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Gel filtration of particle-bound phytochrome

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Abstract

Pelletable phytochrome from hypocotyl hooks of Cucurbita pepo L. seedlings has been separated into two fractions by gel filtration on Sepharose CL-2B. One fraction with a K av of 0.7 was detected only after red irradiation (in vivo or in vitro). This separated from a ribonucleoprotein fraction during gel filtration. The weak interaction with ribonucleo-protein which required magnesium (optimal at 10 mM) was overcome by high salt concentrations and prevented by ribonuclease treatment. The second phytochrome fraction was strongly associated with a high molecular weight material with a K av of less than 0.1. Low levels of this complex were detected in extracts from dark grown tissue but were increased by red irradiation of excised hooks or crude extracts. The binding of phytochrome to the high molecular weight material did not require magnesium, was unaffected by ribonuclease treatment, and was much more resistant to high salt concentrations than was the phytochrome—ribonucleoprotein association. These results suggest that the association of phytochrome with this membrane—containing fraction is not electrostatic.

The separation by agarose-gel filtration offers a useful technique for the preparation of membraneassociated phytochrome for physiological studies.

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Abbreviations

EDTA:

ethylenediaminetetraacetic acid

P r and Pfr :

phytochrome in the red and far-red absorbing forms

RNase:

ribonuclease

RNP:

ribonucleoprotein

References

  1. Butler, W.L., Norris, K.H.: The spectrophotometry of dense light-scattering material. Arch. Biochem. Biophys. 87, 31–40 (1960)

    PubMed  Google Scholar 

  2. Dische, F., Schwartz, K.: Mikromethode zur Bestimmung verschiedener Pentosen nebeneinander bei Gegenwart von Hexosen. Microchim. Acta 2, 13–19 (1937)

    Google Scholar 

  3. Dittmer, J.C., Wells, M.A.: Quantitative and qualitative analysis of lipids and lipid components. In: Methods in enzymology 14, 482–530, Colowick, S.P., Kaplan, N.O., eds. New York: Academic Press 1969

    Google Scholar 

  4. Fiske, C.H., Subbarow, Y.: The colorimetric determination of phosphorus. J. biol. Chem. 66, 375–400 (1925)

    Google Scholar 

  5. Folch, J., Lees, M., Sloane-Stanley, G.H.: A simple method for the isolation and purification of total lipides from animal tissues. J. biol. Chem. 226, 497–509 (1957)

    Google Scholar 

  6. Gressel, J., Quail, P.H.: Particle-bound phytochrome: Differential pigment release by surfactants, ribonuclease and phospholipase C. Plant & Cell Physiol. 17, 771–786 (1976)

    Google Scholar 

  7. Holdgate, D.P., Goodwin, T.W.: Quantitative extraction and estimation of plant nucleic acids. Phytochem. 4, 831–843 (1965)

    Google Scholar 

  8. Laurent, T.C., Killander, J.: A theory of gel filtration and its experimental verification. J. Chromatog. 14, 317–330 (1964)

    Google Scholar 

  9. Locascio, G.A., Tigier, H.A., Batlle, A.M. del C.: Estimation of molecular weights of proteins by agarose gel filtration. J. Chromatog. 40, 453–457 (1969)

    Google Scholar 

  10. Lord, J.M., Kagawa, T., Moore, T.S., Beevers, H.: Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J. Cell Biol. 57, 659–667 (1973)

    Article  Google Scholar 

  11. Lowry, O.H., Roberts, N.R., Leiner, K.Y., Qu, M.L., Farr, A.L.: The quantitative histochemistry of brain. I Chemical methods. J. biol. Chem. 207, 1–17 (1954)

    PubMed  Google Scholar 

  12. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)

    PubMed  Google Scholar 

  13. Quail, P.H.: Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L. Planta 123, 223–234 (1975)

    Google Scholar 

  14. Quail, P.H.: Particle-bound phytochrome: The nature of the interaction between pigment and particulate fractions. Planta 123, 235–246 (1975)

    Google Scholar 

  15. Quail, P.H., Marmé, D., Schäfer, E.: Particle-bound phytochrome from maize and pumpkin. Nature (Lond.) New Biol. 245, 189–191 (1973)

    Google Scholar 

  16. Rice, H.V., Briggs, W.R.: Partial characterization of oat and rye phytochrome. Plant Physiol. 51, 927–938 (1973)

    Google Scholar 

  17. Schmidt, G., Thannhäuser, S.J.: A method for the determination of desoxyribonucleic acid, ribonucleic acid and phosphoproteins in animal tissues. J. biol. Chem. 161, 83–89 (1945)

    Google Scholar 

  18. Slater, T.F., Planterose, D.N.: Studies on the particulate components of rat mammary gland. 5. Comparison of large particles from liver and mammary gland. Biochem. J. 74, 584–591 (1960)

    PubMed  Google Scholar 

  19. Singh, C.: A non-chelating property of EDTA and some of its biochemical and biological implications. Indian J. Biochem. Biophys. 8, 45–49 (1971)

    PubMed  Google Scholar 

  20. Tangen, O., Jonsson, J., Orrenius, S.: Isolation of rat liver microsomes by gel filtration. Analyt. Biochem. 54, 597–603 (1973)

    PubMed  Google Scholar 

  21. Tangen, O., Berman, H.J., Marfey, P.: Gel filtration: A new technique for separation of blood platelets from plasma. Thrombos. Diathes. haemorrh. (Stuttg.) 25, 268–278 (1971)

    Google Scholar 

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  1. Department of Botany, University of Reading, Whiteknights, RG6 2AS, Reading, UK

    Ann M. Jose

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  1. Ann M. Jose
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Jose, A.M. Gel filtration of particle-bound phytochrome. Planta 134, 287–293 (1977). https://doi.org/10.1007/BF00384195

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  • DOI: https://doi.org/10.1007/BF00384195

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