CAH1 contains an intramolecular disulphide bridge. (A) ClustalW2 [47] protein sequence analysis of Arabidopsis α-CA homologues. Cysteine residues at positions 27 and 191 (red) in CAH1 sequence are conserved (*) in seven of the eight homologues described by Fabre et al. [4]. CA8 is substantially larger than CA1-7, possibly indicating that this gene product is not a functional α-CA or belonging to another group of proteins. (B and C) Proteins from Arabidopsis suspension protoplasts expressing HA-tagged wt CAH1 (HC) or C1+C3 double mutant were separated under reducing (+) and non-reducing (−) conditions (with and without 2-mercaptoethanol, 2-ME) and probed with HA antibodies. 2-ME from the reduced sample (+) is diffusing into the middle lane (−), affecting the migration of the non-reduced sample. The C1+C3 double mutant is completely insensitive to reducing agents, since addition of 2-ME to the sample buffer had no effect on the migration pattern of the double mutant, as observed for the HC protein.
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