Phycobilisome-thylakoid Topography on Photosynthetically Active Vesicles of Porphyridium cruentum

Abstract

Conditions are described for isolating functional phycobilisome-thylakoid vesicles from the red alga Porphyridium cruentunL Phycobilisomethylakoid vesicles were prepared by brief sonication and centrifugation in a medium containing 0.5 molar sucrose, 0.5 molar potassium phosphate, and 0.3 molar sodium citrate (pH 7.0). They required ferricyanide as an oxidant and had 02 evolution rates (about 450 micromoles 02 per hour per milligram chlorophyll) higher than whole cells (about 250 micromoles 02 per hour per milligram chlorophyll). Energy transfer to photosystem XI chlorophyUl was evident from a high F695 nanometer (-196 C) emission peak. Preparations could be stored for over 24 hours and were considerably more stable than those from the cyanobacterium Anabaena variabilis (Katoh T, E Gantt 1979 Biochim Biophys Acta 546: 383-393). In electron micrographs of negatively stained material, the active thylakoid vesicles were found covered by closby spaced phycobilisomes on their external surface. The phycobilisome number in negatively stained vesicles was 450 per square micrometer, which was in the same range as the 400 per square micrometer observed in surface sections. A cell containing 1.5 x 10-6 micrograms phycoerythrin and 1.3 x 10-6 micrograms chlorophyll was found to contain 5 to 7 x 10' phycobilisomes on a thylakoid area of 1.1 to 1.6 x 103 square micrometers.The red alga Porphyridium cruentum (Porphyridium purpureum) has long been a favorite photosynthetic organism in investigating the structural and functional interrelationship ofits photosynthetic accessory pigments, the phycobiliproteins, and the thylakoid membrane (2,3,11,15,17,20). Because this alga has very stable PBS,3 their isolation and analysis was first accomplished with this species (8). In whole cells, most ofthe energy absorbed by PE is transferred initially to PSII (16), indicated by a fluorescence emission at F685 and F695 nm (-196 C). In green plants, these emission peaks clearly arise from PSII pigment-protein complexes [see refs. in Rijgersberg et al. (22)]. In phycobiliprotein-containing algae, the interpretation of fluorescence is complicated by the PBS emission in the presence of thylakoid membranes.Isolated PBS have a fluorescence emission at F685 nm which in Porphyridium arises from a special APC, namely, APC-B, the terminal pigment in the energy transfer chain (9,16 18, 25) and can serve as a terminal pigment as well. In Anabaena variabilis, isolated vesicles with bound PBS exhibited a decrease of the F695 nm relative to F685 nm with decreasing 02 evolution in degenerating conditions (14). Thus, the F685 nm emission increase was interpreted as a probable energetic uncoupling between the PBS and PSII.In adpating the isolation procedure to PBS-vesicles of P. cruentum, the following conditions were considered close to the in vivo state: (a) an F695/F685 nm ratio of 1.0 to 1.2; (b) 02 evolution rates representative of whole cells; and (c) full attachments of PBS to thylakoids. Here, we report the application with modifications of t...

Evidence for a Transient Association of New Proteins with the Spirulina maxima Phycobilisome in Relation to Light Intensity

“…Differential centrifugation (48,00Og, 30 min, 4OC) gave a pellet containing mainly thylakoidal fraction functionally associated with PBS (intact vesicles, according to Dilworth and Gantt, 1981) and a supematant containing the major part of soluble proteins, except PBP that remained associated with the thylakoid fragments. This supematant was used as the "soluble protein fraction.…”

Stoichiometry of Photosystem I, Photosystem II, and Phycobilisomes in the Red Alga Porphyridium cruentum as a Function of Growth Irradiance

“…The supernatant fluid was used to assay for PE. PB numbers were calculated from the spectroscopically determined levels of PE by assuming 60 PE hexamers (250,000 g/mol of hexamer) in each PB (9) for LL-grown cultures. It was assumed that PB in ML, HL, and VHL-grown cultures contain only 57, 44.4, and 47.4 PE hexamers, respectively, based on the deficiency of PE relative to APC in these cells compared with LL-grown cells (Table IV).…”

Photosynthetic membrane topography: quantitative in situ localization of photosystems I and II.

“…1 A and B) isolated in a medium that preserves both the capacity for excitation energy transfer from PBsomes to the photosystems and the ability to evolve oxygen at rates comparable to whole cells (20 If the photosystems are distributed throughout the thylakoid membrane, then the number ofPSI and PSII centers per unit area can be calculated (Table 2) from the number of PSI (P700) and PSII (QA) per PBsome estimated from spectral determinations (17). In thylakoids of GL cells, we predict a density of -2520 PSI and =630 PSII per jum2, with -1580 PSI and -1890 PSII per ,&m2 in RL cells.…”