Adenosine and tubercidin binding and transport in Chinese hamster ovary and Novikoff rat hepatoma cells.
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 zero-trans 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 microM, 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 microM and maximum velocities of 10 to 30 pmol/microliters cell H2O . sec, whereas the rate of intracellular phosphorylation was maximal at concentrations between 2 and 8 microM. The first-order rate constant (Vmax/Km) 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 the same extent, whereas pyrazofurin was significantly less effective.
D000241:Adenosine / Q000378:metabolism* / Q000494:pharmacology; D000248:Adenosine Kinase / Q000172:deficiency; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D005260:Female; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D010053:Ovary; D010766:Phosphorylation; D051381:Rats; D012263:Ribonucleosides / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism; D014372:Tubercidin / Q000378:metabolism* / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism
D012263:Ribonucleosides; D000248:Adenosine Kinase; D000241:Adenosine; D014372:Tubercidin; D013936:Thymidine; D014529:Uridine