Structure-function studies on phytochrome. Preliminary characterization of highly purified phytochrome from Avena sativa enriched in the 124-kilodalton species.

Abstract

A new protocol for the purification of phytochrome from etiolated Avena sativa L. seedlings in 15-25% yield is described. This preparation exhibits molecular properties dissimilar to those previously reported from oats. Phytochrome prepared by this method contains a dominant species having a Mr = 124,000 (70% of total) on sodium dodecyl sulfate-polyacrylamide gels, in addition to a chromoprotein species with Mr = 118,000 (30%). A blocked NH2 terminus is indicated by amino acid sequencing of the purified chromoprotein preparation. By ultraviolet-visible spectroscopy, this preparation displays a 730-nm absorption maximum for Pfr, an enhanced APfr730/APfr668 ratio under saturating red light illumination, and a reduced amount of dark reversion. Circular dichroism reveals a new absorption band for Pfr in the blue spectral region. These spectral properties and electrophoretic behavior are consistent with available information on phytochrome in vivo or in rapidly prepared extracts from oat seedlings. Thus, the molecular properties described here may more accurately reflect those of phytochrome in vivo in etiolated A. sativa shoots than those of phytochrome species isolated by previous methodologies.