Abstract
Phytochrome from 10 or 11-d-old oat (Avena sativa L. cv. Garry) leaves, which were harvested just prior to sunset from plants grown in a greenhouse in the absence of supplemental illumination, was purified an estimated 250-fold by sequential poly(ethylenimine) and ammonium-sulfate fractionations, followed by linear-gradient hydroxyapatite chromatography. Compared to earlier protocols, the one presented here is substantially more rapid, provides improved yield and purity, can be used with larger quantities of tissue, and eliminates an apparently immunodominant contaminant with a molecular mass of about 115 kDa (kilodalton). Phytochrome obtained by this procedure has an apparent monomer size of 123 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is estimated to be 0.6% pure. This purity permitted spectral analysis at wavelengths below 500 nm, in which region phytochromes from green and etiolated oat shoots do not differ markedly, as they do at longer wavelengths.
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Abbreviations
- Da:
-
Dalton
- HA:
-
hydroxyapatite
- Pfr, Pr:
-
farredand red-absorbing form of phytochrome, respectively
- SDS:
-
sodium dodecyl sulfate
References
Abe, H., Yamamoto, K.T., Nagatani, A., Furuya, M. (1985) Characterization of green tissue-specific phytochrome isolated immunochemically from pea seedlings. Plant Cell Physiol. 326, 1387–1399
Bradford, M.M. (1976) A rapid and sensitive method for the quantiation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Cordonnier, M.-M. (1989) Monoclonal antibodies: molecular probes for the study of phytochrome. Photochem. Photobiol. 49, 821–831
Cordonnier, M.-M., Pratt, L.H. (1990) Phytochrome from green Avena characterized with monoclonal antibodies directed to it. In: Photobiology, Riklis, E., ed. Plenum Press, New York, in press
Cordonnier, M.-M., Greppin, H., Pratt, L.H. (1986) Phytochrome from green Avena shoots characterized with a monoclonal antibody to phytochrome from etiolated Pisum shoots. Biochemistry 25, 7657–7666
Jabben, M., Deitzer, G.F. (1978) Spectrophotometric phytochrome measurements in light-grown Avena sativa L. Planta 143, 309–313
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Lagarias, J.C., Kelly, J.M., Cyr, K.L., Smith, W.O., Jr. (1987) Comparative photochemical analysis of highly purified 124 kilodalton oat and rye phytochromes in vitro. Photochem. Photobiol. 46, 5–13
Pratt, L.H. (1973) Comparative immunochemistry of phytochrome. Plant Physiol. 51, 203–209
Pratt, L.H. (1983) Assay of photomorphogenic photoreceptors. In: Encyclopedia of plant physiology, N.S., vol. 16A; Photomorphogenesis, pp. 152–177, Shropshire, W., Jr., Mohr, H., eds. Springer, Berlin Heidelberg New York
Pratt, L.H. (1984) Phytochrome purification. In: Techniques in photomorphogenesis, pp. 175–200, Smith, H., Holmes, M.G., eds. Academic Press, London New York
Pratt, L.H., Cordonnier, M.-M. (1987) Phytochrome from green Avena. In: Phytochrome and photoregulation in plants, pp. 83–94, Furuya, M., ed. Academic Press, Tokyo
Pratt, L.H., Wampler, J.E., Rich, E.S., Jr (1985) An automated dual-wavelength spectrophotometer optimized for phytochrome assay. Anal. Instrument. (N.Y.) 13, 269–287
Pratt, L.H., Stewart, S.J., Shimazaki, Y., Wang, Y.-C., Cordonnier, M.-M. (1991) Monoclonal antibodies directed to phytochrome from green leaves of Avena sativa L. cross-react weakly or not at all with the phytochrome that is most abundant in etiolated shoots of the same species. Planta 184, 87–95
Rich, E.S., Jr., Wampler, J.E. (1982) EPR spectrometer control and data acquisition using a general purpose IEEE-488 bus interface and structured software. Am. Lab. 14, 17–28
Shimazaki, Y., Pratt, L.H. (1985) Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots. Planta 164, 333–344
Tokuhisa, J.G., Quail, P.H. (1983) Spectral and immunochemical characterization of phytochrome isolated from light-grown Avena sativa. (Abstr.) Plant Physiol. 72, Suppl., 85
Tokuhisa, J.G., Quail, P.H. (1987) The levels of two distinct species of phytochrome are regulated differently during germination in Avena sativa. L. Planta 172, 371–377
Tokuhisa, J.G., Quail, P.H. (1989) Phytochrome in green tissue: partial purification and characterization of the 118-kilodalton phytochrome species from light-grown Avena sativa L. Photochem. Photobiol. 50, 143–152
Tokuhisa, J.G., Daniels, S.M., Quail, P.H. (1985) Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L. Planta 164, 321–332
Vierstra, R.D., Quail, P.H. (1983) Purification and initial characterization of 124-kilodalton phytochrome from Avena. Biochemistry 22, 2498–2505
Vierstra, R.D., Quail, P.H. (1986) Phytochrome: the protein. In: Photomorphogenesis in plants, pp. 35–60, Kendrick, R.E., Kronenberg, G.H.M., eds. Nijhoff, Dordrecht, The Netherlands
Wampler, J.E., Mulkerrin, M.G., Rich, E.S., Jr. (1979) A flexible, computer-controlled video microscope capable of quantitative spatial, temporal and spectral measurements. Clin. Chem. 25, 1628–1634
Wang, Y-C., Stewart, S.J., Cordonnier, M.-M. Pratt, L.H. (1991) Avena sativa L. contains three phytochromes, only one of which is abundant in etiolated tissue. Planta 184, 96–104
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This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.
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Pratt, L.H., Shimazaki, Y., Stewart, S.J. et al. Large-scale partial purification of phytochrome from green leaves of Avena sativa L.. Planta 184, 81–86 (1991). https://doi.org/10.1007/BF00208240
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DOI: https://doi.org/10.1007/BF00208240