Isolation and properties of mitochondrial aspartate aminotransferase from red muscle of grey mullet, Mugil auratus Risso.
Following the chromatographic separation of the grey mullet (Mugil auratus Risso) red muscle extract, two fractions with aspartate aminotransferase activity were detected. One of the anticipated enzymes was purified to homogeneity. The isolated enzyme was a dimeric protein composed of identical subunits with the overall M(r) of about 65,000. It consisted of three electrophoretically distinct subforms with isoelectric points at pH 8.50, 8.70 and 8.85, respectively. The Michaelis-Menten constants of the substrates L-aspartate and 2-oxoglutarate were estimated to be 0.29 +/- 0.012 mM and 0.45 +/- 0.016 mM, respectively. For the reverse reaction, the Km for L-glutamate was 8.57 +/- 2.1 mM and for oxaloacetate it was 0.13 +/- 0.035 mM. The inhibition of the isolated enzyme by hydroxylamine was of a mixed linear noncompetitive type for L-aspartate as a substrate, whereas with 2-oxoglutarate hyperbolic uncompetitive inhibition was observed. The inhibition by aminooxyacetic acid and D,L-glyceraldehyde 3-phosphate was of a mixed linear noncompetitive type with respect to L-aspartate and 2-oxoglutarate. The isolated enzyme was slightly affected by maleate and succinate and no effects were produced by adipate. According to its subcellular distribution, susceptibility to inhibitors molecular and catalytic properties the isolated enzyme belonged to the mitochondrial form of aspartate aminotransferase.
Petrović
S
S
Center for Marine Research, Ruder Bosković Institute, Rovinj, Croatia.
Krajnović-Ozretić
M
M
Ozretić
B
B
D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008931:Mitochondria, Muscle / Q000201:enzymology*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010071:Oxaloacetates / Q000378:metabolism; D010473:Perciformes
D005971:Glutamates; D006898:Hydroxylamines; D007656:Ketoglutaric Acids; D010071:Oxaloacetates; D019811:Hydroxylamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001219:Aspartate Aminotransferases