Isolation and properties of a liver mitochondrial precursor protein to aspartate aminotransferase expressed in Escherichia coli.
The precursor to rat liver mitochondrial aspartate aminotransferase has been expressed in Escherichia coli JM105 using the pKK233-2 expression vector. This mammalian natural precursor has been isolated as a soluble dimeric protein. The amino-terminal sequence and the amino acid composition of the isolated protein correspond to those predicted from the inserted cDNA (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). The isolated precursor contains bound pyridoxal phosphate and shows catalytic activity with a specific activity equal to that of the mature form of the enzyme. This precursor can also be processed by mitochondria into a form with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of mature enzyme. The isolation of this precursor as a stable and catalytically active entity indicates that the presequence peptide does not necessarily interfere with much of the folding and basic structural properties of the mature protein component.
Altieri
F
F
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110.
Mattingly
J R
JR
Rodriguez-Berrocal
F J
FJ
Youssef
J
J
Iriarte
A
A
Wu
T H
TH
Martinez-Carrion
M
M
D000818:Animals; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000378:metabolism*; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D003067:Coenzymes; D011498:Protein Precursors; D001219:Aspartate Aminotransferases