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Location of Cysteine and Cystine Residues in S ‐Ribonucleases Associated with Gametophytic Self‐Incompatibility

1996; Wiley; Volume: 242; Issue: 3 Linguagem: Inglês

10.1111/j.1432-1033.1996.0627r.x

1432-1033

Takeshi Ishimizu, Shigemi Norioka, Michiko Kanai, Adrienne E. Clarke, Fumio Sakiyama,

Plant tissue culture and regeneration

S ‐Ribonucleases (S‐RNases) that cosegregate with S ‐alleles in the styles of solanaceous and rosaceous plants are associated with gametophytic self‐incompatibility (GSI). The amino acid sequences of many S ‐RNases have been derived from cDNA sequences, but the state of half‐cystines has not been clarified. We report the locations of the two free cysteine residues and four disulfide bridges of tobacco S 6 ‐RNase and of the four disulfide bridges of Japanese pear S 4 ‐RNase. The protein was first S ‐pyridylethylated at a low pH to selectively modify the free cysteines without thiol‐disulfide exchange. The S ‐pyridylethylated protein (PE‐protein) was digested with Achromabucter protease I (API) at pH 6.5 then analyzed by liquid chromatography/electrospray‐ionization mass spectrometry (LC/ESI‐MS). This analysis showed that tobacco S 6 ‐RNase has two free cysteine residues, Cys77 and Cys95, and four disulfide bonds at Cysl6‐ Cys21, Cys45‐Cys94, Cysl53‐Cysl82 and Cys16S‐Cys176. Similarly, four disulfide bonds were identified for pear S 4 ‐RNase, which bears no free cysteine, at Cys15‐Cys22, Cys48‐Cys92, Cys156‐Cys195 and Cys172‐ Cys183. The eight cysteines forming these four disulfide bonds are conserved in all the known S ‐RNases, indicative that these cross‐links are important in stabilizing the tertiary structures of the self‐incompatibility‐associated glycoproteins in the solanaceous and rosaceous families. The LC/ESI‐MS analysis also provided some structural informations regarding sugar chains attached to the S ‐RNases.