3T3 Fibroblasts Transfected with a cDNA for Mitochondrial Aspartate Aminotransferase Express Plasma Membrane Fatty Acid-Binding Protein and Saturable Fatty Acid Uptake

• Published in: Proceedings of the National Academy of Sciences - PNAS 1995-10, Vol.92 (21), p.9866-9870
• Main Authors: Isola, L. M., S.-L. Zhou, C.-L. Kiang, Stump, D. D., Bradbury, M. W., Berk, P. D.
• Format: Article
• Language: English
• Subjects: 3T3 Cells; 3T3 L1 cells; Adipocytes; Animals; Aspartate Aminotransferases - genetics; Aspartate Aminotransferases - metabolism; Biochemistry; Biological Transport - drug effects; Blotting, Western; Carrier Proteins - metabolism; Cell culture techniques; Cell lines; Cell Membrane - metabolism; Cell membranes; Cellular immunity; Cultured cells; Deoxyribonucleic acid; DNA; DNA, Complementary; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty Acids - metabolism; Fibroblasts; Fluorescent Antibody Technique; Membrane Proteins - metabolism; Membranes; Mice; Mitochondria - enzymology; Mitochondria - genetics; Myelin P2 Protein - metabolism; Neoplasm Proteins; Nerve Tissue Proteins; Oils & fats; Plasmids; Proteins; Recombinant Proteins - metabolism; Transfection; Zinc - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.21.9866

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn2+-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 μM bovine serum albumin and 125-1000 μM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax(fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+had no effect in pFR400 controls (P > 0.5). The overall increase in Vmaxbetween pFR400 and pFR400/pMAAT2 in the presence of Zn2+was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpmin plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpmdetectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 μM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.