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Spectral characterization and proteolytic mapping of native 120 kilodalton phytochrome from cucurbita pepo

, : Spectral characterization and proteolytic mapping of native 120 kilodalton phytochrome from cucurbita pepo. Plant Physiology (Rockville) 77(4): 990-998

A spectral, immunochemical and proteolytic characterization of native 120-kilodalton (kD) phytochrome from C. pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena sativa. The molecule was partially purified (.apprx. 200-fold) in the phytochrome.sbd.far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Avena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr.lambda.max at 730 nm a spectral change ratio (.DELTA.Ar/.DELTA.Afr) of 1.05 and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrast with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cucurbita phytochrome is immunologically dissimilar to 124-kD Avena phytochrome. Despite this dissimilarity, immunoblot analyses of proteolytic digests identified at least 3 spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide may for Cucurbita phytochrome was constructed and the role that specific domains play in the overall structure of the photoreceptor was examined. One domain near the NH2 terminus is critical to the spectral integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH2 terminus of Avena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr.lambda.max to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOH-terminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers.

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