Abstract
Cyclic AMP-independent casein kinase-2 from rabbit skeletal muscle has been extensively purified by procedures including phosphocellulose, Bio-Gel A-1.5m, casein-Sepharose, and DEAE-cellulose column chromatographies. The casein kinase and glycogen synthase kinase activities are co-purified throughout the purification. The casein kinase-2 has Mr approximately equal to 135,000 as determined by glycerol density gradient centrifugation and by gel filtration. Analysis of the purified kinase by gel electrophoresis in the presence of sodium dodecyl sulfate reveals the presence of two major protein bands having Mr = 42,000 and 27,000 in a ratio of 0.85:1. The kinase phosphorylates glycogen synthase, casein, and phosvitin either in the presence of ATP or GTP; however, the Km values for GTP are slightly higher than those of ATP. The activity of this kinase is inhibited by heparin but is not affected by the addition of cyclic AMP, heat-stable inhibitor of cyclic AMP-dependent protein kinase, Ca2+, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or the combination of Ca2+ and calmodulin. The phosphorylation of the synthase by the kinase results in the incorporation of approximately 0.8 mol of PO4/subunit and a reduction in the synthase activity ratio from 0.82 to 0.6. The extent of phosphorylation of the synthase catalyzed by casein kinase-2 is similar to that by phosphorylase kinase; however, the sites phosphorylated by these two kinases are different. The relationships of casein kinase-2 to the cyclic AMP-independent synthase kinases that have been described by other laboratories and to the casein kinases isolated from other tissues are discussed.
Highlights
Extraction-Approximately 1.8 kg of fresh rabbit skeletal muscle was homogenized with 5.4 liters of50 m~ Tris/HCl buffer, pH 7.5, containing 1 m~ dithiothreitol, 5 m~ EDTA, 50 m~ KF, 0.05 m~ phenylmethylsulfonyl fluoride in aWaring Blendor for 90 s
Bio-Gel A-1.5m Column Chromatography-The enzyme eluted from the phosphocellulose column was concentrated in an Amicon ultrafiltration cell equipped with a UM-10 membrane
Fractions of 3.3 mlwere collected and the enzyme activities were measured with glycogen synthase, phosphorylase b, and casein assubstrates (Fig. 1).Three synthase kinase activity peaks are separable upon chromatography
Summary
18,185 rylase kinase were generous gifts from Dr J. Methods-Glycogen synthase activity was determined by measuring the incorporation of glucose from UDP-['4C]glucose into glycogen as previously described [32]. The synthase activity ratio is defined as the activity without glucose-6-P divided by the activity with 8 m~ glucose-6-P.I-form glycogen synthase from rabbit skeletal muscle was purified as previously described [33] except that the homogenizing buffer was 50 m~ Tris/HCl, pH 8.2, containing 1 m~ dithiothreitol, 1 n" EDTA, 2 n" EGTA,' and 0.05 m~ phenylmethylsulfonyl fluoride. Synthase kinase and phosphorylase kinase activities were measured in 25 mM Tris/acetate buffer, pH 8.5, Containing 1 m~ dithiothreitol, 8 mM magnesium acetate, 0.1 mM calcium acetate, 0.12 mM [y-"PIATP, 0.1 to 0.2 m g / d of I-form glycogen synthase or 0.5 mg/ml of phosphorylase b, and protein kinase. Protein concentration was determined by the method of Bradford [38]
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