对生玉米幼叶尿卟啉原脱羧酶的纯化及部分性质研究  

The Purification and a Study on Some Properties of Uroporphyrinogen Decarboxylase from Oppositifolious Maize Young Leaves

作　　者：范军[1] 何晓梅[1] 朱娟[1] 朱苏文[1] 程备久[1] 

机构地区：[1]安徽农业大学生命科学学院生物工程系,安徽合肥230036

出　　处：《激光生物学报》2006年第2期148-153,共6页Acta Laser Biology Sinica

基　　金：国家963科技项目资助(2002AA211071)

摘　　要：尿卟啉原Ⅲ脱羧酶是植物血红素和叶绿素合成的一个关键调控酶。对生玉米叶片中叶绿素含量较比互生玉米高。对生玉米幼苗经硫酸铵分级沉淀,DEAE Sepharose CL-6B、Sephacryl S-200、羟基磷灰石和B lueSepharose CL-6B层析,纯化了尿卟啉原脱羧酶。纯化倍数为1 060倍,得率约8%,比活约880 U/mg蛋白。纯化的UROD在SDS/PAGE显示一条带,亚基分子量约为40 kD,Sephacryl S-300测得全酶分子量约为55 kD。IEF-PAGE显示UROD为一条带,等电点约为6.0,酶的最适pH值约7.0,在55℃下保温12 m in,酶活力丧失90%,在100 mm ol/L的巯基乙醇下,UROD的酶活力提高7倍。体外5 mm ol/L的磷酸吡哆醛修饰显示UROD活力下降约30%。Uroporphyrinogen decarboxylase （UROD） is a key regulatory enzyme that plays the important role in the metabolic flux through the branched biosynthetic pathway of chlorophylls, heme and siroheme in plants. The oppositifolious maize is a special mutant that has high contents of chlorophylls in the leaves. In this study, maize UROD was purified to homogeneity from the young oppositifolious maize leaves by several purification procedures including ammonium sulphate fractionation, anion-exchange chromatography, gel filtration, hydroxylapatite absorption and reactive blue affini- ty chromatography. Its final specific activity for uroporphyrinogen III was 880 U/mg protein at pH 7.0 with a fold of 1 060 and a yield of 8 %. The relative molecular weight of UROD holoenzyme was estimated about 60 kD using Sephacryl S-300 and that of UROD subunit was about 40 kD, as shown by SDS/PAGE. Determination of its pI and pH optimum revealed values of 6.0 and 7.0 respectively. The purified UROD decreased 90 % activity incubating for 12 min at 55℃. It increased about seven-fold activity in the presence of 100 mmol/L concertration of 2-mercaptoethanol. Modification with pyridoxal 5-phosphate at a concentration of 5 mmol/L caused a loss of about 40 % enzyme activity.

关 键 词：尿卟啉原脱羧酶 对生玉米 纯化 酶性质 

分 类 号：Q503[生物学—生物化学] Q55