Characterization of the high-affinity uptake of fructose-1,6-bisphosphate by cardiac myocytes
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Previously, we reported that fructose-1,6-bisphosphate (FBP) was taken up by rat cardiac myocytes by two processes: a component that was saturable at micromolar levels and a nonsaturable component that dominated at millimolar levels. Here, we continued to characterize the saturable high-affinity component, with the aim of identifying the physiological substrate and role for this activity. ATP, ADP, and AMP inhibited the uptake of FBP with apparent affinities of 0.2–0.5 mM. Fumarate and succinate were very weak inhibitors. Several phosphorylated sugars (ribulose-1,5-phosphate, fructose-1-phosphate, ribose-5-phosphate, and inositol-2-phosphate) inhibited FBP uptake with apparent affinities of 40–500 μM. As in our previous study, no tested compound appeared to bind as well as FBP. The data suggest that the best ligands have two phosphoryl groups separated by at least 8 Å. The rates of FBP uptake were measured from 3° to 37°. The calculated activation energy was 15–50 kJ/mol, similar to other membrane transport processes. Uptake of FBP was tested in several types of cells other than cardiac myocytes, and compared to the uptake of 2-deoxyglucose and l-glucose. While FBP uptake in excess of that of l-glucose was observed in some cells, in no case was the uptake as high as in cardiac myocytes. The physiological substrate and role for the high-affinity FBP uptake activity remain unknown.
KeywordsCardiac myocytes Fructose-1,6-bisphosphate Transport Adenine nucleotides Dicarboxylate Activation energy
We thank the following colleagues for providing cells: Vilius Stribinskis, Ph.D., and Kenneth Ramos, Ph.D., mouse aortic smooth muscle cells; Barbara Clark, Ph.D., MA-10 cells and Y-1 cells; Sadhak Sengupta, Ph.D., and Thomas Mitchell, Ph.D., mouse splenocytes; Carolyn Klinge, Ph.D., A549, MCF-7, and human umbilical vein epithelial cells; and Stephanie Webb, Ph.D., and Russell Prough, Ph.D., mouse hepatocytes. Preliminary experiments related to measuring uptake in various cells were performed by Dana Ho, who was supported by the Kentucky Biomedical Research Infrastructures Network undergraduate summer research program. We also thank Mary Anne Hauck for technical assistance and Robert D. Gray, Ph.D., for advice concerning molecular structures. This study was supported in part by National Institutes of Health grant HL64186.
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