Cyclic AMP-independent casein/glycogen synthase kinases from pig polymorphonuclear leucocytes.
1. Two cyclic AMP-independent casein/glycogen synthase kinases were purified from pig polymorphonuclear leucocytes by chromatography on phosphocellulose followed by affinity chromatography on casein-Sepharose 4B or gel filtration on Bio-Gel A-1.5m. When the affinity step was used, the specific activities were 86 and 43units/mg of protein for casein kinase 1 and 2, respectively, whereas these values were 94 and 90units/mg of protein when the gel-filtration step was used. 2. These kinases differ as follows: (a) the molecular weight of casein kinase 1 (38000) is very much lower than that of casein kinase 2 (185000); (b) the K(m) for casein (0.46mg/ml) and K(a) for Mg(2+) (0.3mm) of casein kinase 1 are lower than those of casein kinase 2 (0.90mg/ml and 1.7mm respectively); (c) KCl stimulates the phosphorylation of casein by casein kinase 1, whereas it inhibits phosvitin phosphorylation by this enzyme; on the contrary, the effect of KCl on casein kinase 2 is very similar with either casein or phosvitin as substrate; (d) although both kinases phosphorylate rabbit muscle glycogen synthase I, the ratio of glycogen synthase to casein phosphorylation by casein kinase 1 is about 4-fold greater than that by casein kinase 2. Furthermore, (32)P incorporation into glycogen synthase promoted by casein kinase 1 (3.6mol of (32)P/mol of 85000-dalton subunit) is twice that observed with casein kinase 2 (1.8mol of (32)P/mol of 85000-dalton subunit). Such a phosphorylation results in a decrease in the glucose 6-phosphate-independence ratio of glycogen synthase to 10-15 with casein kinase 1 and to 35-45 with casein kinase 2. 3. The activity of both kinases is neither stimulated by cyclic AMP, Ca(2+) and calmodulin nor inhibited by cyclic AMP-dependent protein kinase inhibitor protein. 4. No phosphorylation kinase activity was observed with casein kinase 1 and 2 at either pH6.8 or 8.2 in the presence of Ca(2+). 5. Activities of both kinases on casein and glycogen synthase decreased in parallel when incubated at 50 degrees C.
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D047388:Casein Kinases; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D066298:In Vitro Techniques; D007700:Kinetics; D009504:Neutrophils / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D011189:Potassium Chloride; D011494:Protein Kinases; D047388:Casein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases