A method for the purification of the 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 (182units/mg). The molecular weight determined by sedimentation equilibrium is 87100 ± 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 pI9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. TheKm for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 + 0.20mM and 0.093 ± 0.010mM respectively; the corresponding values for the mitochondrial form were 0.40 ± 0.12mMand0.98 ± 0.14mM. Cytoplasmicaspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH7.4 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 AM (cytoplasmic) and 0.062pM (mitochondrial) and for pyridoxamine 5'-phosphate they were 70AM (cytoplasmic) and 40#M (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37°C were 32A4M for the cytoplasmic isoenzyme and 19.5AuM for the mitochondrial form.
Some Kinetic and Other Properties of the Isoenzymes of AspartateAminotransferase Isolated from Sheep Liver.
ORLACCHIO, Aldo;
1979
Abstract
A method for the purification of the 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 (182units/mg). The molecular weight determined by sedimentation equilibrium is 87100 ± 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 pI9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. TheKm for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 + 0.20mM and 0.093 ± 0.010mM respectively; the corresponding values for the mitochondrial form were 0.40 ± 0.12mMand0.98 ± 0.14mM. Cytoplasmicaspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH7.4 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 AM (cytoplasmic) and 0.062pM (mitochondrial) and for pyridoxamine 5'-phosphate they were 70AM (cytoplasmic) and 40#M (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37°C were 32A4M for the cytoplasmic isoenzyme and 19.5AuM for the mitochondrial form.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
