Parmryd I, Shipton C A, Swiezewska E, Andersson B, Dallner G
Arrhenius Laboratories for Natural Sciences, Biochemistry Department, Stockholm University, Sweden.
Eur J Biochem. 1995 Dec 15;234(3):723-31. doi: 10.1111/j.1432-1033.1995.723_a.x.
Spinach seedlings were found to contain farnesyl protein transferase. The enzyme is activated by Zn2+, but not by Mg2+. The pH optimum is approximately 7.0 and maximal activity is obtained at 40-45 degrees C. The apparent Km for the farnesyl diphosphate substrate is 7 microM. Western blotting of soluble proteins with an antiserum raised against mammalian farnesyl protein transferase demonstrated a specific cross-reactivity with the spinach enzyme. The antiserum preferentially recognises the beta-subunit of the heterodimeric farnesyl protein transferase, and the corresponding spinach polypeptide has a molecular mass of 42 kDa on SDS/PAGE. The enzyme can employ dithiothreitol as an acceptor for the farnesyl moiety and catalyses the formation of a thioether linkage between these substrates. On the basis of this discovery, a new method was developed utilising the hydrophobicity of the reaction product, and its interaction with poly(propylene). During in vivo labelling, the plants took up dithiothreitol, which inhibited the incorporation of [3H]mevalonate metabolites into proteins, indicating that dithiothreitol might be isoprenylated in vivo as well as in vitro. However, isoprenylation of some proteins remains unaffected by dithiothreitol suggesting the existence of different isoprenylation mechanisms. Thus, it is demonstrated that plants possess farnesyl protein transferase, which resembles its mammalian and yeast homologues.
研究发现菠菜幼苗中含有法尼基蛋白转移酶。该酶被Zn2+激活，但不被Mg2+激活。最适pH约为7.0，在40 - 45摄氏度时可获得最大活性。法尼基二磷酸底物的表观Km为7 microM。用针对哺乳动物法尼基蛋白转移酶产生的抗血清对可溶性蛋白进行蛋白质印迹分析，结果表明该抗血清与菠菜酶具有特异性交叉反应。该抗血清优先识别异二聚体法尼基蛋白转移酶的β亚基，在SDS/PAGE上，相应的菠菜多肽分子量为42 kDa。该酶可利用二硫苏糖醇作为法尼基部分的受体，并催化这些底物之间硫醚键的形成。基于这一发现，利用反应产物的疏水性及其与聚丙烯的相互作用开发了一种新方法。在体内标记过程中，植物吸收了二硫苏糖醇，这抑制了[3H]甲羟戊酸代谢产物掺入蛋白质中，表明二硫苏糖醇在体内和体外都可能被异戊二烯化。然而，一些蛋白质的异戊二烯化不受二硫苏糖醇的影响，这表明存在不同的异戊二烯化机制。因此，证明植物拥有法尼基蛋白转移酶，它类似于其哺乳动物和酵母同源物。
