Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei.
In the long-slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2-oxo-acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism [Danson, M. J., Conroy, K., McQuattie, A. & Stevenson, K. J. (1987) Biochem. J. 243, 661-665]. In the present paper, a complete subcellular fractionation of T. brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane. In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction. A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane. The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T. brucei. Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid-hydrolysed extracts of T. brucei contain 1.7 (+/- 0.2) ng of the cofactor/mg protein. The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry.
Jackman
S A
SA
Department of Biochemistry, University of Bath, England.
Hough
D W
DW
Danson
M J
MJ
Stevenson
K J
KJ
Opperdoes
F R
FR
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007425:Intracellular Membranes / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D008063:Thioctic Acid / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure
D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase