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Additional resources for Hormone Action Part F: Protein Kinases
Sample text
For example it has been shown that glucose, a phosphorlyase effector, is a requirement for the removal of phosphate from phosphorylase a catalyzed by the reversal of the phosphorylase kinase reaction. Glucose appears to act by dissociating the tetrameric structure of phosphorylase a into the dimeric form. In the same manner, cAMP is necessary if phosphate is to be removed from the regulatory subunit of cAMP-dependent protein kinase under the action of the catalytic s u b u n i t 1°,12 o r cGMP-dependent protein kinase?
Embi, F. B. Caudwell, and P. Cohen, Eur. J. , in press (1982). 46 x 25 cm) at flow rates of 1 ml/min. Recoveries of 32p-labeled peptides were in the range of 70-85% except for site 2, which was variable between 50 and 70%. Slightly better recoveries of site 2 were obtained using 1-propanol than acetonitrile. Figures 3 and 4 present illustrative HPLC chromatograms of 32p-labeled peptides from synthase phosphorylated by several different kinases. 5 0 20 40 (50 80 I00 120 RETENTION TIME (MIN) FIG.
No albumin is added to the cell suspensions because its presence would distort the polyacrylamide gels eventually used to resolve the phosphoproteins. Carrier-free 32po43- (New England Nuclear) is added to a concentration of 10-20/zCi/mg cell protein and 2-5 ml of cells are incubated in 25-ml plastic flasks under 95% 02-5% CO2 for 45 min to allow the specific activity of the [32p]ATP in the cell to reach a constant value. 5 At 45 min, the hepatocytes are stimulated with the agent(s) of choice for the desired period (usually 3-15 min) and the incubations are stopped as described below.