F. Huijing, J. Larner,
Aug 1, 1966
Citations
0
Influential Citations
42
Citations
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abstract
Experiments with intact tissues indicate that adenosine 3',5' cyclophosphate exerts an important regulatory role in several reactions, including glycogen degradation and synthesisl-4 and lipolysis.' The cyclic nucleotide is formed from ATP together with pyrophosphate by a cyclizing enzyme6 localized primarily in the cell membrane in nucleated erythrocytes. It is hydrolyzed to 5' AMP by a phosphodiesterase.8 In skeletal muscle,1 diaphragm,5 heart,2 I and smooth muscle,9 adenosine 3',5' cyclophosphate concentrations increase rapidly twoto threefold under the influence of epinephrine (for a recent review, see ref. 10). In glycogen degradation, adenosine 3',5' cyclophosphate exerts its control in the phosphorylase b kinase activation reaction.1' In glycogen synthesis, the cyclic nucleotide controls the transferase I kinase which converts the glucose 6-phosphate independent (I) form of (UDPG: a-1,4-glucan a4-glucosyl) transferase (EC 2.4.1.11) to the dependent (D) form.'2 Both the activation of phosphorylase b kinase, and the conversion of the independent (I) form of transferase into the dependent (D) form can also be achieved in two different ways, namely, through the action of Ca++ and a protein factor, or by mild tryptic action."' 13 Krebs et al.'4 have described cell-free systems in which phosphorylase b kinase is minimally activated in the absence of the cyclic nucleotide. When the cyclic nucleotide is added, a marked activation is obtained. In a previous report we determined the sensitivity of the transferase I kinase to adenosine 3',5' cyclophosphate.'5 In the present report, experiments are described in which the effect of varying the concentration (separately and together) of ATP and Mg2+ on the transferase I kinase, as well as the response to adenosine 3',5' cyclophosphate, was studied. Under these conditions it was possible to demonstrate in vitro an apparent regulatory role for the cyclic nucleotide, a virtual on-off effect. Kinetic analysis of these experiments may provide information on the mechanism underlying the action of adenosine 3',5' cyclophosphate in this system. Methods.-The effect of adenosine 3',5' cyclophosphate on the transferase I kinase system was studied using a relatively crude preparation which contained both the transferase and the kinase and which was prepared in the following manner. Fresh rabbit muscle was homogenized in a Waring Blendor in Tris-HCl, 50 mM; KF, 100 mM; EDTA, 5 mM pH 7.8 (300 ml per 100 gm muscle). The homogenate was centrifuged for 20 min at 12,000 X g (2-4oC), the supernatant filtered through glass wool, and the filtrate centrifuged for 3 hr at 78,000 X g (40C). The 78,000 X g precipitate was stored at -60oC. After storage for 1-30 days, one pellet, equivalent to approximately 10 gm of muscle, was homogenized in 5 ml glycerophosphate, 8 mM, pH 7.8, containing mercaptoethanol, 50 mM, and incubated for 45 min at 300C. This incubation solubilized most of the transferase, and the homogenate was then centrifuged at 51,000 X g for 45 min. To the clear supernatant, caffeine was added to a concentration of 5 mM to minimize the phosphodiesterase activity.8 The transferase I kinase was tested as described in principle by Friedman and Larner,16 measuring the decrease in transferase I activity upon incubation with ATE and MgCl2. To 0.1 ml of a