Synthesis of Cytochrome f by Isolated Pea Chloroplasts

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Abstract
Chloroplasts, isolated from the leaves of 7-day-old pea [Pisum sativum] 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 dodecyl sulfate. The cytochrome f band was identified by its intrinsic fluorescence in UV light and was shown to be radioactive by autoradiography or fluorography of dried polyacrylamide gels. 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 synthesized in intact isolated pea chloroplasts.