ミトコンドリア局在および可溶画分局在のトランスアミナーゼの差異について : (III)ミトコンドリアGlutamic-Oxalacetic Transaminaseの精製とその性質

抄録

The existence of two forms of GOT was first shown enzymatically by Katunuma et al. Partially purification and dissimilar properties of mitochondrial GOT (GOT_M) and soluble fraction GOT (GOT_S) have been reported by the previous paper from our laboratory. The present paper describes the procedure of further purification and distinct characteristics of the enzymes. The use of (NH_4)_2SO_4 fractionation, heat treatment and chromatography on DEAE-cellulose effected both about 100-fold purification of GOT_M and GOT_S. Purified preparations obtained were homogeneous column chromatographycally, electrophoretically and ultracentrifugal analytically. Compared to GOT_S, 5.8S, GOT_M had a smaller S_<20>W 3.8S. The purified enzymes also differd in other physicochemical natures. GOT was retained and GOT unretained by DEAE-cellulose, whereas the reverse seemed to be true of the IRC-50. On zone electrophoresis, GOT_M moved to anode (-0.4 cm/hr) and GOT_S to cathode (+0.05 cm/hr) at pH 8 in veronal buffer. GOT_M (GOT_S) gave the following Michaelis constant : for aspartic acid, 1.8×10^<-3> (8.0×10^<-3>), for α-ketoglutaric acid, 5.8×10^<-3> (4.3×10^<-3>). Absorption spectra of the enzyme were recorded and showed typical spectra of vitamin B_6-enzyme without a trace of soret band. Spectrophotometric studies of the enzymes, especially of GOT_M, provided the information that the addition of L-aspartic acid, to pyridoxal phosphate or GOT at pH 5.4 decreased the absorbancy at 430mμ and increased at 340 mμ, showing the possibility of interconversion of the enzyme between pyridoxal phosphate form and pyridoxamine phosphate form even at low pH. Upon addition of L-alanine or L-ornithine, such a shift was not observed.