Spectroscopic characterization of specific phorbol ester binding to PKC-receptor sites in membranes in situ.
Electron paramagnetic resonance (EPR) spectra were measured for the spin labelled phorbol-12,13-diester [5,6]PA bound to membranes of the particulate fraction of mouse brain tissue rich in PKC receptors. [5,6]PA is a bioactive derivative of the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), carrying the spin label in a doxyl group in position 7' of the 12-O-tetradecanoyl residue. A mathematical model based on special algorithms (Griffith, O.H. and Jost, P.C. Spin labeling: theory and applications, 1976) allows a satisfactory reconstruction of the experimental spectrum. It reveals that in the experimental spectrum the signal from the [5,6]PA molecules bound non-specifically to the lipid bilayer of the membranes is superimposed by the signal of [5,6]PA molecules bound specifically, i.e. to the active site of PKC (approximately 10% of total EPR signal intensity). Moreover, interpretation of spectral parameters indicates that in [5,6]PA molecules bound specifically the tetradecanoyl chain exhibits a larger motional freedom compared to that in [5,6]PA bound non-specifically. These new findings are in accordance with views featured independently by two recent molecular models of interaction of PKC with cellular membranes (8,9).
Svetek
J
J
J. Stefan Institute, University of Ljubljana, Slovenia.
Schara
M
M
Pecar
S
S
Hergenhahn
M
M
Hecker
E
E
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002273:Carcinogens / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D008051:Lipid Bilayers; D008433:Mathematics; D051379:Mice; D008962:Models, Theoretical; D011493:Protein Kinase C / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013113:Spin Labels; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000378:metabolism*
C096280:(5,6)PA; D002273:Carcinogens; D008051:Lipid Bilayers; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D013113:Spin Labels; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate