Rubber particles from rubber-producing plant species have many different species-specific proteins bound to their external monolayer biomembranes. To date, identification of those proteins directly involved in enzymatic catalysis of rubber polymerization has not been fully accomplished using solubilization, purification or reconstitution approaches. In an alternative approach, we use several tritiated photoaffinity-labeled benzophenone analogs of the allylic pyrophosphate substrates, required by rubber transferase (RT-ase) to initiate the synthesis of new rubber molecules, to identify the proteins involved in catalysis. Enzymatically-active rubber particles were purified from three phylogenetically-distant rubber producing species, Parthenium argentatum Gray, Hevea brasiliensis Muell. Arg, and Ficus elastica Roxb., each representing a different Superorder of the Dicotyledonae. Geranyl pyrophosphate with the benzophenone in the para position (Bz-GPP(p)) was the most active initiator of rubber biosynthesis in all three species. When rubber particles were exposed to ultra-violet radiation, 95% of RT-ase activity was eliminated in the presence of 50 μΜ Bz-GPP(p), compared to only 50% of activity in the absence of this analog. 3H-Bz-GPP(p) then was used to label and identify the proteins involved in substrate binding and these proteins were characterized electrophoretically. In all three species, three distinct proteins were labeled, one very large protein and two very small proteins, as follows: P. argentatum 287,000, 3,990, and 1,790 Da; H. brasiliensis 241,000, 3,650 and 1,600 Da; F. elastica 360,000, 3,900 and 1,800 Da. The isoelectric points of the P. argentatum proteins were 7.6 for the 287,000 Da, 10.4 for the 3,990 Da and 3.5 for the 1,790 Da proteins, and of the F. elastica proteins were 7.7 for the 360,000 Da, 6,0 for the 3,900 Da, and 11.0 for the 1,800 Da proteins. H. brasiliensis protein pI values were not determined. Additional analysis indicated that the three proteins are components of a membrane-bound complex and that the ratio of each small protein to the large one is 3:1, and the large protein exists as a dimer. Also, the large proteins are membrane bound whereas both small proteins are strongly associated with the large proteins, rather than to the rubber particle proteolipid membrane.

中文翻译：

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不寻常的亚基直接参与多种植物物种天然橡胶生物合成的结合底物

来自产橡胶植物物种的橡胶颗粒具有许多不同的物种特异性蛋白质，它们结合在其外部单层生物膜上。迄今为止，尚未使用增溶、纯化或重构方法对直接参与橡胶聚合酶催化的那些蛋白质进行鉴定。在另一种方法中，我们使用橡胶转移酶 (RT-ase) 所需的几种氚化光亲和标记的烯丙基焦磷酸底物的二苯甲酮类似物来启动新橡胶分子的合成，以识别参与催化的蛋白质。酶活性橡胶颗粒是从三个系统发育较远的橡胶生产物种中纯化的，它们是 Parthenium argentatum Gray、Hevea brasiliensis Muell。Arg, and Ficus elastica Roxb., 每个代表不同的双子叶植物纲。对位二苯甲酮的香叶基焦磷酸酯 (Bz-GPP(p)) 是所有三种橡胶生物合成中最活跃的引发剂。当橡胶颗粒暴露于紫外线辐射时，在 50 μM Bz-GPP(p) 存在下，95% 的 RT-ase 活性被消除，而在没有这种类似物的情况下，只有 50% 的活性被消除。然后使用 3H-Bz-GPP(p) 标记和鉴定参与底物结合的蛋白质，并通过电泳表征这些蛋白质。在所有三个物种中，标记了三种不同的蛋白质，一种非常大的蛋白质和两种非常小的蛋白质，如下所示：P. argentatum 287,000、3,990 和 1,790 Da；H. brasiliensis 241,000、3,650 和 1,600 Da；F. elastica 360,000、3,900 和 1,800 Da。P 的等电点 argentatum 蛋白质为 287,000 Da 的 7.6、3,990 Da 的 10.4 和 1,790 Da 的 3.5，而 F. elastica 蛋白质的 360,000 Da 为 7.7、3,900 Da 的 6,0 和 Da 11,80。蛋白质。H. brasiliensis 蛋白质 pI 值未确定。进一步的分析表明，这三种蛋白质是膜结合复合物的组成部分，每种小蛋白质与大蛋白质的比例为 3:1，大蛋白质以二聚体形式存在。此外，大蛋白质是膜结合的，而两种小蛋白质都与大蛋白质密切相关，而不是与橡胶颗粒蛋白脂质膜相关。0 表示 1,800 Da 蛋白质。H. brasiliensis 蛋白质 pI 值未确定。进一步的分析表明，这三种蛋白质是膜结合复合物的组成部分，每种小蛋白质与大蛋白质的比例为 3:1，大蛋白质以二聚体形式存在。此外，大蛋白质是膜结合的，而两种小蛋白质都与大蛋白质密切相关，而不是与橡胶颗粒蛋白脂质膜相关。0 表示 1,800 Da 蛋白质。H. brasiliensis 蛋白质 pI 值未确定。进一步的分析表明，这三种蛋白质是膜结合复合物的组成部分，每种小蛋白质与大蛋白质的比例为 3:1，大蛋白质以二聚体形式存在。此外，大蛋白质是膜结合的，而两种小蛋白质都与大蛋白质密切相关，而不是与橡胶颗粒蛋白脂质膜相关。