PubMed · 4116782
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1972-09-23. Glucagon.. https://pubmed.ncbi.nlm.nih.gov/4116782/
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The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% alpha-helix, 35% beta-extended structures (including beta-sheet), 14% beta-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in alpha-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.
Numerous studies have shown that catecholamines can modulate lymphocyte migration. This effect may be mediated in part by modulation of lymphocyte-endothelial cell interactions, which is dependent on adhesion molecules expressed on both of these cells. Our results show that catecholamines decreased T-cell binding to IL-1 activated endothelial cells in vitro. The decrease in adhesion was not mediated by a change in adhesion molecule expression as LFA-1 and VLA-4 expression on T-cells and ICAM-1 and VCAM-1 expression on endothelial cells were not changed by catecholamine stimulation. T-cells flatten and enlarge the area of surface contact as they adhere to endothelial cells. Image analysis of the number of T-cells bound and the amount of cell spreading over several time points suggests that catecholamines alter the kinetics of T-cell-endothelial cell adhesion. These results support the hypothesis that catecholamines can alter lymphocyte-endothelial interactions in vivo, which in turn would affect lymphocyte migration.
An end-column electrical decoupler contructed with perfluorosulfonated ionomer (Nafion) is described. This decoupler was fabricated at the cathodic end of the separation capillary by casting liquid Nafion ion exchange powder over a copper-plated tungsten wire. The internal diameter of the flow channel was controlled by adjusting the thickness of the copper plating. This design overcomes problems of conventional end-column detection such as low sensitivity due to low collection efficiency of analytes at the detection electrode, difficulty in precise placement of the detection electrode, and the need to use small-bore capillaries (<-25 micron). The loss of cationic analytes observed with long-cast Nafion on-column decouplers was significantly reduced. The high current shunting capability of the long Nafion decoupler was maintained in this configuration. Elimination of the detection capillary required for on-column electrical decouplers provided higher separation efficiency by maintaining plug-type flow throughout the system. Four model catecholamines were well separated within 5 min with separation efficiency of up to 230 000 plates and migration time reproducibilities of <0.6% RSD. With the optimized experimental conditions, detection limits of 3 nM for the catecholamines were achieved in a Ringer's solution matrix (to model a microdialysis sample).