Identification of surface-exposed parts of red-light- and far-red-light-absorbing forms of native pea phytochrome by limited proteolysis.
Peptide fragments were obtained by limited proteolysis with trypsin and Staphylococcus aureus V 8 protease from either the PR or the PFR form of 121-kDa phytochrome purified from etiolated pea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamide gel electrophoresis in the presence of SDS of the digests were different, with some bands appearing preferentially when the digestions were carried out with the PR or the PFR form. Amino-terminal sequences of the fragments were analyzed to determine the exact locations of the amino-termini of the fragments within the amino acid sequence of the apoprotein of pea phytochrome. The amino acid compositions of some of the sequenced fragments were determined in order to confirm the carboxy-terminal amino acids. Three cleavage regions were identified as kinetically favored sites of cleavage of PFR (Arg-746 to Lys-752, around Glu-877 and around Arg-1010), whereas only one was identified for PR (Glu-38 to Arg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 and Lys-1093 to Glu-1115 were also identified as potential sites of proteolytic cleavage in both forms of the phytochrome. Other cleavage sites, the specificities of which have not yet been determined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the PR and PFR forms are discussed.
Department of Biology, Yokohama City University, Japan.
M
M
Nakazawa
H
H
Hayashi
Y
Y
Yoshida
K
K
Manabe
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010834:Phytochrome / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D010946:Plants, Medicinal; D011336:Probability; D017433:Protein Structure, Secondary; D013499:Surface Properties; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D010834:Phytochrome; D014357:Trypsin