Abstract
A single polypeptide is immunospecifically precipitated by monospecific antiphytochrome from the total translation products of both wheat-germ and rabbit-reticulocyte cell-free protein synthesizing systems programmed with oat (Avena sativa L.) poly(A) RNA. The mobility of this polypeptide is slightly lower on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than that of immunoaffinity-purified, 118 kdalton phytochrome and corresponds to an apparent molecular weight of 124 kdalton. Evidence against the possibility that this mobility difference results from intracellular processing of the 124-kdalton protein is provided by extraction of freeze-dried tissue directly into boiling SDS-containing buffer. This procedure yields a phytochrome species with a mobility on SDS polyacrylamide gel electrophoresis indistinguishable from that of the in-vitro translation product. Together the data indicate that the phytochrome polypeptide is synthesized in its mature form in the cell but is subject to modification to a form with lower apparent molecular weight during immunopurification.
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Abbreviations
- IgG:
-
immunoglobulin G
- PAGE:
-
polyacrylamide gel electrophoresis
- SDS:
-
sodium dodecyl sulfate
References
Bantle, J.A., Maxwell, I.H., Hahn, W.E. (1976) Specificity of oligo(dT)-cellulose chromatography in the isolation of polyadenylated RNA. Anal. Biochem. 72, 413–427
Blackburn, P. (1979) Ribonuclease inhibitor from human placenta: rapid purification and assay. J. Biol. Chem. 254, 12484–12487
Blobel, G. (1980) Intracellular protein topogenesis. Proc. Natl. Acad. Sci. USA 77, 1496–1500
Bolton, G.W., Quail, P.H. (1981) Cell free synthesis of Avena phytochrome. (Abstr.) Plant Physiol. 67, Suppl., 130
Heijne, G. von (1980) Trans-membrane translocation of proteins. Eur. J. Biochem. 103, 431–438
Hunt, R.E., Pratt, L.H. (1979) Phytochrome immunoaffinity purification. Plant Physiol. 64, 332–336
Hunt, R.E., Pratt, L.H. (1980) Partial characterization of undegraded oat phytochrome. Biochemistry 19, 390–394
Ivarie, R.D., Jones, P.P. (1979) A rapid sensitive assay for specific protein translations: use of Staphylococcus aureus as an adsorbent for immune complexes. Anal. Biochem. 97, 24–35
Kessler, S.W. (1975) Rapid isolation of antigens from cells with a Staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol. 115, 1617–1624
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685
Lagarias, J.C., Rappaport, H. (1980) Chromopeptides from phytochrome. The structure and linkage of the Pr form of the phytochrome chromophore. J. Am. Chem. Soc. 102, 4821–4828
Laskey, R.A. (1980) The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. Methods Enzymol. 65, 363–371
Lizardi, P.M. (1980) Isolation of giant silk fibroin polysomes and fibroin mRNP particles using a novel ribonuclease inhibitor, hydroxystilbamidine. J. Cell Biol. 87, 292–296
Lizardi, P.M., Engelberg, A. (1979) Rapid isolation of RNA using proteinase K and sodium perchlorate. Anal. Biochem. 98, 116–122
Marcu, K., Dudock, D. (1974) Characterization of highly efficient protein synthesizing system derived from commercial wheat germ. Nucl. Acids Res. 1, 1385–1397
McLeester, R.C., Hall, T.C. (1977) Simplification of amino acid incorporation and other assays using filter paper techniques. Anal. Biochem. 79, 627–630
Mohr, H. (1972) Lectures on photomorphogenesis. Springer, Berlin Heidelberg New York
Pelham, H.R.B., Jackson, R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247–256
Pratt, L.H. (1979) Phytochrome: function and properties. Photochem. Photobiol. Rev. 4, 59–124
Pratt, L.H. (1980) Phytochrome pelletability induced by irradiation in vivo. Test for in vitro binding of added [35S]phytochrome. Plant Physiol. 66, 903–907
Pratt, L.H. (1982) Phytochrome: the protein moeity. Annu. Rev. Plant Physiol. 33, 557–582
Quail, P.H., Briggs, W.R. (1979) Immunoaffinity purification of phytochrome. Carnegie Inst. Washington Yearb. 78, 126–128
Rice, H.V., Briggs, W.R., Jackson-White, C.J. (1973) Purification of oat and rye phytochrome. Plant Physiol. 51, 917–926
Sabatini, D.D., Kreibich, G., Morimoto, T., Adesnik, M. (1982) Mechanisms for the incorporation of proteins in membranes and organelles. J. Cell Biol. 92, 1–22
Scheele, S., Blackburn, P. (1979) Role of mammalian RNase inhibitor in cell-free protein synthesis. Proc. Natl. Acad. Sci. USA 76, 4898–4902
Shields, D., Blobel, G. (1978) Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. J. Biol. Chem. 253, 3753–3756
Smith, H. (1975) Phytochrome and photomorphogenesis. McGraw-Hill, London
Vierstra, R.D., Quail, P.H. (1982) Native phytochrome: inhibition of proteolysis yields a homogeneous monomer of 124 kdalton from Avena. Proc. Natl. Acad. Sci. USA (in press)
Wickner, W. (1979) The assembly of proteins into biological membranes: the membrane trigger hypothesis. Annu. Rev. Biochem. 48, 23–45
Zurfluh, L.L., Guilfoyle, T.J. (1982) Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl. Plant Physiol. 69, 332–337
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Bolton, G.W., Quail, P.H. Cell-free synthesis of phytochrome apoprotein. Planta 155, 212–217 (1982). https://doi.org/10.1007/BF00392718
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DOI: https://doi.org/10.1007/BF00392718