Purification And Characterization Of Pyridoxamin-Pyridoxal Conversion Reaction Enzymes From Plants (Tobacco)

Vitamin B6(VB6) is a group of water-soluble vitamin, including the free forms of Pyridoxine(PN), Pyridoxamine(PM), Pyridoxal(PL) and the phosphate forms of Pyrido-xine-5’-phosphate(PNP), Pyridoxamine-5’-phosphate(PMP), Pyridoxal-5’-phosphate(PLP). PLP acts as the essential coenzyme in many metabolic transition of amino acids and plays an important role in a variety of synthetic and metabolic reactions.Pyridoxamine-pyruvate transaminase and Pyridoxamine-Oxaloacetic transaminase can catalyze conversion between PM and PL. And GOT, which without coenzyme PLP, also can catalyze PM to PL. But these are proved only in animals and microorganism. In the higher plant, there is a conversion between PM and PL, too. But the related enzymes are indeterminacy.As a model plant, tobacco is widely used in plant physiological and biochemical research. We used the tobacco as the material in this paper, and purified two kinds of transaminases. It can lay the foundation for the study of the metabolic pathway of VB6in higher plants.GOT was purified from tobacco by ammonium sulfate, DEAE-Sepharose Fast Flow ion exchange chromatography, Sephadex G-100gel filtration. The results showed that the GOT was purified102.66-fold. And as a PLP-independent enzyme, GOT couldn’t catalyze the reaction which used PM and Oxaloacetic acid as substrate when there was PLP existing. However when deleted the PLP, it could. But, without PLP, it couldn’t catalyze the reaction which used PM and Pyruvic acid as substrate. While used the L-Aspartate and a-ketoglutarate as the substrate, the activity of GOT was decreased with the increased of PLP’s release amount. The characteristic was as same as the GOT in animal.And then, we used the same purification method to purify the PPAT, and studied its characteristics. The results showed that the PPAT was purified82.79-fold. Used the PM and Pyruvate acid as substrate, the purified enzyme showed a high activity. So we considered preliminary that PPAT was the enzyme which could catalyze PM to PL in plants. The study of PPAT’s characteristics showed that β-Mercaptoethanol and EDTA would inhibit the activity of PPAT. The maximum reaction rate of the enzymatic reactions was4.545μmol·L-1·min-1, and the Michaels constant of PM and Pyruvate were2.465μmol·L-and1.537umol·L-1.