Spectrophotometric and molecular properties of mutated rice phytochrome A.
A cDNA (PHYA) for the phytochrome A apoprotein (PHYA) of rice and three mutated sequences (phyA S/A, the first ten serine residues in the N-terminal domain of PHYA were changed to alanine residues; phyA ND, the first 80 N-terminal amino acids were deleted; phyA CD, the amino acids of the C-terminal domain from 689 to 1,128 were deleted) were expressed in yeast, and the wild-type and mutant apophytochromes were allowed to combine in vitro with the chromophore phycocyanobilin (PCB). The PCB-attached product of phyA S/A gave very similar spectrophotometric peaks to the PhAr and PhyAfr forms of wild-type product. By contrast, the peak of the product of phyA CD in the Pfr form was significantly shifted towards a shorter wavelength, an indication that, whereas the C-terminal domain is not crucial for the PCB attachment, it greatly influences the absorption maximum of PhyAfr. The rate of 50% reversion from PhyAfr to PhAr in darkness was 3 h at 27 degrees C with all of the samples, showing that the S/A and CD mutations did not affect this property. No photoreversibility was detected with the product of phyA ND. Gel-filtration analysis of the wild-type PHYA and the product of phyA S/A showed that the apparent molecular mass of each was 330 kDa, suggesting that both exists as dimers in solution.
Tomizawa
K
K
Frontier Research Program, Laboratory of Plant Biological Regulation, RIKEN Institute, Saitama, Japan.
Stockhaus
J
J
Chua
N H
NH
Furuya
M
M
D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D010834:Phytochrome / Q000737:chemistry* / Q000302:isolation & purification; D051942:Phytochrome A; D011994:Recombinant Proteins / Q000235:genetics; D013053:Spectrophotometry
D001059:Apoproteins; D017931:DNA Primers; D018076:DNA, Complementary; D051942:Phytochrome A; D011994:Recombinant Proteins; C071907:apophytochrome; D010834:Phytochrome