Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids

Ligand-receptor interaction rates in the presence of convective mass transport.

The rate of binding of a ligand to receptors on the cell surface can be diffusion limited. We analyze the kinetics of binding, diffusion-limited in a stationary liquid, in the presence of convective mass transport. We derive a formula that expresses the reaction kinetics in terms of the mass transfer coefficient. A moderately transport-limited kinetics is not readily recognizable from the shape of the binding curve and may lead to erroneous estimates of the rate coefficients. We apply our results to practically important cases: a cell suspension in a stirred volume of liquid and a confluent cell colony under a laminar stream. Using typical numbers characterizing the ligand-receptor interactions, we show that stirring and perfusion can be important factors determining the reaction rates. With the confluent colony, the early reaction kinetics requires a different treatment, and we provide it for the case of low receptor occupancy. We show that, even with a fast perfusion, a cell monolayer can transiently generate a zone of depletion of the ligand, and that would affect the early stages of the reaction. Our results are expressed in a simple analytical form and can be used for the design and interpretation of experimental data

H2O2-Induced Increases in Cellular F-Actin Occur without Increases in Actin Nucleation Activity

Previous work has shown that H2O2 causes an increase in polymerized actin (F-actin) inside cells. To test the hypothesis that increased polymerization resulted from a mechanism involving increased actin nucleation activity, we employed methods utilizing pyrene-labeled actin to quantify the actin nucleation activity of cell lysates and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallacidin binding assays to quantify the amount of F-actin in P388D1 cells. H2O2 increased polymerized actin (NBD-phallacidin assay) in a dose-dependent manner with an effective dose giving 50% response (ED50) [asymp] 1 mM. Five millimolar H2O2 caused a 1.6-fold increase in NBD-phallacidin staining. In contrast, actin nucleation activity decreased in a dose-dependent manner with a similar ED50. Five millimolar H2O2 caused a 30-40% decrease in actin nucleation activity. The effect was rapid, occurring within 5 min of H2O2 addition. The results indicate that H2O2 causes cytoskeletal changes that enhance NBD-phallacidin binding without increasing actin nucleation activity. Fractionation studies showed that the nucleation activity in H2O2-treated cells and controls sedimented with the Triton X-100-insoluble cytoskeleton, and the cytosolic fraction appeared to contain an inhibitor of actin polymerization.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31818/1/0000764.pd