Some kinetic and other properties of the isoenzymes of aspartate aminotransferase isolated from sheep liver

Abstract

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.

Kinetic and Crystallographic Analysis of Active Site Mutants of Escherichia coli γ-Aminobutyrate Aminotransferase

“…The larger slopes at higher concentrations of α-KG in Figure B and the upwardly curving data in Figure C are due to inhibition of the PLP enzyme by α-KG, which is competitive with respect to GABA. Similar inhibition by α-KG has been observed with aspartate aminotransferase ( − ).…”

Discovery and validation of serum biomarkers expressed over the first twelve weeks of Fasciola hepatica infection in sheep

“…(Sexton et al, 1990). Sheep AST exists as two isoforms of 87.1 kDa (pI of 9.14) (Orlacchio et al, 1979) and 86-88.9 kDa (Campos-Cavieres and Munn, 1973). The up-regulated biomarkers at 88.0 and 89.7 kDa in the pH 9 fraction observed in week 9 sera may represent AST ( Fig.…”

Cysteine sulfinate aminotransferase and aspartate aminotransferase isoenzymes of rat brain. Purification, characterization, and further evidence for identity

“…Purified aspartate aminotransferase (EC 2.6.1.1) (AAT) isoenzymes [for a review, see Braunstein (1973)] were able to utilize, besides their "normal" substrates L-glutamic acid and L-aspartic acid, L-cysteine sulfinic acid (CSA) and other amino acids (Singer & Kearney, 1956; Ellis & Davies, 1961;Novogrodsky & Meister, 1964). Although AAT has been extensively investigated, most of the available information on its purification and its miscellaneous properties has been derived from studies on the liver and heart of various species (Morino et al, 1963;Boyd, 1966;Bertland & Kaplan, 1968; Shrawder & Martinez-Carrion, 1973;Schlegel & Christen, 1974;Orlacchio et al, 1979). As far as we know, only a single report (Magee & Phillips, 1971) describes the molecular properties of rat brain aspartate aminotransferase isoenzymes.…”