The uptake of adenosine and tubercidin by control and ATP-deleted wild-type and adenosine kinase-deficient cells was measured by rapid kinetic techniques. Adenosine deamination was inhibited by pretreatment with 2-deoxycoformycin. Control wild-type cells phosphorylated adenosine so rapidly that the kinetics of transport per se could not be assessed unambiguously. ATP depletion and adenosine kinase deficiency did not abolish the conversion of adenosine to nucleotides, but reduced it to such an extent that initial velocities of uptake could be safely construed as transport velocities in both zerotrans and equilibrium exchange modes. The same was true for tubercidin, which was not phosphorylated in adenosine kinase-deficient cells. It accumulated intracellularly, however, to concentrations 50 to 120% higher than those in the extracellular space, apparently due to binding to some intracellular component(s). Binding was not saturated up to a concentration of 200 pM, but seemed to be slow relative to transport. Fits of appropriate integrated rate equations based on the simple carrier model to uptake time courses obtained under these conditions yielded Michaelis-Menten constants for adenosine and tubercidin transport of 100 to 200 pM and maximum velocities of 10 to 30 pmollpl cell H20 . sec, whereas the rate of intracellular phosphorylation was maximal at concentrations between 2 and 8 pM. The first-order rate constant (Vmax/K,,,) for adenosine phosphorylation, however, seemed to be appreciably higher than that for its transport. This indicates that at physiological concentrations, which fall in the first-order range for both processes, adenosine trapping is very efficient. Adenosine, tubercidin, tricyclic nucleoside, 2'-deoxyadenosine, and 3'-deoxyadenosine all inhibited uridine and thymidine transport to about t h e same extent, whereas pyrazofurin was signficantly less effective.
In a previous study (Lum et al., 1979) we have determined the kinetics of adenosine transport' in P388 mouse leukemia cells in which adenosine phosphorylation and deamination were blocked by depletion of cellular ATP and treatment with 2-deoxycoformycin (dCF), respectively. Rapid kinetic techniques were applied to determine detailed time courses of adenosine equilibration across the membrane in both zero-trans2 and equilibrium exchange2 protocols, and the kinetic parameters for transport were computed by fitting appropriate integrated rate equations based on the simple carrier model to these time courses. The validity of this approach has been amply demonstrated for a number of nucleosides (Plagemann and Wohlhueter, 1980; Wohlhueter and Plagemann, 1980, 1982). We find that nucleosides are transported by a single, symmetrical carrier with broad specificity. The Michaelis-Menten constant for adenosine transport in P388 cells has been estimated as 120-140 pM. Adenosine transport was found to be very rapid; at adenosine concentrations in the first-order range, the half time for transmembrane equilibration is only 3-6 sec at 25Β°C.