Abstract
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.
Highlights
We describethe purification and the characteriza- able to use lipoate plusMgATP as a substrate in E. coli
Al-We report herethe further purification and characterization of lipoyltransferase from bovine liver mitochondria
Purification of Lipoyltransferase-Bovine liver mitochondria protein-Sepharosecolumnchromatographiesasdescribed for lipoyl- were suspended in potassium phosphate buffer, and extracts transferase I.Active fractions were applied to TaSK-gel HA-1000 were prepared anadpplied directlyto hydroxylapatite columns
Summary
Materials dodecyl sulfate-polyacrylamidegel electrophoresisand BSA’ (fatty acid-free),AMP,ATP, hexanoic anhydride, decanoic angel exclusionchromatography (40m a ) ;and ( c )catalytic hydride, and butyl-agarose were obtained from Sigma. 2-Propylpenproperties The phagemid vectorspTZ-BH and pTZ-mBH were linearized, transcripted, and translated in the presence of ~-[~~Slmethioniansedescribed previously[5].The lipoylation reaction was carried out8ipnl of solution containing 1 pl of the translated products, 40 mM potassium phosphate buffer, pH 7.4, p1~0MnCI,, 0.2mg/ml BSA, 0.1m~ lipoylAMP, and purified lipoyltransferase a t 37 “C for 2 h. Lyzed against buffer C(20mM potassium phosphatebuffer, pH 7.4,l mM DTT, 10 PM amidino-PMSF, 10 PM MnCI,, 10% glycerol, 0.15% CHAPS), and applied to an apoH-protein-Sepharose column (0.8x 3 cm) equilibrated with buffer C. The active fractions (about 120 mM NaCI) were pooled, concentrated with a n Amicon Y M l O membrane, applied to a Sephacryl S-200HR column (1.5x 90 cm) equilibratedwithbufferD (30mM potassium phosphate buffer, pH 7.4,1 mM DTT, 10 pv amidino-PMSF, 10 p~ MnCI,, 10% glycerol, 200 mM NaCI, 0.15% CHAPS), and developed with buffer D.
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