Photoaffinity labeling of fatty acid-binding proteins involved in long chain fatty acid transport in Escherichia coli.
The photoreactive fatty acid 11-m-diazirinophenoxy-[11-3H]undecanoate was shown to be taken up specifically by the 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 was labeled by the probe, confirmed to be exclusively in the outer membrane and to exhibit the heat modifiable behavior typical of outer membrane proteins. The apparent pI of the incompletely denatured form of the protein having the mobility of a 33-kDa protein was 4.6 while that of the fully denatured form was consistent with the calculated value of 5.2. The denaturation was reversible depending upon the protein to detergent ratios. The photoreactive fatty acid partitions into the outer membrane, resulting in extensive photolabeling of the lipid; a high affinity fatty acid-binding site is not apparent in total membranes labeled using free fatty acids due to this large binding capacity of the outer membrane. However, when the free fatty acid concentration was controlled by supplying it as a bovine serum albumin complex, the fadL protein exhibited saturable high affinity fatty acid binding, having an apparent Kd for the probe of 63 nM. The methods described very readily identify fatty acid-binding proteins: the fact that even when the sensitivity was increased 500-fold, no evidence was found for the presence of a fatty acid-binding protein in the inner membrane is consistent with the proposal that fatty acid permeation across the plasma membrane is not protein mediated but occurs by a simple diffusive mechanism.
Mangroo
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Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Gerber
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D000345:Affinity Labels; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009363:Neoplasm Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism*
D000345:Affinity Labels; D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009829:Oleic Acids; D019301:Oleic Acid; D005947:Glucose