A kinetic characterization of putrescine and spermidine uptake and export in human erythrocytes

Abstract

Using human erythrocytes as a model system for the study of mammalian polyamine transport, detailed kinetic parameters regarding the uptake and export of putrescine and spermidine were determined. The putrescine uptake data indicated a multi-component uptake system comprised of a low-capacity saturable component and a non-saturable component. The saturable putrescine uptake component demonstrated a calculated K m of 21.0 μM and a V max of only 6.52 × 10 −13M/s. The non-saturable linear putrescine uptake rate was defined by a significant pH dependence, a lack of uptake inhibition by related polyamines, and a permeability π of 3.19 × 10 −8s −1. These findings suggested that non-saturable putrescine uptake involved a process of simple diffusion. Spermidine uptake exhibited Michaelis-Menten kinetics with a K m and V max of 12.5 μM and 1.36 × 10 −12M/s, respectively. Spermidine uptake did not demonstrate pH dependence and was not significantly inhibited by any of the tested polyamines. The Arrhenius plot of spermidine uptake was determined to be biphasic with calculated activation energies of spermidine uptake of 135.2 kJ/mol for 19–21°C and 59.3 kJ/mol for 21–35°C. These data suggest the possibility of multiple spermidine uptake processes which are not mediated by simple diffusion across the cell membrane. The putrescine export process demonstrated both saturable and non-saturable components. The calculated K m, V max and π for putrescine export were 33.8 μM, 1.19 × 10 −11M/s and 2.81 × 10 −7 s −1, respectively. The spermidine export process was non-saturable up to intracellular spermidine concentrations of 4 μM. At similar intracellular and extracellular concentrations of putrescine and spermidine, however, export processes displayed rates which were an order of magnitude greater than their respective uptake rates. This finding supports the possible presence of mediated putrescine and spermidine export processes different than simple diffusion.