Fractionation and characterization of rapidly phosphorylated nuclear proteins in salivary gland cells of Chironomus tentans

Abstract

Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with 32Pi. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of 32P) and not sedimentable (supernatant) material. About 90% of 32P in the supernatant fraction was sedimentable at 100,000 g (“disperse chromatin”). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of 32P. The 32P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the 32P-labeled nuclear proteins into 12 major bands in the Mr range of 12,000–120,000. The incorporation of 32P into most bands reached a steady-state within 5–10 min of incubation with 32Pi and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an Mr 95,000 phosphoprotein. Two of the fastest moving 32P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving 32P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.