A BETA-GLUCOSIDASE FROM LODGEPOLE PINE XYLEM SPECIFIC FOR THE LIGNIN PRECURSOR CONIFERIN
A BETA-GLUCOSIDASE FROM LODGEPOLE PINE XYLEM SPECIFIC FOR THE LIGNIN PRECURSOR CONIFERIN
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DOI：
10.1104/pp.107.2.331
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发表时间：
1995-02-01
期刊：
影响因子：
7.4
通讯作者：
CARLSON, JE
中科院分区：
文献类型：
--
作者：
DHARMAWARDHANA, DP;ELLIS, BE;CARLSON, JE
松柏苷是木质醇松柏醇的葡萄糖苷，在春季形成层重新激活期间在裸子植物中积累到高水平。肉桂醇葡萄糖苷/β-葡萄糖苷酶系统被认为通过释放木质素单体糖苷在木质化中发挥关键作用。对大叶松 (Pinus contorta var latifolia Engelm) 木质部中这种酶系统的研究。揭示了两种主要的β-葡萄糖苷酶。一种能有效水解天然底物松柏苷，另一种对合成糖苷更具活性。使用阴离子交换、疏水相互作用和尺寸排阻色谱法将松柏素 β-葡萄糖苷酶纯化至表观同质性。表观天然分子量估计为 60,000。经过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，在纯化制剂中检测到主要的 28-kD 蛋白质和次要的 24-kD 蛋白质。针对 24-kD 蛋白合成 N 端肽的多克隆抗体的免疫学证据表明，天然蛋白是 28-kD 亚基大小的二聚体。 N端序列表明，松柏素β-葡萄糖苷酶与已知的植物β-葡萄糖苷酶具有高度同源性。松柏素、丁香苷和合成的松柏素类似物是松柏素β-葡萄糖苷酶的优选底物。使用显色松柏素类似物进行的原位定位显示，分化木质部中仅存在β-葡萄糖苷酶活性，与过氧化物酶活性相似。
Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/beta-glucosidase system is thought to play a key role in lignification by releasing the monolignol aglycones. Investigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major beta-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. The coniferin beta-glucosidase was purified to apparent homogeneity using anion exchange, hydrophobic interaction, and size-exclusion chromatography. The apparent native molecular weight was estimated to be 60,000. A dominant 28-kD protein and a minor 24-kD protein were detected in the purified preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological evidence from polyclonal antibodies directed against the synthetic N-terminal peptide of the 24-kD protein suggested that the native protein is a dimer of 28-kD subunit size. The N-terminal sequence showed that coniferin beta-glucosidase has high homology to known plant beta-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin beta-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of beta-glucosidase activity in the differentiating xylem, similar to peroxidase activity.