Biosynthetic incorporation of fluorescent carbazolylundecanoic acid into membrane phospholipids of LM cells and determination of quenching constants and partition coefficients of hydrophobic quenchers.
A fluorescence quenching method was developed for determining partition coefficients and diffusional rates of small molecules in cell membranes. This method involves quenching the fluorescence of carbazole-labeled membranes by hydrophobic molecules that partition into membranes. Cell membrane phospholipids of mouse LM cells in tissue culture were biosynthetically labeled with the carbazole moiety by supplementing the growth media with 11-(9-carbazolyl)undecanoic acid. Plasma membranes, microsomes, and mitochondria were isolated free of nonmembranous neutral lipids, and the incorporation of the fluorescent probe was characterized. Quenching studies of the carbazole moiety by a series of N-substituted picolinium perchlorate salts showed that the carbazole moiety was located in the hydrophobic interior of the membrane bilayer. The carbazole fluorescence also was quenched by the hydrophobic quenchers lindane, methoxychlor, and 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene, indicating that these compounds partitioned into the membrane. Stern-Volmer quenching constants determined by fluorescence lifetime and intensity measurements were identical, as expected for dynamic quenching. The effects of different lipid compositions on quenching constants and partition coefficients were determined by comparing different membrane fractions. These parameters also were measured in membranes from cells in which the phospholipid composition was altered by substituting ethanolamine for choline in the growth medium. Changes in the lipid composition produced changes in the bimolecular quenching constants. For example, bimolecular quenching constants for 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene were higher in mitochondrial membranes than in plasma membranes and microsomes. They were also higher in dispersions made from membrane phospholipids as compared with intact membranes or total lipid dispersion.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002227:Carbazoles / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D008563:Membrane Lipids / Q000096:biosynthesis*; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010743:Phospholipids / Q000096:biosynthesis*; D013050:Spectrometry, Fluorescence
D002227:Carbazoles; D008563:Membrane Lipids; D010743:Phospholipids; C043881:11-(9-carbazolyl)undecanoic acid