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R J Walkenbach

Publications and source records attributed to R J Walkenbach.

25 records · Page 2Linked to original sources
PubMed1980

Hormonal regulation of glycogen synthase: insulin decreases protein kinase sensitivity to cyclic AMP.

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1--2 microM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 microM. Insulin (0--100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.

Animals↗
PubMed1980

Characterization of adenylate cyclase activity in bovine and human corneal epithelium.

The properties of adenylate cyclase from bovine and human corneal epithelium were investigated. Adrenergic drugs were the most effective stimulatory agents tested in bovine tissue, causing greater activation than did fluoride. Isoproterenol was the most potent agonist, followed by epinephrine and norepinephrine. Phenylephrine and dopamine also stimulated adenylate cyclase through beta-adrenergic receptors at relatively high concentrations. Enzyme stimulation by all the adrenergic drugs tested was completely inhibited by 1 microM propranolol or 0.1 microM timolol. The GTP analogue, GppNp, produced considerable activation and caused an augmented response when combined with isoproterenol, but not with fluoride. Prostaglandins E1, E2, or F2 alpha produced a small but significant stimulation over control which was not sensitive to propranolol inhibition. Adenylate cyclase from human corneal epithelium exhibited qualitatively similar characteristics to those of the bovine enzyme. Fluoride was the most effective stimulatory agent, followed by isoproterenol, phenylephrine, and dopamine. Prostaglandins failed to stimulate adenylate cyclase activity in human corneal epithelial preparations.

Adenylyl Cyclase Inhibitors↗
PubMed1978

Reversible inhibition of cyclic AMP-dependent protein kinase by insulin.

Extracts of fasted rat diaphragms, previously treated with or without insulin were assayed for glycogen synthase, protein kinase and cyclic [3H]-AMP binding. Treatment with insulin produced an elevation in the % of glycogen synthase I and a concurrent decrease in cyclic AMP-dependent protein kinase activity and cyclic [3H]-AMP binding. Analysis of extracts by disc gel electrophoresis demonstrated the inhibition of cyclic [3H]-AMP binding to involve the Type I protein kinase holoenzyme. Inhibition of protein kinase activity was most apparent in the presence of 0.2 micrometer cyclic AMP, with enzymatic activity of the insulin-treated extracts typically 60--65% of control. Higher assay concentrations diminished the difference between control and insulin-treated extracts and concentrations greater than 20 micrometer abolished it. The inhibition of cyclic AMP-dependent protein kinase activity after insulin was a transient and labile phenomenon. The effect was independent of ATP concentration in the assay, but was sensitive to the pH of tissue extraction, requiring a pH of 7.0 to 8.4 to be observed. Insulin-mediated inhibition of protein kinase activity was reversed upon preincubation of extracts at 0--2 degrees. Relatively concentrated homogenates (less than 4 microliter buffer/mg tissue) yielded extracts which exhibited little or no inhibition of protein kinase activity compared to extracts prepared from more dilute (6--10 microliter/mg) homogenates. A model for the inhibition of the cyclic-AMP dependent protein kinase by an insulin-generated inhibitor which becomes directly associated with the Type 1 holoenzyme is proposed.

Animals↗
PubMed1977

Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.

Affinity Labels↗