Proteolytic removal of the C-terminal transmembrane region of cytochrome f during extraction from turnip and charlock leaves generates a water-soluble monomeric form of the protein.
Water-soluble, monomeric cytochrome f purified from leaves of turnip (Brassica rapa) and charlock (Sinapis arvensis) is approximately 3 kDa smaller than the protein in chloroplast thylakoid membranes determined by SDS/PAGE. Sequencing the N-terminal and C-terminal regions of the monomeric protein, by automated Edman degradation and carboxypeptidase P digestion, suggested the loss of 33 amino acid residues at the C-terminus by comparison to sequences of cytochrome f from other higher plants. This was confirmed by the isolation and nucleotide sequencing of the turnip petA gene and by determination of the molecular mass of the monomeric turnip protein by electrospray mass spectrometry. The turnip petA gene encodes a protein of 320 amino acid residues consisting of a presequence of 35 amino acid residues and a mature protein of 285 amino acid residues. The molecular mass of the monomeric turnip protein was 28,160.2 +/- 5.4 Da, indicating cleavage after Gln252 of the mature protein. Electrospray mass spectrometry of the monomeric charlock protein indicated the presence of two main forms with molecular masses of 28,135.1 +/- 5.5 Da and 27,750.7 +/- 4.3 Da corresponding to cleavage after Gln252 and Leu249, respectively. Cleavage in this region of the cytochrome f polypeptide during extraction with butanone removes the single transmembrane span of the protein and liberates the water-soluble globular domain of cytochrome f.
Gray
J C
JC
Cambridge Centre for Molecular Recognition, University of Cambridge, England.
Rochford
R J
RJ
Packman
L C
LC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology*; D002736:Chloroplasts / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D045348:Cytochromes f; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012995:Solubility
D003580:Cytochromes; D045348:Cytochromes f; D007930:Leucine; D005998:Glycine