In situ binding of fatty acids to the liver fatty acid binding protein: analysis using 3-[125I]iodo-4-azido-n-hexadecylsalicylamide

David W. Waggoner, Joan A. Manning, Nathan M. Bass, David A. Bernlohr

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21 Scopus citations

Abstract

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 solublize fatty acids as a step in their metabolic utilization.

Original languageEnglish (US)
Pages (from-to)407-415
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume180
Issue number1
DOIs
StatePublished - Oct 15 1991

Bibliographical note

Funding Information:
We wish to thank Tom Krick for his expert technical assistance with the gas chromatographic and mass spectral analysis of AHS. We also thank Dr. Harvey Sharp and Kathy Cleary (Department of Pediatrics, University of Minnesota Medical School) for providing the primary rat hepatocytes, and Dr. Clifford Steere (Department of Medicine, University of Minnesota Medical School) for providing Hep G2 cells. This work was supported in part by NIH grants P30-DK3493t andDK32926 to N.M.B. and

Funding Information:
grants from the American Diabetes Association and the National Science Foundation (DMB 8552942) to D.A.B.

Keywords

  • 4-azidohexadecyl salicylamide
  • AHS
  • ALBP
  • CoA
  • DMEM
  • Dulbecco's modified Eagle's medium
  • EDTA
  • FABP
  • FAS
  • PAGE
  • PMSF
  • SDS
  • adipocyte lipid binding protein
  • coenzyme A
  • ethylene diaminetetraacetic acid
  • fatty acid binding protein
  • fatty acid synthase
  • phenylmethylsulfonylfluoride
  • polyacrylamide gel electrophoresis
  • sodium dodecyl sulfate

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