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

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Abstract
Phytochrome purified from Avena as the red-absorbing form, Pr, by an established immunoaffinity column procedure is heterogeneous. Two major polypeptides and 1 minor polypeptide with apparent MW of 118, 114 and 112 kdaltons (kD), respectively, were observed on sodium dodecylsulfate [SDS]/polyacrylamide gel electrophoresis. Only a single band of 124 kD was obtained when phytochrome was rapidly immunoprecipitated after extraction either as the far-red absorbing form, Pfr, in detergent-free buffer or in either spectral form in a 100.degree. C SDS4-containing buffer. On 2-dimensional gel electrophoresis, the 3 column-purified species have pI of 5.8, 6.0 and 6.0, whereas 124-kD phytochrome is a single spot with a pI of 5.9. Incubation as Pr in extracts causes progressive conversion of the 124-kD polypeptide to the 118- and 114-kD 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 kD (Bolton, et Quail 1982), indicate that the native monomer from Avena is a single species of 124 kD. The heterogeneous preparations of slightly lower MW (large or 120-kD 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.