65-kilodalton protein phosphorylation in human peripheral blood eosinophils

To evaluate the presence of protein phosphorylation in peripheral blood eosinophils, venous blood was drawn from normal healthy volunteers. Eosinophils were isolated on a Percoll gradient and were incubated with [gamma32P]ATP in the presence of Mg2+. After stopping the reaction, SDS-PAGE was performed and autoradiographs were prepared to determine the incorporation of 32P into proteins. Eosinophils developed at least 24 protein bands below 116.25 kD by SDS-PAGE. In the autoradiographs, one distinct radioactive band was observed with a molecular weight of 65 kD. 32P incorporation into the 65-kD band was dependent on Mg2+ concentration and maximal response was observed at concentrations of 2-6 mM MgCl2. 32P incorporation into the band was dependent on the reaction time and temperature of the reaction system. Acid hydrolysis showed that [32P]phosphate radioactivity in the cells was present primarily as phosphoserine, indicating the presence of 65-kD protein phosphorylation in human peripheral blood eosinophils.