抄録
Asparate aminotransferase was isolated from the supernatant fraction of pig heart muscle according to the method of Morino, Itoh and Wada. Holoenzyme was resolved to apoenzyme and coenzyme by incubation with aspartate and phosphate, followed by chromatography with Sephadex column. Km values for α-ketoglutarate of holo- and apo-aspartate aminotransferases were 4×10^<-3>M and 2.5×10^<-4> M, respectively. With apo-aspartate aminotransferase, Lineweaver・Burk plots of the coenzyme concentration curves gave Km value of 1.33mμM for pyridoxal phosphate and 10mμM for pyridoxamine phosphate. The aspartate aminotransferase reaction was markedly inhibited by the preincubation of apoenzyme with pyridoxal phosphate or pyridoxamine phosphate in the presence of low concentration of α-ketoglutarate, but was not inhibited in the presence of other substrates, such as L-aspartate, L-glutamate or oxalacetate. This inhibition was typically competitive and Ki values for α-ketoglutarate were 2.6×10^<-6>M against pyridoxal phosphate and 6.28×10^<-5>M against pyridoxamine phosphate. Of these organic acids tested, such as pyruvate, citrate, malate, succinate, and lactate, only citrate inhibited competitively and its Ki values were calculated as 5.6×10^<-7>M against pyridoxal phosphate and 8.0×10^<-7>M against pyridoxamine phosphate from Lineweaver・Burk plots. Phosphate ion and some nucleotides also inhibited competitively, but pyridoxal and pyridoxamine did not inhibit. Ki values for phosphate were calculated as 2.15×10^<-7>M against pyridoxal phosphate and 3.9×10^<-7>M against pyridoxamine phosphate.
