Sagami H, Lennarz W J, Ogura K
Chemical Institute of Non-Aqueous Solutions, Tohoku University, Sendai, Japan.
Biochim Biophys Acta. 1989 Apr 3;1002(2):218-24. doi: 10.1016/0005-2760(89)90290-7.
Using improved conditions with rat liver microsomes in the presence of 20% glycerol and 2% Triton X-100 at pH 8.5 it was shown that dehydrodolichyl diphosphate and dehydrodolichyl phosphate were synthesized from isopentenyl diphosphate and farnesyl diphosphate. Small amounts of geranylgeranyl diphosphate and geranylgeranyl phosphate were also formed. The carbon chain lengths of the dehydrodolichyl diphosphate and dehydrodolichyl phosphate were identical (C80-C85). A kinetic study showed that dehydrodolichyl diphosphate formed from farnesyl diphosphate and isopentenyl diphosphate was subsequently hydrolyzed to dehydrodolichyl phosphate. As the concentration of isopentenyl diphosphate was increased from 1 to 50 microM, the chain-length distribution of dehydrodolichyl products shifted from C75-C80 to C80-C85. Addition of MgCl2 into the assay mixture decreased product formation, but did not affect the chain-length distribution (C80-C85). The shift of the chain-length distribution to the same as that observed in naturally occurring dolichol derivatives (C90-C95) was observed when Triton X-100 was omitted from the assay mixture, although deletion of the detergent decreased the enzyme activity. These results, which provide insight into optimal conditions for enzymatic synthesis of the dolichol chain, are discussed in the context of the in vivo pathway for dolichol biosynthesis.
在pH 8.5条件下，使用含有20%甘油和2% Triton X-100的大鼠肝微粒体的改进条件，结果表明，异戊烯基二磷酸和法尼基二磷酸可合成脱氢多萜醇二磷酸和脱氢多萜醇磷酸。还生成了少量的香叶基香叶基二磷酸和香叶基香叶基磷酸。脱氢多萜醇二磷酸和脱氢多萜醇磷酸的碳链长度相同（C80 - C85）。动力学研究表明，由法尼基二磷酸和异戊烯基二磷酸形成的脱氢多萜醇二磷酸随后水解为脱氢多萜醇磷酸。随着异戊烯基二磷酸的浓度从1 microM增加到50 microM，脱氢多萜醇产物的链长分布从C75 - C80转变为C80 - C85。向测定混合物中添加MgCl2会减少产物形成，但不影响链长分布（C80 - C85）。当从测定混合物中省略Triton X-100时，观察到链长分布的转变与天然存在的多萜醇衍生物（C90 - C95）中观察到的相同，尽管去除去污剂会降低酶活性。这些结果为多萜醇链的酶促合成提供了最佳条件的见解，并在多萜醇生物合成的体内途径背景下进行了讨论。
