Purification of Oat and Rye Phytochrome

Abstract

Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10-to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.

Partial Characterization of Oat and Rye Phytochrome

“…As noted previously (51,53), rye preparations reported by Correll et al (19) 58,000 to 62,000 for oat (38). Correll et al.…”

“…Evidence has been presented elsewhere (12,24,51,53) that apparent differences between purified oat and rye phytochrome are at least partly a consequence of proteolysis during the isolation and purification of oat phytochrome. Further evidence favoring this thesis is provided in the present work by additional comparison of oat phytochrome with rye phytochrome subjected to mild proteolysis, either with commercial proteases or with an oat shoot protease described by Pike and Briggs (43).…”

Biliproteins

“…Monomeric phytochrome from oats and rye has a molecu lar weight of about 120000* 200,2011 and it probably has a dumbbell shape* 203,2041 (for reviews see * L ,4 -18 -211 ). The hither to best characterized pigment from oats is readily cleaved by endogenous proteases (at the incision?…”

Western blot analysis of a lytic process in vitro specific for the red light absorbing form of phytochrome

“…Phytochrome, which has been isolated from several species as a soluble protein [1][2][3][4], is regarded as the photoreceptor of all photoresponses induced by red light and reversed by far-red light, as well as of photoresponses with the characteristics of the so-called high intensity reaction (review [5]). Several examples of phytochrome-regulated gene expression have been recognized [6][7][8].…”

“…Perhaps loss of the native protein structure soon after extraction is the reason of these failures. Undoubtedly, this applies to the 60 000 Mr species purified after extensive proteolysis [1]. But also preparations termed 'undegraded phytochrome' seem to have undergone limited degradation, for after discontinuous electrophoresis, they showed a heterogeneous band pattern with main bands at 114000 and 118000 Mr [2,4,9].…”