CCL

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001-es BibID:BIBFORM006025
Első szerző:Balázs Margit (sejtbiológus, molekuláris genetikus)
Cím:Fluorescent tetradecanoylphorbol acetate : a novel probe of phorbol ester binding domains / Balázs, M., Szöllösi, J., Lee, W. C., Haugland, R. P., Guzikowski, A. P., Fulwyler, M. J., Damjanovich, S., Feuerstein, B. G., Pershadsingh, H. A.
Dátum:1991
Megjegyzések:Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
analysis
Animal
Antigens,Viral
Binding Sites
Brain Chemistry
Cell Cycle
chemical synthesis
Cytosol
Dyes
Female
Flow Cytometry
Fluorescence
Fluorescence Polarization
Fluorescent Dyes
Gene Expression
Herpesvirus 4,Human
Image Processing,Computer-Assisted
immunology
metabolism
Oxadiazoles
pharmacology
Phorbol Esters
Protein Kinase C
Rats
Rats,Inbred Strains
Signal Transduction
Support,Non-U.S.Gov't
Support,U.S.Gov't,P.H.S.
Tetradecanoylphorbol Acetate
Tumor Cells,Cultured
Megjelenés:Journal of Cellular Biochemistry. - 46 : 3 (1991), p. 266-276. -
További szerzők:Szöllősi János (1953-) (biofizikus) Lee, W. C. Haugland, R. P. Guzikowski, A. P. Fulwyler, Mack J. Damjanovich Sándor (1936-2017) (biofizikus) Feuerstein, Burt G. Pershadsingh, Harrihar A.
Internet cím:elektronikus változat
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