Antisera to the Coupling Factor of Photophosphorylation and Its Subunits

Abstract

Abstract Stroma-freed chloroplasts were extracted with sucrose palmitate-stearate containing buffer. After the addition of dodecyl sulfate and mercaptoethanol to the extract a series of polypeptides was isolated from the mixture by gel filtration. These polypeptides were later used for immunization. Antisera to four polypeptides reacted in the Ouchterlony double diffusion test with authentic coupling factor yielding a precipitation band. According to the observed apparent molecular weights the polypeptides are the α, β , δ and ε subunits of the coupling factor. An antiserum to the γ subunit has been obtained already previously. All antisera inhibit photophosphorylation reactions and electron transport considerably. Addition of gramicidin inhibits photophosphorylation completely whereas gramicidin restores electron transport in the assays with the antisera to the α, β , γ and δ subunit. In the case of the antiserum to the ε subunit gramicidin does not regenerate electron transport. As in the presence of the serum to the ε subunit pH changes in the suspension medium are not observed, this serum seems to open a proton channel. Also, upon addition of dicyclohexyl carbodiimide (DCCD) pH changes in the suspension medium in the assay with antiserum do not reoccur. According to these unexpected results the identity of the antigen with the ε subunit of the coupling factor is not certain. ATP-ase reactions are only inhibited by the antisera to the α and γ subunit and what is thought to be the ε subunit. The antiserum to the α subunit uncouples electron transport as the only one when used in sufficient concentrations. The dosis-effect curves of the inhibition of the electron transport exhibits a maximum. The dosis-effect curves for the other components rise after a lag phase in an approximately hyperbolic manner. The inhibitory action on electron transport is exerted by all antisera in the region of the reaction center I or in its immediate vicinity. This is thought to be due to the fact that a protein of the reation center I is inhibited in its function by the increasing proton concentration inside the thylakoid. The inhibition of electron transport by the antiserum to the ε subunit is considered to be a direct serum effect. Besides the increase in fluorescence yield, due to the inhibition of electron transport in the region of photosystem I, decreases of the fluorescence yield are observed in the presence of DCMU, which do not depend on the redox state of Q but rather on the condition of the thylakoid mem­brane. Moreover, the antisera affect in a differing manner the energy spill-over of excitation from photosystem II to photosystem I.