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

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Apical shoot of pea seedling contains two immunochemically distinct species of phytochrome, type I (PI) and type II (PII) [Abe et al. (1985) Plant Cell Physiol. 26: 1387-1399]. A PII sample was prepared from crude extracts from apical shoot of 7-day-old, light-grown pea (Pisum sativum L. cv. Alaska) seedlings, using brushite, DEAE-agarose and an agarose linked monoclonal anti-pea PII antibody (mAP11) column chromatography. More than 75% of the PII sample consisted of one major 115 kDa chromopeptide, and less than 25% of PII was composed of two minor non-chromophoric 49 and 48 kDa fragments, as judged by SDS-polyacrylamide gel electrophoresis and zinc-induced chromophore fluorescence analysis. Limited proteolysis of PII was performed with either trypsin or Staphylococcus aureus V8 protease, and the resulting fragments were separated by SDS-polyacrylamide gel electrophoresis and blotted electrophoretically onto a polyvinylidene difluoride membrane. Amino-terminal amino acid sequences of the four fragments of PII cut from the membrane were determined by a gas-phase sequencer. The sequence of the determined regions (76 amino acid residues in total), reveals that PII was 63% homologous with PI. Hence the primary structure of PII is distinct from that of PI.
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