Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.
Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.
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血红素硫醇盐酶细胞色素 P-450TYR 的分离，该酶催化高粱 (L.) Moench 中氰基葡萄糖苷杜胡蛋白生物合成的关键步骤。
DOI：
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发表时间：
1994
影响因子：
11.1
通讯作者：
B. Møller
中科院分区：
文献类型：
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作者：
O. Sibbesen;B. Koch;B. Halkier;B. Møller
从高粱黄化苗制备的微粒体中分离出了催化l -酪氨酸n -羟基化为n -羟基酪氨酸的细胞色素P-450酶（hemethiolate酶），该酶是生成氰糖苷苦苷生物合成的重要步骤。Moench。细胞色素P-450酶用清洁剂Renex 690、还原Triton X-100和3-[（3-胆酰胺丙基）二甲酰胺]-1-丙磺酸溶解，通过离子交换（DEAE-Sepharose）和染料（Cibacron蓝和活性红120）柱层析分离。为了防止细胞色素P-450酶的不可逆聚集，分离过程没有任何浓缩步骤。，用凝胶过滤材料稀释离子交换凝胶。分离的酶，我们将其命名为细胞色素P-450TYR酶，在l -酪氨酸存在下产生I型底物结合谱。微粒体制剂含有0.2 nmol总细胞色素P-450/mg蛋白质。细胞色素P-450TYR酶估计约占微粒体膜总细胞色素P-450含量的20%，约占其总蛋白质含量的0.2%。细胞色素P-450TYR酶的表观分子质量为57 kDa， n端氨基酸序列为ATMEVEAAAATVLAAP。Western blot检测发现，针对分离的细胞色素P-450TYR酶产生的多克隆抗体具有特异性，可抑制微粒体系统催化l -酪氨酸向对羟基杏仁腈的体外转化。细胞色素P-450TYR酶具有高底物特异性，并在单一内源性底物上作为n -羟化酶。所报道的基于染料柱的分离方法是细胞色素P-450酶的一种温和的分离方法，它能够从催化对羟基苯乙腈c -羟基化的细胞色素P-450酶和肉桂酸4-羟化酶中分离出细胞色素P-450TYR，具有普遍的用途。
The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step--i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450TYR enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450TYR enzyme is estimated to constitute approximately 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450TYR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TYR enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450TYR enzyme exhibits high substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its ability to separate cytochrome P-450TYR from the cytochrome P-450 enzyme catalyzing the C-hydroxylation of p-hydroxyphenylacetonitrile and from cinnamic acid 4-hydroxylase.