Doherty A, Gray J C
Eur J Biochem. 1979 Jul;98(1):87-92. doi: 10.1111/j.1432-1033.1979.tb13164.x.
Chloroplasts, isolated from the leaves of 7-day-old pea seedlings, were incubated in the light with [35S]methionine or [3H]leucine. After extraction from the washed chloroplast membranes using a mixture of ethyl acetate, ethanol and ammonia, cytochrome f was precipitated with a monospecific antiserum and resolved by gradient polyacrylamide gel electrophoresis in sodium dodecylsulphate. The cytochrome f band was identified by its intrinsic fluorescence in ultraviolet light and was shown to be radioactive by autoradiography or fluorography of dried polyacrylamide gel. One-dimensional peptide mapping of the products of papain hydrolysis confirmed that the radioactivity was an integral part of cytochrome f. The incorporation of [35S]methionine into cytochrome f was inhibited by D(-)threo-chloramphenicol but not by cycloheximide and did not occur in the dark. The synthesis was resistant to ribonuclease. It is concluded that cytochrome f is synthesised in intact isolated pea chloroplasts.
从7日龄豌豆幼苗叶片中分离出的叶绿体，在光照下与[35S]甲硫氨酸或[3H]亮氨酸一起孵育。使用乙酸乙酯、乙醇和氨的混合物从洗涤过的叶绿体膜中提取后，细胞色素f用单特异性抗血清沉淀，并用十二烷基硫酸钠梯度聚丙烯酰胺凝胶电泳进行分离。细胞色素f条带通过其在紫外光下的固有荧光进行鉴定，并通过干燥聚丙烯酰胺凝胶的放射自显影或荧光自显影显示具有放射性。木瓜蛋白酶水解产物的一维肽图谱证实放射性是细胞色素f的一个组成部分。[35S]甲硫氨酸掺入细胞色素f受到D(-)苏式氯霉素的抑制，但不受环己酰亚胺的抑制，并且在黑暗中不发生掺入。该合成对核糖核酸酶具有抗性。得出的结论是，细胞色素f是在完整的离体豌豆叶绿体中合成的。
