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Fluorescent tetradecanoylphorbol acetate: a novel probe of phorbol ester binding domains
M Balázs1, J Szöllösi, W C Lee
1Department of Laboratory Medicine, University of California, San Francisco 94143.
Journal of Cellular Biochemistry
|July 1, 1991
Summary
Researchers developed a fluorescent phorbol ester, N-C12-Ac(13), to study protein kinase C (PKC) in cells. While it shows specific binding and biological activity, high non-specific binding limits its use in live cells but is valuable for purified PKC studies.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Pharmacology
Background:
- Protein kinase C (PKC) is crucial for signal transduction pathways activated by various bioactive substances.
- Understanding PKC localization and activity in living cells is vital for deciphering cellular responses.
- Phorbol esters are known activators of PKC, making them valuable tools for research.
Purpose of the Study:
- To synthesize and characterize a novel fluorescent phorbol ester derivative, N-C12-Ac(13), for monitoring phorbol ester binding sites.
- To evaluate the potential of N-C12-Ac(13) as a fluorescent probe for protein kinase C (PKC) in viable cells.
- To assess the specificity, cellular uptake, and biological activity of the synthesized probe.
Main Methods:
- Synthesis of N-C12-Ac(13), a fluorescent derivative of 12-O-tetradecanoylphorbol-13-acetate (TPA).
- Cellular uptake and fluorescence measurements in P3HR-1 Burkitt lymphoma cells using flow cytometry and imaging cytometry.
- Competitive displacement assays using [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) to determine binding affinity (Kd).
- Assessment of biological activity by inducing viral glycoprotein expression, cell differentiation, and mitotic cycle changes.
Main Results:
- N-C12-Ac(13) exhibited excitation maxima near 488 nm, suitable for laser-based cytometry.
- Rapid cellular accumulation and significant fluorescence increase (20-fold above autofluorescence) were observed in P3HR-1 cells within 25 minutes.
- Specific binding was confirmed by dose-dependent displacement with unlabeled TPA and a Kd of 11 nM for purified PKC.
- N-C12-Ac(13) demonstrated biological activity comparable to TPA, inducing differentiation and viral glycoprotein expression at lower concentrations.
- Intracellular localization was observed, primarily in cytoplasmic structures, with minimal nuclear fluorescence, suggesting exclusion from the nucleus.
Conclusions:
- N-C12-Ac(13) is a functional fluorescent probe with specific binding characteristics and biological activity relevant to PKC.
- High non-specific binding limits its utility for visualizing PKC in intact, viable cells.
- The probe is valuable for kinetic studies of purified PKC and provides insights for designing future phorbol ester probes with reduced non-specific binding.