Isolation and characterization of Photosystems I and II from the red alga Porphyridium cruentum

Abstract

Photosystem I and Photosystem II complexes were isolated from the red alga Porphyridium cruentum ( P. purpureum) following solubilization of thylakoid membranes with dodecyl-β- d-maltoside. More than 90% of the total chlorophyll was recovered in two green bands on a sucrose gradient. PS I and PS II complexes, present in the lower green band, were separated by anion exchange chromatography. The purified PS II complex, with an absorption maximum at 674 nm and a fluorescence emission maximum at 692 nm (77 K), contained the reaction center polypeptide D2 and the core antenna proteins CP43 and CP47 as shown by immunoblot analysis. Two bands (< 14 kDa), presumably the cyt b-559 subunits were also observed after silver-staining SDS-PAGE gels. The photoactivity rate of 58 μmol DCPIP/mg Chl per h with DPC for PS II core complexes, and 112 μmol DCPIP/mg Chl per h for thylakoids (minus phycobilisomes), was sensitive to DCMU inhibition. The purified PS I holocomplex had approx. 130 chlorophyll per P700, an absorption maximum at 680 nm and a fluorescence emission maximum at 730 nm (at 77 K). Prominent silver-stained bands in SDS-PAGE gels at 62 and 57 kDa polypeptides were immunologically identified as the P700 apoproteins. Polypeptides of 23.5, 23, 22, 20, 19.5, 18, 16.5, 15.5, and 11 kDa and at least three low molecular mass polypeptides (< 11 kDa) were also observed. Of particular interest are the 18–23.5 kDa polypeptides in the PS I complex, since such polypeptides were previously unknown in phycobilisome-containing organisms. In fact, we have found that the PS I complex of P. cruentum is structurally similar to that of higher plants, especially since polypeptides of 18–23.5 kDa were shown to be immunologically related to LHC I of higher plants (Wolfe, G. et al. (1994) Nature 367, 566–568). These results clearly suggest that the thylakoid structure of this red alga is more complex than in cyanobacteria, which like rhodophytes, also have phycobilisomes as major antenna complexes.