Photochemistry of 124 Kilodalton Avena Phytochrome In Vitro

Abstract

The photochemical proprties of purified 124 kilodalton (kD) Avena cv Garry phytochrome are examined and compared with those of the proteolyticaily degraded 118/114 kD species. The proportion of the chromoprotein in the far red absorbing form, Pfr, foflowing saturating red irradiation is 0.86 for 124 kD phytochrome, substantially higher than the vahes of 0.79 determined here and 0.75 reported in the literature for 118/114 kD preparations. The ratio of the quantum yields for Pr to Pfr phototransformation and for Pfr to Pr phototransformation (Or/f1r) is 1.76 for the 124 kD molecule and 0M for the 118/114 kD species. Based on extintion coefficients determined using the Lowry assay as a measure of protein weight, the individual phototransformation quantum yields for 124 kD phytochrome are 0.17 for Pr -) Pfr (or) and 0.10 for Pfr -* Pr (#fr).Comparison of these quantum yields with those of the 118/114 kD species (where 4r = ofr --0.11) indicates that proteolytic degradation of the 124 kD molecule to the 118/114 kD species signifcantiy affects only #r.

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

“…Conversion to the 118 000/114 000 M r species is inhibited by a saturating dose of red light (665 nm) given either before ( fig.2c) or after extraction ( fig.2d) and restored by a subsequent 730 nm light pulse ( fig.2e). This is in agreement with a recent note that a 124 000 Mr species was extracted from shortly irradiated seedlings [10].…”

“…Crude extracts of fresh dark-grown seedlings showed one major band at 123 000 Mr ( fig. l) that is presumably identical with the native phytochrome species observed after extraction in the presence of SDS [10] or after cell-free translation [11].…”

“…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]. These bands, however, were neither observed after extraction of seedlings with denaturing buffer [10] nor after cell-flee translation of the phytochrome message [11]. In both Abbreviations: PMSF, phenylmethyl-suifonyl fluoride; SDS, sodium dodecyl sulfate; Pr, red light-absorbing form of phytochrome; Pfr, far-red light-absorbing form of phytochrome cases a single phytochrome band at -124 000 Mr was reported.…”

Comparison of Spectral Properties of Phytochromes from Different Preparations

“…The spectral parameters of purified phytochrome determined in recent studies (3,8,15,17,18) are different from those reported by Butler et al (2). Two factors are probably responsible for these differences.…”

“…Two factors are probably responsible for these differences. The phytochrome samples used in the old studies (1,2) contained a high proportion of proteolytically degraded 60 kD phytochrome, while most recent preparations consist mainly of undegraded ('native') 124 kD phytochrome (8,17,18). Recent preparations are purer, as suggested by values of the A665/A280 specific absorbance ratio for Pr much higher than that reported for the old phytochrome preparations (1,2,14).…”