We will extend our previous work on NAD+-dependent dehydrogenases by (1) completing the primary structure of human 17Beta-estradiol dehydrogenase, (2) obtaining full length cDNA clones for pig cytoplasmic(s) and mitochondrial (m) malate dehydrogenase (MDH), (3) obtaining a full length cdNA clone for pig Beta-hydroxyacyl Coa dehydrogenase (BetaHADH), (4) identifying and sequencing the corresponding genes for these three proteins, and (5) isolating, characterizing and obtaining a full length cDNA clones for B. subtilis MDH and the two MDH isozymes for Tetrahymena pyriformis. We will also continue sequence studies on porcine heart fumarase with the view of obtaining sufficient sequence data to construct oligonucleotide probes to obtain a full length cDNA and ultimately a genomic clone for this molecule as well. In addition to extending our structural knowledge of these molecules, the cloning experiments will provide the opportunity for the preparation of precursor molecules and for genetically altered (in vitro mutagenized) molecules for further characterization of the enzymes and proteins involved in intracellular transfer. Two enzymes that play major roles in the co- and post-translational modification of the majority of proteins are methionine aminopeptidase, which removes the initiator methionine, and protein N-acetyltransferase, which adds and Nalpha-acetyl group to most proteins. Both enzymes will be isolated, characterized and eventually cloned. Studies on their respective specificities will utilize a variety of synthetic peptides and active site structures will be elucidated. Proteins destined to be imported into the mitochondria also have distinctive features including, in many cases, an amino terminal extension. Initial interaction with the mitochondrion occurs via a """"""""receptor"""""""" located in the outer membrane followed by translocation to the inner membrane and matrix. Excision of the extra sequence occurs by a metalloprotease located in the matrix. We will identify and characterize both the receptor molecule(s) and the protease(s) involved in the processing and translocation of mMDH, BetaHADH and fumarase. Monoclonal antibodies that will be prepared against the receptor molecules and ultimately cDNA clones will be identified. We will also continue to sequence chicken phosphoglycerate mutase, C. vinosum hemoprotein and spinach chloroplast fructose-P2-aldolase.
Showing the most recent 10 out of 28 publications
