小麦胆色素原脱氨酶的纯化及部分性质研究

Purification and Some Properties of Porphobilinogen Deaminase from Wheat( Triticum aestivum, L. )

生物中四吡咯化合物合成的共同途径是由δ－氨基酮戊酸（δ－ａｍｉｎｏｌｅｖｕｌｉｎｉｃａｃｉｄ，ＡＬＡ）在δ－氨基酮戊酸脱水酶（δ－ａｍｉｎｏｌｅｖｕｌｉｎａｔｅｄｅｈｙｄｒａｔａｓｅ，ＡＬＡＤ）作用下合成胆色素原（ｐｏｒｐｈｏ－ｂｉｌｉｎｏｇｅｎ，Ｐ...

Porphobilinogen deaminase (PBGD,EC 4.3.1.8) was isolated and purified from wheat seedling, which was used for studying wheat PBGD mutant 'stage albinism line winter wheat'. The purification steps included extract, heat treatment, ammonium sulphate fractionation, Sephadex G 100 gel filtration and Red Sepharose CL 6B affinity chromatography. It′s final sepecific activity was 3 960 U/mg protein at pH 8.0 with a yield of 19%. The SDS PAGE and IEF confirmed that the enzyme was homogeneous. The deaminase was a monomer of M r 36 300 as shown by SDS PAGE, and 33 000 using Sephadex G 100. The pure enzyme had a V max of 6.67 μmol/h·mg protein and a K m of 7 μmol/L for uroporphyrin. Determination of the p I and pH optimum revealed 4.8 and 8.0 respectively. The PBGD activity decreased 95% in 4℃ for a month in 20 mmol/L Tris HCl buffer (pH 8.0) including 7 mmol/L 2 mercaptoethanol and 10 % glycerine after the enzyme was concentrated. It was inhibited by divalent metal ions, but activated by 2 mercaptoethanol.