Ammo-Terminal Amino Acid Sequences of Pea Phytochrome II Fragments Obtained by Limited Proteolysis

Apical shoot of pea seedling contains two immunochemically distinctspecies of phytochrome, type I (PI) and type II (PII) [Abe etal. (1985) Plant Cell Physiol. 26: 1387-1399]. A PII samplewas prepared from crude extracts from apical shoot of 7-day-old,light-grown pea (Pisum sativum L. cv. Alaska) seedlings, usingbrushite, DEAE-agarose and an agarose linked monoclonal anti-peaPII antibody (mAPll) column chromatography. More than 75% ofthe PII sample consisted of one major 115 kDa chromopeptide,and less than 25% of PII was composed of two minor non-chromophoric49 and 48 kDa fragments, as judged by SDS-polyacrylamide gelelectrophoresis and zinc-induced chromophore fluorescence analysis.Limited proteolysis of PII was performed with either trypsinor Staphylococcus aureus V8 protease, and the resulting fragmentswere separated by SDS-polyacrylamide gel electrophoresis andblotted electrophoretically onto a polyvinylidene difluoridemembrane. Amino-terminal amino acid sequences of the four fragmentsof PII cut from the membrane were determined by a gas-phasesequencer. The sequence of the determined regions (76 aminoacid residues in total), reveals that PII was 63% homologouswith PI. Hence the primary structure of PII is distinct fromthat of PI. (Received July 21, 1989; Accepted September 1, 1989)