Characterization of a soluble carnitine acetyltransferase from Candida tropicalis.
Carnitine acetyltransferase was purified from the cytoplasmic fraction of Candida tropicalis grown on alkanes in continuous culture. By ion-exchange chromatography the enzyme was resolved in two fractions with the same specific activity of 80 U/mg. The molecular mass of both enzyme forms, determined by non-denaturing gradient gel electrophoresis, was 540 kDa. After SDS electrophoresis only one band of 64 kDa was detected indicating that both enzymes are oligomers each containing eight subunits. Isoelectric focusing in agarose under non-denaturing conditions demonstrated the presence of at least four different charged species in the pH range between 5.6 and 6.7. After isoelectric focusing in 9 M urea/1% Nonidet P-40 gels, both enzyme forms were resolved into four bands. Peptide mapping, performed by cyanogen bromide cleavage of polypeptides separated by denaturing isoelectric focusing followed by second-dimension SDS electrophoresis, revealed a very high degree of homology between these polypeptides. The presence of the octameric form of carnitine acetyltransferase already in the starting material was demonstrated by non-denaturing gradient gel electrophoresis and immunoblotting. Antibodies against carnitine acetyltransferase from C. tropicalis ATCC 32113 formed precipitation lines with extracts from several Candida species but not with extracts of Candida utilis, Candida ethanothermophilum and an another strain of C. tropicalis.
Kozulić
B
B
Institute of Biotechnology, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Käppeli
O
O
Meussdoerffer
F
F
Fiechter
A
A
D000123:Acetyltransferases / Q000302:isolation & purification*; D000473:Alkanes / Q000378:metabolism; D002175:Candida / Q000201:enzymology*; D002332:Carnitine O-Acetyltransferase / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D007118:Immunoassay; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010449:Peptide Mapping; D012995:Solubility; D013045:Species Specificity
D000473:Alkanes; D000123:Acetyltransferases; D002332:Carnitine O-Acetyltransferase