Native phytochrome: Inhibition of proteolysis yields a homogeneous monomer of 124 kilodaltons from Avena

Abstract

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 NaDodSO(4)/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 NaDodSO(4)-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.