Purification and Characterization of Tauropine Dehydrogenase from the Marine Sponge Halichondria Japonica Kadota (Demospongia)*1
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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 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 substrate 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 enzymes 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.