Purification and characterization of lipoyl-AMP:N epsilon-lysine lipoyltransferase from bovine liver mitochondria.
Lipoyl-AMP:N epsilon-lysine lipolytransferase (lipolytransferase) catalyzes the transfer of the lipoyl group from lipoyl-AMP to a lysine residue of the specific enzyme proteins. We have shown previously that the lipoyltransferase activities locate in mitochondria using apoH-protein of the glycine cleavage system as an acceptor of the lipoyl group (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1990) J. Biol. Chem. 265, 17463-17467). Here we describe the purification and the characterization of two isoforms of lipolytransferase termed lipoyltransferase I and lipoyltransferase II from bovine liver mitochondria. Lipoyltransferase II was purified to apparent homogeneity, whereas the final product of lipoyltransferase I still contained a minor contaminant. Although the two forms could be resolved on a hydroxylapatite column chromatography, they were indistinguishable, as judged by: (a) behavior during purification on ion exchange, hydrophobic, or affinity columns; (b) molecular mass determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography (40 kDa); and (c) catalytic properties (substrate specificity, kinetic constants, and optimal pH). Both lipoyltransferase I and II could not use lipoic acid plus MgATP as a substrate in place of lipoyl-AMP. Surprisingly, the lipoyltransferases transferred not only the lipoyl group but also the acyl groups from hexanoyl-, octanoyl-, and decanoyl-AMP to apoH-protein to a similar extent.
Fujiwara
K
K
Institute for Enzyme Research, University of Tokushima, Japan.
Okamura-Ikeda
K
K
Motokawa
Y
Y
D000217:Acyltransferases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D017886:Durapatite; D000217:Acyltransferases; C077290:H-protein lipoyltransferase