PROTEIN PHOSPHORYLATIONS IN GRANULOCYTE/MACROPHAGE COLONY-STIMULATING FACTOR-STIMULATED HUMAN PERIPHERAL BLOOD EOSINOPHILS

Abstract

Peripheral blood eosinophils from normal healthy subjects were isolated on a Percoll gradient and were incubated with [^<32>P] orthophosphoric acid. ^<32>P-labeled eosinophils were washed and stimulated with GM-CSF under different conditions. After reaction was stopped, SDS/PAG electrophoresis was performed along with autoradio-graphs to determine the incorporation of ^<32>P into proteins. GM-CSF-stimulated eosinophils produced an increase of ^<32>P incorporation into the bands in the 31 kDa and 35 kDa areas after 30 sec and 1 min of stimulation. The increase of ^<32>P incorpolation was dependent on Mg^<2+> concentration and temperature, suggesting that proteins in the eosinophils with apparent molecular weights of 31 kDa and 35 kDa can be phosphorylated with the stimulation of GM-CSF.