Use of sulfhydryl-directed inhibitors in vitro to distinguish activities of the mitochondrial and plastidic forms of pyruvate dehydrogenase.
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Amino Acid Sequence↗
Biomedical subjects
M L Johnston
Publications and source records attributed to M L Johnston.
Import, processing, and assembly of the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase.
Sequence comparisons were used to identify cDNAs potentially encoding the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase. Coupled in-vitro transcription plus translation was used to synthesize radiolabeled pyruvate dehydrogenase alpha- and beta-subunit precursor proteins. When the precursors were incubated with intact pea (Pisum sativum L.) seedling chloroplasts in the presence of ATP, they were imported and proteolytically processed. In contrast, there was no import or processing of the precursors by isolated, intact pea seedling mitochondria. Monospecific antibodies to the recombinant pyruvate dehydrogenase alpha-subunit were additionally able to co-precipitate radiolabeled pyruvate dehydrogenase beta-subunit, indicating association between subunits after import and processing. Furthermore, size-exclusion chromatography was used to identify an alphabeta heterodimer that is an intermediate in the assembly of the native alpha2beta2 heterotetrameric enzyme.
Chloroplasts↗
Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits.
Herein we report the first molecular description of the pyruvate dehydrogenase component of the higher plant plastid pyruvate dehydrogenase complex. The full-length cDNAs for the E1 alpha (1530 bp) and E1 beta (1441 bp) subunits of the Arabidopsis thaliana plastid pyruvate dehydrogenase contain open reading frames that encode polypeptides of 428 and 406 amino acids, respectively, with calculated molecular weight values of 47,120 and 44,208. The deduced amino acid sequences for Arabidopsis plastid E1 alpha and E1 beta have 61% and 68% identity to the odpA and odpB genes of the red alga Porphyra purpurea, respectively, but only 31% and 32% identity to the plant mitochondrial counterparts. Results of Southern analyses suggest that each subunit is encoded by a single gene. Northern blot analyses indicate expression of mRNAs of the appropriate size in Arabidopsis leaves.
Amino Acid Sequence↗