PubMed · 5405972
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V Cagli. 1969-10-06. [The catecholamines (biosynthesis and physiologic action)].. https://pubmed.ncbi.nlm.nih.gov/5405972/
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This paper reports the development of a method based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of catecholamines and excitatory amino acids on submicroliter microdialysis samples, with short analysis times (3 min or less), high sensitivity (nanomolar range, i.e., attomoles detected) and high separation efficiency (up to 1.10(6) theoretical plates). A continuous flow derivatization of small volumes of microdialysate (500 nl) using naphthalene-2,3-dicarboxaldehyde as derivatizing reagent is described. Thereafter, two subsequent off-line analyses are performed on each of the 30-s dialysates to determine catecholamines and amino acids. The performances of the present method are demonstrated in vitro by monitoring rapid fluctuations in the concentration of catecholamines and amino acids in the external microdialysis medium.
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.