65-KILODALTON PROTEIN-PHOSPHORYLATION IN HUMAN PERIPHERAL-BLOOD EOSINOPHILS

To evaluate the presence of protein phosphorylation in peripheral bloo d eosinophils, venous blood was drawn from normal healthy volunteers. Eosinophils were isolated on a Percoll gradient and were incubated wit h [gamma(32)P]ATP in the presence of Mg2+. After stopping the reaction , SDS-PAGE was performed and autoradiographs were prepared to determin e the incorporation of P-32 into proteins. Eosinophils developed at le ast 24 protein bands below 116.25 kD by SDS-PAGE. In the autoradiograp hs, one distinct radioactive band was observed with a molecular weight of 65 kD. P-32 incorporation into the 65-kD band was dependent on Mg2 + concentration and maximal response was observed at concentrations of 2-6 mM MgCl2. P-32 incorporation into the band was dependent on the r eaction time and temperature of the reaction system. Acid hydrolysis s howed that [P-32]phosphate radioactivity in the cells was present prim arily as phosphoserine, indicating the presence of 65-kD protein phosp horylation in human peripheral blood eosinophils.