PURIFICATION AND CHARACTERIZATION OF ISOFORMS OF CINNAMYL ALCOHOL-DEHYDROGENASE FROM EUCALYPTUS XYLEM
PURIFICATION AND CHARACTERIZATION OF ISOFORMS OF CINNAMYL ALCOHOL-DEHYDROGENASE FROM EUCALYPTUS XYLEM
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DOI：
10.1007/bf00198938
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发表时间：
1992-08-01
期刊：
影响因子：
4.3
通讯作者：
BOUDET, AM
中科院分区：
文献类型：
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作者：
GOFFNER, D;JOFFROY, I;BOUDET, AM
从蓝桉（Eucalyptus gunii Hook）富含木质部的组分中纯化出了两种不同的肉桂醇脱氢酶异构体CAD1和CAD2，并对其进行了部分特性分析。它们在物理和生化性质方面差异很大，可以通过苯基琼脂糖凝胶CL - 4B的疏水作用色谱法进行分离。通过在Superose 6上进行凝胶过滤色谱法测定，CAD1的天然分子量为38 kDa，并且由于在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中产生35 kDa的多肽，该异构体可能是一个单体。它对松柏醇和对香豆醇及其相应的醛具有较低的底物亲和力。未检测到对芥子醛和芥子醇有活性。含量更丰富的异构体是CAD2，其天然分子量为83 kDa，是由两个分子量略有不同（42 - 43 kDa）的亚基组成的二聚体。这些亚基在用N - 氯代琥珀酰亚胺消化后显示出相同的肽段模式。异构体CAD2对所有测试的底物都具有高底物亲和力。这两种异构体在免疫学上是不同的，因为针对CAD2产生的多克隆抗体与CAD1不发生交叉反应。对表现出如此显著差异的两种CAD形式的特性分析表明它们参与了单木质醇利用的特定途径。
Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions of Eucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl and p-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dimer composed of two subunits of slightly different molecular weights (42-43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.