Aminotransferases in Grapes. Isolation and Characterization of Aspartate Aminotransferase

¹ Laboratoire de Biochimie Metabolique et Technologie, Institut des Produits de la Vigne, INRA, Place Viala, 34060 Montpellier, France.

Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase, and -aminobutyrate transaminase were the three aminotransferases tested in grape extracts. GOT was the main enzyme detected. This enzyme extracted at pH 8.5 in a protective buffer was purified 195-fold after Sephadex LH-20 decoloration, ammonium sulfate precipitation, and successive steps of gel-filtration and ion-exchange and adsorption chromatographies. The protein had a molecular weight of 83000 ± 3000 daltons and was presumably constituted by two identical subunits. Native polyacrylamide gel electrophoresis exhibited four isozymic bands. Isoelectrofocusing in liquid medium of a total purified extract did not differentiate these four forms and showed a single peak with an isoelectric point of 4.9 ± 0.2. The optimum pH of the enzyme reaction was found to be 7.8. Ionic and thermic stabilities were also examined. Kinetic data obtained with the more purified fraction indicated the commonly found ping-pong mechanism for transaminases with a K_m of 4.8 and 0.2 mM for aspartase and -ketoglutarate, respectively, the pyridoxal phosphate not being required for the catalysis. Transaminase activity should be considered for a better understanding of the modification of the amino acid pool observed in carbonic maceration of grape. Furthermore, the inhibition study of GOT using ethanol showed that this compound was not involved in the arrest of the intracellular fermentation associated with carbonic maceration.

Key words: grape, aspartate aminotransferase, aminotransferases, purification

Submitted on June 18, 1990