Light D R, Dennis M S
Department of Medicinal and Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.
J Biol Chem. 1989 Nov 5;264(31):18589-97.
We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.
我们从商业橡胶树巴西橡胶树的乳胶中纯化出了“橡胶转移酶”，发现它是一种二聚体，单体分子量为38,000道尔顿，需要Mg2+，并且通过硫醇稳定，这与先前描述的部分纯化制剂的研究结果一致（Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476 - 480）。纯化的异戊烯基转移酶将[1-14C]异戊烯基焦磷酸掺入脱蛋白橡胶颗粒中，其中超过90%被添加到高分子量聚异戊二烯（大于20,000道尔顿）中。在橡胶含量匹配的样品中，纯化的异戊烯基转移酶和脱蛋白橡胶颗粒可重构全乳胶生物合成活性的40 - 60%。掺入量与添加的橡胶颗粒呈线性关系，直至至少10 mg/ml橡胶或20 μM橡胶分子（基于数均分子量500,000道尔顿）。全乳胶中估计的异戊烯基转移酶浓度（0.37%或160 nM）足以饱和全乳胶中的所有延伸位点，添加纯化的异戊烯基转移酶不会增加[1-14C]异戊烯基焦磷酸的掺入量。脱蛋白橡胶颗粒可用纯酶进行滴定（Kd = 9 nM），表明可用于添加的橡胶分子比例很低（约0.01%）。在典型测定中，每个复合物每秒以1的速率添加约7,000个异戊二烯单元。巴西橡胶树异戊烯基转移酶在橡胶颗粒存在下催化顺式异戊二烯的形成。然而，在没有橡胶颗粒且存在二甲基烯丙基焦磷酸的情况下，纯化的异戊烯基转移酶通过薄层色谱、气相色谱和分子排阻色谱证明催化香叶基焦磷酸和全反式法呢基焦磷酸的形成。
