The rapid induction by phytohemagglutinin of increased α-aminoisobutyric acid uptake by lymphocytes

Abstract

The effect of phytohemagglutinin (PHA) on the ability of human lymphocytes to transport the nonutilizable amino acid, α-aminoisobutyric acid (AIB) has been studied. PHA binds rapidly to plasma membrane receptor sites with half maximal binding requiring approximately 7.5 min. During the first 30 min after PHA addition to lymphocytes no change was detected in AIB transport, but then a linear increase in the initial rate of AIB transport occurred over the next 9 hr. Subsequently, the rate of AIB transport stabilized at a level 6-7 times greater than that found in control lymphocytes. The change in membrane function developed even when de novo protein synthesis was inhibited by 85-90% with puromycin or cycloheximide. However, the PHA effect did not occur when the lymphocytes were maintained at 4°C. Studies of the kinetics of AIB uptake by control and PHA-treated lymphocytes demonstrated that PHA increases the V_max of AIB uptake by 6-7-fold (0.7 mμmole AIB per 10⁶ lymphocytes/15 min versus 0.1 mμmole per 10⁶ lymphocytes/15 min) without affecting the Km (Michaelis constant) of the transport system (2mM in both cases). When fetuin was added to lymphocyte cultures to remove bound PHA, the PHA-induced increase in the rate of AIB uptake was arrested at the rate achieved during the time of prior incubation with PHA. This level of AIB transport persisted for at least 3 hr after 80% of the PHA was removed from the cell membrane. These data demonstrate that PHA rapidly induces a change in a lymphocyte cell membrane transport function, and that the continued presence of PHA on the cell membrane is required for the full stimulatory effect to be reached. The data do not distinguish between a direct action of PHA upon the lymphocyte membrane or the possibility that PHA slowly enters into the cell where it then exerts its effect.