Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Abstract

Tauropine dehydrogenase (tauropine : NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethy1)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38000 f 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42000 f 150 by electrophoresis on 5 -10% polyacrylamide gels in the presence of 1 YO sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the 0x0 acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent K , values for the substrates NADH, pyruvate and taurine are 0.022 f 0.003 mM, 0.64 5 0.07 mM and 64.7 f 5.4 mM, respectively, at pH 7.0 and for the products, NAD' and tauropine, are 0.29 L-0.01 mM and 9.04 f 1.27 mM, respectively, at pH 8.3. Apparent K , values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD' was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.Exposure during low tide or reduced oxygen tension at the bottom of muddy, shallow, eutrophic waters force many marine and fresh water invertebrates such as bivalves, gastropods and various annelids to survive hypoxic or even anoxic conditions in the environment [l -31. These species can withstand prolonged periods of environmental anaerobiosis and, similarly, exposure to nitrogen in the laboratory [4 -61. Investigations on the main pathways of intermediary metabolism in these species have shown that the simultaneous breakdown of aspartate and carbohydrates lead to the production of multiple end products such as alanine, acetate, succinate and propionate in varying species-specific concentrations [l -3, 7 -91. Degradation of carbohydrates via the Embden-Meyerhof-Parnas pathway to lactate, i. e. anaerobic glycolysis, appeared to be of minor importance, since accumulation of lactate has been rarely demonstrated under hypoxic conditions [8, 11, 121. Several enzymes, however, have been discovered in molluscs and annelids, other than lactate dehydrog...

Can the End Products of Anaerobic Metabolism, Tauropine and D-lactate, Be Used As Metabolic Stress Indicators During Transport of Live South African AbaloneHaliotis Midae?

“…As found for H. lamellosa (Gäde 1986), D-LDH is the predominant pyruvate dehydrogenase in the foot muscle of H. midae and TDH the main one in shell adductor muscle. In New Zealand abalone, however, TDH is also more active in the foot muscle (Baldwin et al 1992, Wells andBaldwin 1995).…”

“…The South African abalone, like other abalone from Europe (H. lamellose; Gäde 1986Gäde , 1988 and New Zealand (H. iris and H. australis; Baldwin et al 1992, Wells andBaldwin 1995), contains only TDH and D-LDH in their main muscle tissues. The very low, but seemingly measurable, activities of octopine-, strombineand alanopine dehydrogenases using the substrates arginine, glycine and alanine respectively in the cuvette are easily explained by a non-specific reaction of TDH.…”

“…It has been shown numerous times for opine dehydrogenase that they exhibit low activity with other amino acid substrates (see Gäde and Grieshaber 1986 for a review). For example, TDH of H. lamellosa shows 5% of its maximal activity measured with taurine when the amino acid alanine was used (Gäde 1986).…”

“…Neutralized perchloric acid extracts were prepared from the frozen tissues of H. midae according to previously published methods (Gäde et al 1978, Carlsson andGäde 1986). D-lactate and tauropine concentrations were determined as outlined in Gäde (1988); the enzyme tauropine dehydrogenase was purified from the shell adductor muscle according to a method slightly modified from that of Gäde (1987).…”

“…Haliotis discuss hannai, H. iris and H. lamellosa exhibit both tauropine and D-lactate dehydrogenase in the foot and shell adductor muscle (Gäde 1986, Sato and Gäde 1986, Baldwin et al 1992.…”