Kinetic analysis of uptake and phosphorylation of 5,6-dichlororibofuranosylbenzimidazole (DRB) by salivary gland cells of Chironomus tentans.

The kinetics of transport and phosphorylation of the nucleoside analogue 5,6-dichloro-l-~-~-ribofuranosylbenzimidazole (DRB) and the nature of monophosphate metabolites obtained were studied in salivary gland cells of Chironomw tentans. DRB, labeled by exchange with tritiated water, is transported across the plasma membrane, but only monophosphate derivatives emerge in the ethanol-soluble cell extract. Kinetic analysis of DRB uptake shows a rapid initial transport rate through the plasma membrane and an acquirement of equilibrium state between external and internal substrate concentrations within about 2 min. After that time, it is the phosphorylation of DRB within the cells which constitutes the rate-limiting step in the overall uptake process. The nonlinear dependence of the transport step on extracellular DRB concentrations is consistent with the occurrence of saturable reaction capacities and thus a facilitated diffision mechanism, in accordance with the Michaelis-Menten kinetics. The apparent K,,, value for the transport reaction falls in the region of 125 PM, while the apparent V value is about 160 pmol/liter of cell volume X min. The labeled DRB monophosphate fraction derived from the cells is not uniform, but rather it consists of monophosphate molecules in which the phosphate group is linked at different positions in the sugar moiety. Digestion of labeled DRB metabolite(s) with 3’and 5’-nucleotidases and separation by thin layer electrophoresis in borate buffer indicate the presence of 2’and B’-DRB monophosphate isomers. No measurable quantities of 3”DRB monophosphate could be detected. The enzymes involved in phosphorylation of DRB display lower affinities, with K,,, values in the range of 250 t o 400 PM, and considerably lower capacities, with V values of 3 t o 5 pmol/liter of cell volume X min, than the mediator proteins in the transport reaction. The results are discussed in relation to the question whether phosphorylated DRB metabolites may constitute the active form of DRB in the inhibition of the transcription of heterogeneous nuclear RNA producing genes in living cells.