In situ binding of fatty acids to the liver fatty acid binding protein: analysis using 3-[125I]iodo-4-azido-N-hexadecylsalicylamide.
A photoactivatable radioiodinated fatty acid analogue, 3-[125I]iodo-4-azido-N-hexadecylsalicylamide (125I-AHS) has been synthesized and used to investigate the involvement of cellular lipid carriers in hepatic fatty acid utilization. Photoactivation of Hep G2 internalized 125I-AHS revealed that several cellular proteins were crosslinked with the radiolabeled fatty acid analogue. Three predominant proteins in the membrane fraction of the cell with molecular masses 17, 50 and 127 kDa were crosslinked with the lipid analogue, as determined using autoradiography after SDS-PAGE. Three other proteins in the soluble fraction of the cell, with molecular masses 14, 24 and 35 kDa, were also labeled in situ. In contrast to the other labeled proteins, the fatty acid analogue accumulated on the cytoplasmic 14 kDa protein in a time and temperature dependent fashion. The in situ-labeled 14 kDa protein was identified from primary rat hepatocytes as the liver fatty acid binding protein by partial purification and its ability to be immunoprecipitated with immunospecific L-FABP antiserum. Collectively the results indicate that fatty acids traverse the plasma membrane and are bound cytoplasmically by the liver fatty acid binding protein, as well as other proteins in the cell. This represents the first demonstration in intact hepatocytes that the liver fatty acid binding protein participates in the process of intracellular fatty acid trafficking, and supports a model in which cytoplasmic lipid carriers solubilize fatty acids as a step in their metabolic utilization.
Department of Biochemistry, University of Minnesota, St. Paul 55108.
D W
DW
Waggoner
J A
JA
Manning
N M
NM
Bass
D A
DA
Bernlohr
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001386:Azides; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D051381:Rats; D012457:Salicylamides
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D007457:Iodine Radioisotopes; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D012457:Salicylamides; C064906:3-iodo-4-azido-N-hexadecylsalicylamide