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|Title:||Fatty acid uptake in Escherichia coli: Development of a photoaffinity labeling approach for identifying proteins involved in the transmembrane movement of long chain fatty acids|
|Advisor:||Gerber, Gerhard E.|
|Abstract:||<p>The photoreactive fatty acid 11-m-diazirinophenoxy-[11-³H]-undecanoate was shown to be taken up specifically by the long chain fatty acid transport system expressed in Escherichia coli grown on oleate. This photoreactive fatty acid analogue was therefore used to identify proteins involved in fatty acid uptake in E. coli. The fadL protein, required for fatty acid permeation of the outer membrane of E. coli was labeled by the probe, confirmed to be exclusively in the outer membrane and to exhibit the heat modifiable behaviour typical of outer membrane proteins. The fatty acid uptake activity in E. coli was reduced upon starvation whereas proline uptake was not affected. The inhibitory effect of starvation was rapidly reversed by the addition of D-lactate, L-lactate, succinate or acetate. The stimulatory effect of D-lactate on oleate uptake increased the affinity of the transport system for fatty acid and the maximal rate of uptake, the Kt values being 37.0 and 10.6 μM and the maximal rates being 0.29 and 2.30 nmole/min/mg of protein in the absence and in the presence of D-lactate, respectively. Investigation into the mechanism of regulation of fatty acyl-CoA synthetase showed that D-lactate did not affect the activity of the enzyme directly. It was shown that fatty acyl-CoA synthetase is co-isolated with the inner membrane in the presence of D-lactate but not in its absence; these results suggest that recruitment of the enzyme to the inner membrane by D-lactate results in its activation and consequently in the increased level of fatty acid uptake. The involvement of fatty acyl-CoA synthetase in fatty acid uptake was studied directly using inner membrane vesicles. The vesicles were characterized functionally by assessing proline uptake. The uptake of proline in vesicles prepared from cells grown on oleate, glucose and palmitate was dependent on the state of energization of the membrane, indicating that these vesicles are functionally comparable to those prepared by others. It was determined that vesicles prepared from cells grown on oleate were destabilized by freeze-thawing which is required for trapping CoASH and ATP, while those prepared from glucose and palmitate grown cells were not. Fatty acid uptake in palmitate vesicles containing fatty acyl-CoA synthetase was dependent on exogenously trapped ATP and CoASH. These results demonstrate that fatty acyl-CoA synthetase is directly involved in fatty acid uptake in E. coli and suggest that the observed effects of starvation and D-lactate on oleate uptake in whole cells are related to regulation of fatty acyl-CoA synthetase. This in vitro system will allow for the study of the recruitment of fatty acyl-CoA synthetase to the inner membrane by D-lactate, succinate, L-lactate, acetate and their metabolites. (Abstract shortened by UMI.)</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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