of isoelectric focusing in thin layer polyacrylamide gels to the study of opine dehydrogenases in marine invertebrates. Fish

eries Sci., 62, 122-125 (1996).22) R. W. Blakeslay and J. A. Boezi: A new staining technique for pro

teins in polyacrylamide gels using Coomassie brilliant blue G250.

Anal. Biochem., 82, 580-582 (1977).23) U. K. Laemmli: Cleavage of structural proteins during the assembly

of the head of bacteriophage T4. Nature, 227: 680-685 (1970). 24) S. M. Read and D. H. Northcote: Minimization of variation in the

response to different proteins of the coomassie blue G dye-binding assay for protein. Anal, Biochem., 116, 53-64 (1981).

25) J. Barrett and P. E. Butterworth: A novel amino acid linked dehydrogenase in the sponge Halichondria panicea (Pallas). Comp.

Biochem. Physiol., 70B, 141-146 (1981).26) U. Kreutzer, B. R. Siegmund, and M. K. Grieshaber: Parameters

controlling opine formation during muscular activity and environmental hypoxia. J. Comp. Physiol. B, 159, 617-628 (1989).

27) G. Gade: Energy metabolism during anoxia and recovery in shell adductor and foot muscle of the gastropod mollusc Haliotis lamellosa: formation of the novel anaerobic end product tauropine. Biol.

Bull., 175, 122-131 (1988).28) B. S. Storey and P. R. Dando: Substrate specificities of octopine de

hydrogenases from marine invertebrates. Comp. Biochem. Physi ol., 73B, 521-528 (1982).

29) K. B. Storey and J. M. Storey: Kinetic characterization of tissue specific isozymes of octopine dehydrogenase from mantle muscle

and brain of Sepia officinalis: functional similarities to the M4 and H4 isozymes of lactate dehydrogenase. Eur. J. Biochem., 93, 545

-552 (1979).