Amplification and substantial purification of cardiolipin synthase of Escherichia coli.
A simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed. This measures the radioactivity of glycerol formed from phosphatidyl [2-3H]glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme. Cardiolipin synthase was amplified 760-fold upon induction with isopropyl beta-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter. Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency. The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10,000-times higher than that of wild-type whole cell lysate. This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography. This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E. coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.
Hiraoka
S
S
Department of Biochemistry, Saitama University.
Nukui
K
K
Uetake
N
N
Ohta
A
A
Shibuya
I
I
D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D005784:Gene Amplification; D005798:Genes, Bacterial; D007700:Kinetics; D008564:Membrane Potentials; D008565:Membrane Proteins; D017830:Octoxynol; D010743:Phospholipids / Q000032:analysis / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011092:Polyethylene Glycols / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013104:Spheroplasts / Q000201:enzymology / Q000502:physiology; D017855:Transferases (Other Substituted Phosphate Groups)
D003902:Detergents; D008565:Membrane Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase