Inactivation In Response Research Articles

Transcriptional mapping of human respiratory syncytial virus.

A transcriptional map for human respiratory syncytial virus was determined by measuring the kinetics of viral gene inactivation in response to UV irradiation. Monolayer cell cultures of respiratory syncytial virus-infected HEp-2 cells were exposed to UV light, and residual viral RNA synthesis was monitored both by gel electrophoresis and by hybridization to dot blots of cloned cDNAs of the 10 known viral genes. Target sizes for the 10 individual viral genes were calculated relative to the UV sensitivity of intracellular viral genome replication. Target size analysis indicated that the 10 viral genes were transcribed as a single transcriptional unit and that the transcription of an individual gene was dependent on the prior transcription of all the preceding genes. The order of gene transcription (with nomenclature according to encoded proteins) was determined to proceed from the promoter as follows: 14K, 11K, N, P, M, 9.5K, 36K, F, 24K, L.

Glycogen synthase from rabbit skeletal muscle. State of phosphorylation of the seven phosphoserine residues in vivo in the presence and absence of adrenaline.

Glycogen synthase has been shown to be phosphorylated in vitro by five protein kinases, cyclic‐AMP‐dependent protein kinase (sites la, 1b and 2). phosphorylase kinase (site 2), glycogen synthase kinase 3 (sites 3a,3b and 3c), glycogen synthase kinase 4 (site 2) and glycogen synthase kinase 5 (site 5) [Cohen,P. et al. (1982)Eur. J. Biochem. 124, 21–35; Picton, C. et al. (1982) Eur. J. Biocliem. 124, 37–45]. All seven serine residues have now been found to be phosphorylated in vivo.Glycogen synthase purified from rabbits that had been injected with l‐propranolol contained 0.33 mol of phosphate/mol subunit in site la, 0.38 in site Ib, 0.39 in site 2, 1.27 in sites 3a+3b+3c and 0.65 in site 5, totalling 3.0 mol/mol subunit. The activity ratio (∓ glucose‐6 P) for these preparations was 0.21 and their Ka for glucose‐6‐P was 1.4mM.Glycogen synthase purified from animals that had been injected with I‐adrenaline contained 0.59 mol phosphate/mol subunit in site la, 0.65 in site lb, 1.03 in site 2, 2.43 in sites 3a + 3b + 3c and 0.65 in site 5, totalling 5.3 mol/mol subunit. The activity ratio of these preparations was 0.04 and their Ka for glucose‐6‐P was 6.0mM.The results show that cyclic‐AMP‐dependent protein kinase, glycogen synthase kinase 3 and glycogen synthase kinase 5 act as glycogen synthase kinases in vivo. Glycogen synthase kinase 4 may be largely responsible for the phosphorylation of site 2 in propranolol‐treated animals, and cyclic‐AMP‐dependent protein kinase and phos‐ phorylase kinase for the increased phosphorylation of site 2 in response to adrenaline.The activity of glycogen synthase in vivo in propranolol‐treated animals is largely determined by the state of phosphorylation of sites 3a, 3b and 3c, while the inactivation in response to adrenaline is due to increased phosphorylation at site 2 as well as at sites 3a, 3b and 3c. The finding that the phosphorylation of sites 3a, 3b and 3c is increased from 1.27 mol/mol subunit to 2.43 mol/mol subunit by adrenaline was unexpected, since this enzyme is not regulated by cyclic AMP. Possible explanations for this result are discussed.Phosphofructokinase was isolated as a byproduct of the purification of glycogen synthase. The phosphate content of this enzyme did not differ significantly between l−propranolol‐treated (0.15 mol/mol subunit) and adrenaline‐treated (0.18 mol/mol subunit) animals. These results indicate that muscle phosphofructokinase is not a substrate for cyclic‐AMP‐dependent protein kinase in vivo.

Insulin regulation of hepatic glycogen synthase and phosphorylase

The relative roles of insulin and glucose in the regulation of hepatic glycogen synthase and phosphorylase were studied in hepatocytes from fed rats. Elevation of extra-cellular glucose led to a rapid decrease in phosphorylase a activity followed by a slower increase in glycogen synthase I activity. A reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose was observed; following initial glucose-induced inactivation of phosphorylase, there was a highly significant linear inverse relationship between residual phosphorylase activity and glycogen synthase activation. Insulin led to a further decrease in phosphorylase activity and a 30-50% additional increase in glycogen synthase activity over that caused by glucose. The effects of insulin required the presence of glucose and served to augment acute glucose stimulation of glycogen synthase and inhibition of phosphorylase. Insulin did not perturb the reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose. The results suggest that the ability of insulin to activate hepatic glycogen synthase can be entirely accounted for by its ability to inactivate phosphorylase.