Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli, and, Identification and characterization of a cDNA clone coding for rat testis pyruvate dehydrogenase E1-alpha isoform
Abstract
The first part of the dissertation deals with studies of the precursor of rat liver mitochondrial aldehyde dehydrogenase (p-ALDH) expressed in E. coli. The E. coli recombinant expressed p-ALDH was isolated as a soluble tetrameric protein and exhibited specific activity and Kms for substrates similar to that of the mature ALDH (m-ALDH). The p-ALDH was less stable than was m-ALDH both in E. coli and in vitro. Thus, the presence of signal peptide served as a destabilizing factor. The p-ALDH expressed in E. coli could bind to and be translocated into rat liver mitochondria, however, with lower efficiency when compared to the import of p-ALDH synthesized in reticulocyte lysate. Both p-ALDH and m-ALDH synthesized in a rabbit reticulocyte lysate were also studied. Both of them were monomers and exhibited no catalytic activity. The second part of this dissertation deals with the cloning of the cDNA coding for rat testis PDH E1$\alpha$. A cDNA clone encoding for the rat testis PDH E1$\alpha$ isoform was isolated and characterized (RTA2.1). This cDNA contained an open reading frame of 1482 nucleotides which coded for a 494 amino acids (54.4 Kd) protein. The RTA2.1 showed a 75.4% similarity to the rat liver PDH E1$\alpha$ isoform from nt484 to nt1659 (corresponding to the coding region of the rat liver PDH E1$\alpha$). No homology was observed beyond this region. An in-frame upstream ATG initiation codon was found which would add an extra 103 amino acids to the N-terminus of the rat testis PDH E1$\alpha$. The proposed TPP-binding motif and the phosphorylation site for PDH-kinase/PDH-phosphatase were conserved. PCR amplifications of rat genomic DNA revealed that the gene coding for RTA2.1 contained no intron in the coding region. The RTA2.1 was subsequently expressed in E. coli. The expressed recombinant protein product was then purified and used to raise antibodies. A 54 Kd protein was recognized by this antibody in a partially purified PDH E1$\alpha$ preparation from rat testis mitochondria. This 54 Kd protein was also recognized by two anti-peptide antibodies raised against the phosphorylation site and a N-terminus 27-mer peptide, respectively.
Degree
Ph.D.
Advisors
Weiner, Purdue University.
Subject Area
Molecular biology|Biochemistry
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