Phycobilisome isolation

Fig. 2. Electron micrographs of phycobilisomes in the red alga Rhodella violacea (A) and phycobilisomes isolated from Porphyridium cruentum (B). (C) shows a membrane model consisting of the electron-transfer complexes of PS I, PS II, the cytochrome bet complex, the ATP synthase, CFq CFi, and the phycobilisomes. (A) and (C) from MOrschel and Rhiel (1987) Phycobilisomes and thylakoids The light-harvesting system of cyanobacteria and red algae. In JR Harris and RW Horne (eds) Membranous Structure, pp 216, 248. Acad Press (A) kindly furnished by Dr. Erhard Mbrschei and (B) kindly furnished by Dr. Alexander Glazer.

Fig. 9. Absorption spectrum of isolated Fig. 10. Fluorescence emission spectra of phycobilisomes. isolated phycobilisomes at -196°C.

NPQ (Rakhimberdieva et al. 2004) exactly matches the absorption spectrum of the carotenoid, 3 -hydrox yech i nenone (Polivka et al. 2005) in the OCP. The OCP is now known to be specifically involved in the phycobilisome-associated NPQ and not in other mechanisms affecting the levels of fluorescence such as state transitions or D1 damage (Wilson et al. 2006). Studies by immunogold labeling and electron microscopy showed that most of the OCP is present in the interthylakoid cytoplasmic region, on the phycobilisome side of the membrane, Figure 1.2 (Wilson et al. 2006). The existence of an interaction between the OCP and the phycobilisomes and thylakoids was supported by the co-isolation of the OCP with the phycobilisome-associated membrane fraction (Wilson et al. 2006, 2007). 

Extensive fractionation of the dissociation products from A. variabilis phycobilisomes led to the isolation of three PC complexes, (a(3)3-LRc , (aP)3-LR - and (aP)3 LR , and one phycoerythrocyanin complex (aP)3 L °. With suchtrimer-linker complexes available, in vitro reconstitution experiments could be performed to help clarify the sequence of the components in the rods and how the linker polypeptides mediate rod assembly. As already shown in Pig. 8, a large portion of the linker polypeptide is buried inside the trimer, and only the small segment ofthe linker that is projecting outside the trimer is apparently able to link with the next trimer or hexamer. 

G Porter, CJ Treadwell, GFW Searle and J Barber (1978) Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga. Biochim Biophys Acta 501 232-245 M Mimuro, I Yamazaki, N Tamai and T Katoh (1989) Excitation energy transfer in phycobilisomes at-196 C isolated from the cyanobacterium Anabaena variabilis (M-3) evidence for the plural transfer pathways to the terminal emitters. Biochim Biophys Acta 973, 153-162... 

The chlorophyll-protein (CP) and polypeptide pattern of PSII complexes were determined by two dimensional gel electrophoresis. Prominent polypeptides could be attributed to the biliprotein subunits of AP (14> 16 kDa) and PC (15f 18 kDa) and at least four phycobilisome-linker polypeptides (29f 31, 34 and 120 kDa. The Chl-proteins were characterized by apoproteins of 47 (CPIIa,b) and 41 (CPIIc) kDa. CP Ila and b were similar in their spectroscopic properties and showed fluorescence maxima at 685 nm and shoulders at 693 nm, whilst CP lie exhibited only one peak at 686 nm, when excited at 445 nm. Thus the isolated Chl-proteins had similar emission properties as the in situ" PSII antennae. 

Experimental Phycobilisomes were isolated accord-ing Eo [S ]. The intact phycobilisomes were dissociated against 5mM potassium phosphate buffer pH 7.0. Absorption Spectra were recorded on Shimadzu UV-visible recording spectrophotometer. Static fluorescence excitation and emission spectra were recorded on Shimadzu RF-540 recording Spectrofluorophotometer. The fluorescence life times were measured by the time correlated single photon counting set up capable of measuring life... 

Rhodosorus marinus is a unicellular, marine, red alga common in tropical and subtropical waters. We recently isolated it for the first time in Australia, and in the course of a routine ultrastructural investigation found that it had phycobilisomes of most unusual structure and arrangement. 

The spectral quality of the growth li t greatly affects the cell content and stoichiometry of PSI, PSII, and PBS in P. cruentum (Fig. 2). PSII centers (measured as Q ) are three times as numerous in RL cells than in GL cells (Table 2). WL cells contain an intermediate amount. Conversely, PSI centers in RL cells (measured as P7Q0) are only two-thirds as numerous as in GL or WL cells (Table 2). Cell content of PBS is about the same whether cultures are grown under continuous low intensity red, green or white li t (Table 2), and their size and composition are constant (based on a fixed stoichiometry of PE, PC, and APC in whole cells and on comparable absorption spectra for isolated phycobilisomes data not shown). 

C-Phycocyanin, a biliprotein isolated from blue-green and red algae [10], was tested in the chloroplast extract BLM system [4]. This protein has been identified as an extrinsic membrane protein in structures known as phycobilisomes [10]. The well established function of this protein is as an accessory energy transfer pigment for Photosystem II [11]. 

Thylakoid membranes were isolated from Porphyridium cruentum cells which were washed free of phycobilisomes and other surface binding proteins as described by Redlinger and Gantt (1982b),... 

FIGURE 2. a) Polypeptide profiles of P. laminosum membranes (MF), PSI particles (PSI), isolated phycobilisomes (PB), PSII-enriched LDAO supernatant (PSII Snt), POPS and Fractions 1-5 from the sucrose density gradient, using the SDS-PAGE buffer system of Chua (1980). Chlorophyll loadings per track were 5 hg (MF) / 10 ig (PSI) or 2 ig (PSII Snt and gradient fractions). b) Polypeptide profile of POPS (3 ig Chi) using the buffer system of Laemmli (1970). standards (St) were BSA... 

Gantt E, Lipschultz CA, Grabowski J and Zimmerman BK (1979) Phycobilisomes from blue-green and red algae. Isolation criteria and dissociation characteristics. Plant Physiol. 63, 615-620. 

Nies M and Wehrmeyer W (1980) Isolation and biliprotein characterization of phycobilisomes from the thermophilic Cyanobacterium Mastigocladus laminosus Cohn. Planta 150, 330-337. 

Anacystis nidulans was cultured in a modified medium D under 1% CO2 in air. Cultures were grown under white fluorescent light or under a far-red light (>650 nm) provided by tungsten-halogen lamps with Corning No. 2030 filter as described by Myers et al. (1980). Phycobilisomes were isolated in 0.75 M K-PO4 buffer (pH 8.0) as previously described (Gantt et al., 1979). 

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