期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1982
卷号：79
期号：17
页码：5272-5276
DOI：10.1073/pnas.79.17.5272
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Phytochrome purified from Avena as the red-absorbing form, Pr, by an established immunoaffinity column procedure is heterogeneous. Two major polypeptides and one minor polypeptide with apparent molecular masses of 118, 114, and 112 kilodaltons (kDal), respectively, are observed on NaDodSO4/polyacrylamide gel electrophoresis. In contrast, only a single band of 124 kDal is obtained when phytochrome is rapidly immunoprecipitated after extraction either (i) as the far-red absorbing form, Pfr, in detergent-free buffer or (ii) in either spectral form in a 100{degrees}C NaDodSO4-containing buffer. On two-dimensional gel electrophoresis the three column-purified species have pIs of 5.8, 6.0, and 6.0, whereas 124-kDal phytochrome is a single spot with a pI of 5.9. Incubation as Pr in extracts causes progressive conversion of the 124-kDal polypeptide to the 118- and 114-kDal species. This process is inhibited by phenylmethylsulfonyl fluoride, suggesting that Pr is susceptible and Pfr resistant to limited proteolysis during extraction. These data, and the fact that the cell-free translation product of phytochrome mRNA is also 124 kDal [Bolton, G. W. & Quail, P. H. (1982) Planta, in press], indicate that the native monomer from Avena is a single species of 124 kDal. Thus the heterogeneous preparations of slightly lower molecular weight ("large" or "120-kilodalton" phytochrome) previously extensively characterized appear to have consisted of a mixture of partially degraded molecules that have undergone limited proteolysis during purification as Pr, as is established practice. A reexamination of the molecular properties of phytochrome appears necessary.
关键词：hot NaDodSO4 extraction ; protease inhibitors ; solid-phase immunoprecipitation ; one- and two-dimensional gel electrophoresis
