Abstract
A method to purify the phytochrome photoreceptor from the unicellular green alga Mesotaenium caldariorum is presented. Preparative scale formation of algal protoplasts and controlled osmotic cell lysis have permitted separation of intact organelles from the phytochrome-enriched soluble protein fraction. We have utilized the observation that red light-absorbing (Pr) and far-red light-absorbing (Pfr) forms of phytochrome are differentially retained on an anion exchange matrix to purify M. caldariorum phytochrome to apparent homogeneity. M. caldariorum phytochrome preparations with A650/A280 ratios greater than 0.78 exhibit a single 120-kDa band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Immunoblot analyses using a cross-reactive pea phytochrome monoclonal antibody reveal that 1) the 120-kDa band represents the full-length polypeptide, 2) phytochrome is predominantly localized in the algal cytoplasm, and 3) there are 150,000-250,000 phytochrome molecules/cell. Steric exclusion high pressure liquid chromatography analysis under nondenaturing conditions indicates that M. caldariorum phytochrome has an apparent mass of 355 kDa. The absorption maxima for Pr and Pfr are 650 and 722 nm, respectively. Both are blue-shifted compared with those of phytochromes from dark-grown angiosperm tissue. The molar absorption coefficient for Pr at 650 nm is 86,800 +/- 2800 liter mol-1 cm-1, which is lower than that of higher plant phytochromes. The significance of the similarities and differences of the molecular properties of phytochromes from M. caldariorum and higher plant sources is discussed.
Highlights
THE JOURNAL OF BKILOCICAL CHEMISTRY rC;1990 hy The American Society for Biochemistry and Molecular Biology, Inc
We have utilized the observation that red light-absorbing (Pr) and far-red light-absorbing (Pfr) forms of phytochrome are differentially retained on an anion exchange matrix to purify M, caldariorum phytochrome to apparent homogeneity
Enriched preparations of phytochrome have been obtained from green plant tissues including a lower plant species (5, 25, 26, 2%30), but attempts to purify phytochrome from chlorophyll-containing plant tissues have failed to yield homogeneous spectrally native photoreceptor
Summary
P,, red light-absorbing form of phytochrome; Pf,, far-red light-absorbing form of phytochrome; AAA, spectral difference assav units for phvtochrome defined as (A(P,.650). Whole M. caldariorum cells and protoplasts obtained with this method are shown in Fig. 1 (A and B, respectively). Since phytochrome cannot be spectrally assayed in whole cells, in protoplasts, or in crude soluble protein extracts, yields for these initial steps have been determined by quantitative immunoblot analysis (see Fig. 2). For these determinations, spectrally assayed algal phytochrome preparations obtained from the latter steps of the purification procedure were used to construct standard curves. These values represent molar absorption coefficients for the phytochrome monomer which has an apparent
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