INTERACTION OF ALDH WITH MITOCHONDRIAL IMPORT APPARATUS

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DESCRIPTION (from Applicant's Abstract): Unlike ethanol which is primarily oxidized in liver cytosol, acetaldehyde formed during the metabolism of ethanol is oxidized in the matrix space of the mitochondria by the action of aldehyde dehydrogenase. The nuclear coded enzyme, after being synthesized on free ribosomes, must be translocated into the mitochondria where it is processed to the mature protein by the action of a protease. The information for targeting and processing is located in an N-terminal extension of amino acids, called a leader sequence. The structure of the leader has been determined by 2DNMNR in a micellar milieu. Because of the inherent structural flexibility of the leader sequence, it is not known if this is the structure that is presented to each of the targets that binds the peptide. These are the outer membrane, the outer membrane receptor/translocator and the processing protease. It has been possible to identify some components of the import machinery using antigenic and cross-linking experiments. The search for additional components will be continued. The proteins will be cloned, expressed and reconstituted into liposomes. The interaction of synthetic peptides corresponding to those from human, beef, and rat liver aldehyde dehydrogenase will be studied when bound to the liposomes, reconstituted translocator/receptor components and processing peptidase. The structure and dynamics of the peptides, when bound, will be deduced from NMR spectroscopic data using transferred NOE experiments and relaxation rates. The effect of ethanol on mitochondrial import will be reinvestigated to determine if it inhibits import of proteins other than aldehyde dehydrogenase and how it inhibits the import of aldehyde dehydrogenase. A complete understanding of ethanol and acetaldehyde oxidation required that all aspects of the enzymology be understood, including gene regulation, reaction mechanisms and targeting to the proper organelle.