Spin-labeled phorbol esters and their interactions with cellular membranes—V. Electron paramagnetic resonance of spin-labeled phorbol-12,13-diesters bound to their receptors in mouse brain particulate fraction

• Published in: Carcinogenesis (New York) 1992-02, Vol.13 (2), p.211-216
• Main Authors: Svetek, J., Hergenhahn, M., Schara, M., Pecar, S., Hecker, E.
• Format: Article
• Language: English
• Subjects: analogs; Animals; binding; Binding, Competitive; Biological and medical sciences; brain; Brain - metabolism; Caenorhabditis elegans Proteins; Carcinogenesis, carcinogens and anticarcinogens; Carrier Proteins; cell membranes; Chemical agents; E.P.R; Electron Spin Resonance Spectroscopy; Kinetics; Mathematics; Medical sciences; Mice; phorbol 12-myristate 13-acetate diester; Phorbol Esters - metabolism; Protein Kinase C; Receptors, Drug - metabolism; Spin Labels; Tumors
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/13.2.211

The relatively small concentrations required for in vivo bioactivity of diterpene ester skin irritants and promoters (∼10 nmol per animal; ∼10 nM in cell cultures) has discouraged studies of EPR spectra of bioactive, TPA-analogous, spin-labeled phorbol-12,13-diesters [(n,m)PA] bound to their membrane receptors, protein kinases C (PKC). To meet the requirements of present EPR spectrometers, particulate fraction from mouse brain containing at least 25 × 10−12 mol of receptors/mg protein (PKC species) were employed together with certain (n,m)PA selected to give an optimal ratio of specific to non-specific binding. For selection and optimization of experimental conditions, a theoretical model was developed that considers all characteristic parameters of the system. By fitting the model calculations to the experimental data of competitive agonist displacement from the particulate fraction of tritium-labeled TPA, the dissociation constants Kd for four selected (n,m)PA used as antagonists were determined. Optimal experimental conditions are met by (5,6)PA and by (5,8)PA, in that for both compounds the relative amount of displaced (n,m)PA is in accordance with the predictions derived from the model. Moreover, the model turned out also to be reliable for samples containing either small or large amounts of membranes. To obtain an EPR spectrum of an agonist bound to brain particulate fraction, the (5,6)PA was used. It shows a broad EPR spectrum typical for an immobilized molecule. The spectrum changes if an excess of TPA is added to the system; the slight differences in shape are due to displacement of (5,6)PA from specific receptor sites by non-labeled TPA and show up as a descreased central peak amplitude. This is the first time that the agonist/receptor interaction of a diterpene ester type irritant and tumor promoter has been demonstrated by direct spectroscopic measurement.