The Tricarboxylate Carrier
The citrate carrier has recently been purified from rat liver mitochondria by several groups using different methods. A 37–38 kDa protein has been prepared by Silica Gel 60 chromatography by our group (Claeys and Azzi, 1989; Glerum et al., 1990). The specific citrate transport activity of this preparation is not significantly different from that measured in mitochondria and it is inhibitable by 1, 2, 3- benzenetricarboxylic acid. Bisaccia et al. (1990) have reported the isolation of a 30 kDa protein by Celite 535 chromatography, and Kaplan et al. (1990) have isolated a 32.5 kDa protein by Matrex Orange, Matrex Blue and Affi-Gel chromatography. The clarification of the structural diversities of the above-described preparations has been one of the objectives of the present work. Peptide mapping, after cleavage of the different protein preparations with Endoproteinase Glu-C (Staphylococcus aureus V8 protease), cyanogen bromide and hydroxylamine, has failed to support any structural homologies. The 38 kD protein is N-terminally blocked. The peptides obtained from enodproteinase Glu-C or hydroxylamine lysis, after separation on SDS-PAGE, and those obtained after CNBr cleavage and HPLC purification have been partially sequenced. The protein sequence information is being used to obtain a full-length cDNA, which will allow further characterisation of the carrier at the molecular level.
KeywordsLiver Mitochondrion Cyanogen Bromide Citrate Transport Protein Sequence Information CNBr Cleavage
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