Abstract
Pyridoxal 5'-phosphate(PLP)is known to combine with bovine serum albumin to form a (1:1) complex which scarcely dissociates, even when subjected to intensive dialysis. When this complex was incubated with apo-aspartate aminotransferase (apoCOT)for an appropriate time and the preincubated mixture then submitted to the usual GOT assay, the appearance of GOT activity was obviously confirmed, indicating that PLP was transferred from the albumin-PLP complex to apoGOT. The affinity (Km)of the albumin-PLP complex for apoGOT was 1.56μM. Simultaneous addition of α-ketoglutarate to the preincubation mixture decreased markedly the transfer of PLP. Albumin-PLP (1:1) com-plex showed its absorption peak at 332nm. Although no appreciable change was observed in the absorption spectrum of the complex when incubated with apoGOT, the fluorescence spectrum of the mixture excited at 330nm was remarkably different from that of the complex alone. On addition of DL-erythro-β-hydroxyaspartate to the incubated mixture of albumin-PLP complex with apoGOT, a new absorption peak at 492 nm, assignable to a dead-end deprotonated Schiff base between holoGOT and the substrate analog, appeared immediately. These facts strongly suggest that PLP was transferred by interaction of the albumin-PLP complex With apoGOT to yield holoGOT-substrate Schiff base on addition of substrate.
