Purification and characterization of catalytically active precursor of rat liver mitochondrial aldehyde dehydrogenase expressed in Escherichia coli.
The cDNA coding for the precursor (p-ALDH) or mature (m-ALDH) rat liver mitochondrial aldehyde dehydrogenase was cloned in an expression vector pT7-7 and expressed in Escherichia coli strain BL21 (DE3)/plysS. The p-ALDH expressed in E. coli was a soluble tetrameric protein. It exhibited virtually the same specific activity and KmS for substrates as m-ALDH. N-terminal sequencing of isolated p-ALDH provided the evidence that the catalytic activity was not derived from a partially processed mature-like enzyme. The assembly states of both p-ALDH and m-ALDH synthesized in a rabbit reticulocyte lysate were also determined. Both of them were monomers and could not bind to a 5'-AMP-Sepharose column, showing that the monomeric form of the enzyme is inactive. The stabilities in vivo and in vitro were compared between p-ALDH and m-ALDH expressed in E. coli. p-ALDH was less stable than was m-ALDH both in vivo and in vitro. Thus, although the conformations of p-ALDH and m-ALDH are similar, the presence of signal peptide is a destabilizing factor to the p-ALDH. 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.
Jeng
J J
JJ
Departmen of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats
D004792:Enzyme Precursors; D004247:DNA; D000444:Aldehyde Dehydrogenase