Functional Characterization of Four Thylakoid Membrane Polypeptides with Apparent Molecular Weights

Four polypeptides which exhibited apparent molecular weights from 40 000 to 48 000 were isolated from the thylakoid membrane. The isolation was achieved by gel filtration of polypeptides, solubilized by means of sodium dodecyl sulfate and, after removal of detergent by anion exchange chromatography in ethanolic solution. The polypeptide of the molecular weight 40000 was iden­ tified as the 7-component of the coupling factor of photophosphorylation via the effect of its antiserum and by its serological cross reactions. The antiserum not only inhibits photophosphory­ lation reactions by 90% but also to the same extent the photoreduction of anthraquinone-2-sulfonate with tetramethyl benzidine as the electron donor, provided ADP, Pi and Mg2+ are present in the assay. The inhibition of electron transport is accompanied by a corresponding increase in the fluorescence yield. The other three antisera inhibit photosystem I reactions. In addition, the antiserum designated 45 000 PSI-1 inhibits phenazine methosulfate-mediated cyclic photophos­ phorylation. This antiserum as well as the two other sera do not contain any detectable anti­ coupling factor activity. Antiserum 45 000 PSI-4, in contrast to 45 000 PSI-1, does not inhibit cyclic photophosphorylation. Moreover, it is the only one of the four antisera which does not affect the ratio of the fluorescence yields measured at 735 and 685 nm (F 735/F 685) at 77 °K. Hence, the antigen 45 000 PSI-4 probably plays a role on the acceptor side of photosystem I. On the other hand, the antiserum 48 000 PSI-3 seems to exert its effect on the donor side of photosystem I, because it inhibits the photoreduction of anthraquinone-2-sulfonate with low con­ centrations of the electron donor dichlorophenol indophenol. The prominent property of this anti­ serum is that the photophosphorylation with ferricyanide as the electron acceptor is stimulated more than two-fold. This, however, it not accompanied by an apparent change of the electron flux between water and ferricyanide. The antiserum causes a decrease of fluorescence yield which is probably due to an increased energy spill-over. All antisera exert their activity only after an il­ lumination of the chloroplast preparations for several minutes. The dosis-effect curve is hyperbolic for the antiserum 48 000 PSI-3 and is sigmoidal with the other three antisera.