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

Abstract

Phytochrome, which has been isolated from several species as a soluble protein [1-4], 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 [6-8]. The photochemical interconversion between the 2 spectrally distinct forms of phytochrome, Pr and Pfr, is the basis of photoreception in vivo and occurs also in cell-flee preparations. However, reactions that might be related to signal transmission have never been observed in vitro. 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]. 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