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Inhibition of Queuine Uptake in Cultured Human Fibroblasts by Phorbol-12,13-didecanoate¹

Department of Microbiology and Immunology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 [M. S. E., J. R. K.], and Department of Physiological Chemistry, Ohio State University, Columbus, Ohio 43210 [R. W. T.]

The modified base queuine is inserted posttranscriptionally into the first position of the anticodon of tyrosine tRNA, histidine tRNA, asparginine tRNA, and aspartic acid tRNA. Phorbol-12,13-didecanoate (PDD) effects a decrease in the queuine content of tRNA in cultured human foreskin fibroblasts. The present data suggest that this results from a PDD-mediated inhibition of queuine uptake. Nonsaturable uptake was observed for tritiated dihydroqueuine (rQT₃) for up to 2 hr at 10 to 1000 nM concentrations, while saturation of uptake was observed after 3 to 4 hr. Lineweaver-Burke analysis of concentration versus uptake revealed biphasic uptake kinetics with high and low K_m components of approximately 350 and 30 nM, respectively. Competition by queuine of rQT₃ uptake indicated that both compounds have equal affinity for the uptake mechanism. PDD inhibited rQT₃ uptake but required 30 to 60 min of exposure before the uptake was completely blocked. The rQT₃ efflux rate from cells was found to be 3 to 4 times greater than that of uptake, and PDD also inhibited the efflux reaction. The potential inhibitors furosemide, nitrobenzylthioinosine, ouabain, 7-methylguanine, 7-deazaguanine, guanine, guanosine, adenine, adenosine, hypoxanthine, and epidermal growth factor had no effect on rQT₃ uptake. However, dipyridamole was immediately effective at reducing rQT₃ uptake.

¹ Supported in part by grants from the Air Force Office of Scientific Research. A preliminary account of this work was presented at the annual meeting of the American Society of Biological Chemists, St. Louis, MO, June 1984 (9).

² To whom requests for reprints should be addressed.

³ Recipient of a grant from the Department of Defense (AFOSR-80-0283).

⁴ Recipient of a grant from the National Cancer Institute, Department of Health and Human Services (CA 20919).

Received 7/17/84. Accepted 11/19/84.