Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Abstract

cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 x g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 M NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 M NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 M NaCl, the peak B enzyme had Mr 250 000, 7.2S and the peak A enzyme Mr 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl2. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase. Synthase kinase had Kmapp 4.2 microM for muscle glycogen synthease I and Kmapp 45 microM for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca2+, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.