Purification of Native Glutamate Dehydrogenase from Eel Liver

Native glutamate dehydrogenase (GDH) was purified from the eel liver through procedures including fractionation of mitochondria, absorption chromatography with hydroxyapatite, ion-exchange chromatography with DEAE-Sephacel, affinity chromatography with GTP-Sepharose, and gel filtration with Toyopearl HW-55. Molecular weight of native GDH was 340,000 and the subunit of the GDHconsisted of one kind of protein with molecular weight' of 53,000 to 55,000. Isoelectric point of the GDH was pH 5.9. Amino acid composition of the GDH wasanalyzed andhada certainsimilarity to thatof bovine liver GDH. The composition of glycine, histidine, methionine, tryptophan, tyrosine and valine in both GDHs was very similar. When glutamate dehydrogenase (GDH) was purified from the livers of eel (Anguilla japonica), two kinds of GDHs were obtained. One was native GDH and the other was GDH sufferedlimited proteolysis. However, at the beginning period of the study on GDH, it was not noticed that the purified GDH had suffered limited proteolysis1-3*. We concluded at that time that the eel liver GDH consisted of two kinds of subunits with different molecular weights. Namely one was 53,000-55,000, and the other was 50,000-52,000. It was found later that the ratio of two kinds of subunits was altered due to the livers used for purification. When the livers preserved at —20*0 for several weeks were used forpurification, theratioof the protein with molecular weight of 53,000-55,000 to the protein with that of 50,00-52,000 increased. Furthermore, it was revealed that when leupeptin known as an inhibitor of trypsin or thiol proteases was used during the purification of GDH, the purified GDH consisted of only one kind of subunit of which molecular weight was 53,000-55,000. That was native GDH. This report describes the method for the purification of the native GDH and some of its properties.