Increase In Fluorescence Polarization Research Articles

Elevation of apparent membrane viscosity in ovarian granulosa cells by follicle-stimulating hormone

Continued exposure of cultured granulosa cells to follicle-stimulating hormone (FSH) induced: (i) a rise in apparent membrane microviscosity, as reflected by an increase in fluorescence polarization of the lipid-soluble probe, 1,6-diphenyl-1,3,5,-hexatriene; and (ii) a progressive decline in the cyclic AMP response to renewed challenge with the same hormone. Both changes were reduced or prevented by pretreatment of the cells with oleic or linoleic acid, agents which reduce membrane viscosity, but not by elaidic or palmitic acid which increase the rigidity of membrane lipids. Other agents that inhibited FSH-induced changes in membrane fluidity (gonadotropin-releasing hormone, actinomycin D and cycloheximide) also prevented desensitization to FSH. Cyclic AMP and cyclic GMP derivatives did not mimic the effects of FSH on apparent membrane viscosity or desensitization. Changes in membrane fluidity are unlikely to be the sole cause of desensitization since (i) pretreatment of the cells with fatty acids that increase lipid viscosity did not induce desensitization to FSH, and (ii) desensitization of granulosa cells to lutropin and prostaglandin E 2 by exposure to the homologous hormone was not attended by increased membrane viscosity. The experiments described provide the first example of a hormonally induced increase in the target cell apparent membrane viscosity.

Fluorescence-spectroscopic investigations of cell membranes: Characterization of cellular subpopulations in different states of proliferation

Primary processess in cell-density dependent regulation of proliferation are mediated by the outer cell membrane. We have characterized the physical state of the plasma membrane by labelling it with the lipophilic fluorescence indicator 1,6-diphenyl-1,3,5-hexatriene and measuring its fluorescence polarization. Normal and tumorvirus-transformed permanent mouse-cell cultures were investigated while still being attached to the growth substrate or after suspension. Extrinsic fluorescence polarization was shown to increase substantially with increasing cell-growth density. For normal mouse cells this increase of fluorescence polarization could be shown to be correlated with a parallel increase of cell number in the subpopulation of irreversibly growth-inhibited cells.

Spectrophotometric and fluorescence polarization studies of the binding of ethidium, daunomycin and mepacrine to DNA and to poly(I · C)

The changes in the visible absorption spectrum and the degree of fluorescence polarization of daunomycin and mepacrine on binding to DNA have been compared with those shown by the archetypical intercalating agent ethidium. The changes are qualitatively similar for the three drugs, namely a bathochromic shift and hypochromicity (giving an isosbestic point), and an increase in polarization of fluorescence when irradiated with polarized light. These effects are typical of intercalation. The effect of changing the nucleic acid conformation has been investigated by repeating the studies with poly(I · C) (a nucleic acid in the A conformation). Upon interaction with poly(I · C) only ethidium shows effects typical of intercalation, neither daunomycin nor mepacrine intercalates into the poly(I · C) helix.

Binding of aflatoxins B 1 and G 1 to rat liver chromatin

Direct evidence for aflatoxin-chromatin interaction was obtained using fluorescence quenching as a measure of the interaction. Interaction of aflatoxin B 1 with components of chromatin increased in the following order: nonhistone residue, DNA, histone, histone-free chromatin, and intact chromatin. Fluorescence change due to interaction of aflatoxin B 1 with various complexes of chromatin constituents was not significantly different from that obtained with DNA. Association of aflatoxin B 1 with chromatin components was very weak whereas that with reconstituted deoxyribonucleoprotein (consisting of histone, nonhistone residue, and DNA) and with native chromatin was strong as shown by increase in fluorescence polarization of aflatoxin. Such data suggest the importance of structural integrity of chromatin in the binding with aflatoxin. Interaction of aflatoxin G 1 with chromatin was less than that of aflatoxin B 1.

Polarization of the Fluorescence of Solutions Subjected to an Electric Field

The dipole moments of fluorescent molecules in nonpolar solvents in the lowest singlet excited state has been previously calculated from observations of the increase in fluorescence polarization when the molecules are subjected to an electric field on the assumption that the Langevin distribution prevails at the time of the emission. By the use of Benoit's theory of the rotational diffusion of a dipole placed in an electric field, and under conditions of isotropic depolarization, an equation is obtained which allows the calculation of the excited dipole moment when the equilibrium distribution is not reached during the excited state.