Cytosolic aspartate aminotransferase from the grey mullet ( Mugil auratus Risso) red muscle: Isolation and properties

Abstract

Aspartate aminotransferase isoenzymes play a central role in the metabolism of amino acids and participate in maintaining redox balance via an aspartate-malate shuttle in aerobic tissues such as red muscle. The aim of this study was to purify and characterize cytosolic isoenzyme of aspartate aminotransferase from the red muscle of grey mullet, Mugil auratus Risso. The properties of the fish enzyme and its possible physiological role are discussed and compared with homotopic isoenzymes from other species. The enzyme was purified to homogeneity by using different column chromatography techniques and further characterized with gel electrophoresis. It was found to be composed of two identical subunits, M r = 41,000 ± 882 (SEM, n = 3). Electrophoretic analysis revealed several active subforms of isolated cytosolic isoenzyme with pI in the pH range 5.2–5.6. The isolated enzyme was stable in the pH range 5.0–10.0 and between 0 and 30°C. It showed optimum activity at pH 7.5–9.0. K m values of substrates L-aspartate and α-ketoglutarate were 1.01 ± 0.082 and 0.031 ± 0.0035 mM, respectively. In the reverse reaction K m values of substrates L-glutamate and oxaloacetate were estimated to be 4.65 ± 0.90 and 1.22 ± 0.41 mM, respectively. The isolated enzyme was responsible for the transamination of taurine intermediate, L-cysteine sulfinate, in the cytosol of the red muscle with apparent K m of 3.08 ± 0.35 mM. This study demonstrated that cytosolic aspartate aminotransferase isoenzyme from the grey mullet red muscle resembles vertebrate cytosolic isoenzymes in its general physicochemical and catalytic properties.