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Biomedical subjects

E L Elson

Publications and source records attributed to E L Elson.

At least 91 records · Page 5Linked to original sources
PubMed1977

Lateral transport of a lipid probe and labeled proteins on a cell membrane.

Diffusion coefficients (D) of a lipid probe and labeled proteins on L-6 myoblast membranes have been measured giving D(protein) approximately 2 X 10(-10) square centimeter per second and D (lipid probe) approximately 9 X 10(-9) square centimeter per second. Some of the membrane proteins are immobile, but the lipid probe diffuses freely over macroscopic distances. Cytochalasin B slows protein but not lipid probe diffusion.

Azides↗
PubMed1977

Lateral diffusion in planar lipid bilayers.

Direct measurements by fluorescence correlation spectroscopy of lateral diffusion coefficients of fluorescent lipid analogs in lipid bilaryer membranes indicate self-diffusion coefficients D greater than 10(-7) square centimeters per second for various lipid systems above their reported transition temperatures. Cholesterol in egg lecithin at mole ratio of 1 : 2 reduces D by about twofold, while retained hydrocarbon solvent can increase it by two- to threefold.

Cholesterol↗
PubMed1977

Receptor diffusion on cell surfaces modulated by locally bound concanavalin A.

In order to test the anchorage modulation hypothesis, the fluorescence photobleaching recovery method was used to measure the global inhibition of cell surface receptor mobility induced in 3T3 mouse fibroblasts by local binding of platelets labeled with concanavalin A (Con A). By measuring the diffusion of antibody-labeled cell surface receptors at various points on the cell surface, two states, immobile and mobile, were distinguished in the receptor population. Bound Con A-platelets, occupying between 4% and 30% of the cell surface, decreased the diffusion coefficient of the mobile population by a factor of 6. The magnitude of this effect was independent of distance from the sites of the bound Con A-platelets, demonstrating the propagated and nonlocal properties of the modulation effect. The immobile fraction of the population was not changed by Con A-platelet binding. Modulation of the diffusion constant of mobile receptors was partially reversed by treatment with microtubule-disrupting agents such as Colcemid and Vinca alkaloids. High doses of soluble Con A induced even higher levels of modulation than Con A-platelets, but reversal by microtubule-disrupting drugs was observed. These experiments provide additional support for the anchorage modulation hypothesis and provide a measure of the nature and degree of mobility at the molecular level. They also put important constraints on the hypothesized interactions among submembranous components (microtubules and microfilaments) of surface modulating assemblies.

Antigen-Antibody Complex↗
PubMed1977

Mobility and distribution of a cell surface glycoprotein and its interaction with other membrane components.

Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the lateral mobility and topographical distribution of a major cell surface glycoprotein (CSP). Both endogenous CSP and fluorescent-labeled exogenous CSP bind to the cell surface in a fibrillar pattern and are immobile on the experimental time scale. Azide, vinblastine, and cytochalasin B do not alter the immobility and cell surface distribution of the CSP molecules. Therefore, oxidative phosphorylation and the cytoskeleton do not seem to be responsible for the properties of the bound glycoprotein. The presence of immobile CSP fibrils does not, however, impede the diffusion of a lipid probe, a ganglioside analogue, or various surface antigens. Therefore, the fibrils apparently do not form a "barrier" across the lipid phase of the plasma membrane. In contrast, concanavalin A binds to CSP and is largely immobile in regions rich in CSP. The presence of immobile concanavalin A receptors in areas or on cells lacking CSP indicates that other types of immobile concanavalin A receptors also exist.CSP does not bind to lipid bilayers composed of phosphatidylcholine or oxidized cholesterol. It does bind to dextran-coated bilayers as a diffuse distribution of mobile molecules that can patch after addition of antibodies to CSP. The latter result suggests that CSP molecules do not interact strongly with other CSP molecules under these conditions. Exogenous CSP binds to regions on the cell surface that already bear CSP. In view of the apparent weakness of CSP-CSP interactions on the lipid bilayer, it seems possible that the assembly of CSP fibrils is nucleated by cell surface components in addition to CSP.

Antigens↗
PubMed1976

Dynamics of fluorescence marker concentration as a probe of mobility.

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.

Biological Transport↗
PubMed1976

Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.

We have made direct, quantitative measurements of the lateral motion and age-dependent distribution of acetylcholine receptors (AChR) on the surface of rat myotubes in primary culture. AChR were fluorescently marked with tetramethylrhodamine-labeled alpha-bungarotoxin and AChR lateral motion was measured by the fluoresence photobleaching recovery technique. We found two coexisting distinct classes of AChR: (i) mobile, uniformly distributed AChR that appear on all myotubes shortly after fusion from myoblasts; and (ii) immobile, dense, highly granular AChR in patches of 10-60 mum size that appear shortly after fusion and disappear after myotubes have become extensively interconnected. In addition, evidence of turnover of AChR labeled with tetramethylrhodamine-alpha-bungarotoxin is seen in the gradual internalization of surface fluorescence within 36 hr after labeling. The relevance of these results to an understanding of the membrane dynamics and localization of muscle AChR is discussed.

Age Factors↗
PubMed1976

Lateral transport on cell membranes: mobility of concanavalin A receptors on myoblasts.

We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor complexes on the plasma membrane of cultured myoblasts of rat. Transport rates were measured by observing the recovery of fluorescence in a small region of the cell surface initially photobleached irreversibly by an intense, focused laser light pulse. Under different conditions we measured effective diffusion coefficients of the receptor complexes in the range 8 x 10(-12) less than D less than 3 x 10(-11) cm2/sec which is two orders of magnitude lower than we found for a fluorescent lipid probe, D approximately (8 +/- 3) x 10(-9) cm2/sec. This large difference and the presence of apparently immobile concanavalin A receptors suggests that factors beyond the fluoidity of the phospholipid bilayer membrane matrix control the rate of lateral transport of the complexes. Effective mobilities of the complexes decrease with increases in the valence, dose, and occupation time of the lectin on the membrane. These properties imply an aggregation of the lectin-receptor complexes. Mobilities are not influenced by azide, colchicine or preincubation at low temperature. Cytochalasin B and low temperatures, during the time of measurement, decrease the lateral transport rate.

Azides↗
PubMed1976

Measurement of lateral transport on cell surfaces.

This paper describes the principles and recent applications of two new methods for measuring rates of macroscopic lateral transport of fluorescent-labeled particles on the surface of individual cells. Both methods are based on microfluorimetric measurements of fluorescence intensity from a small open region (greater than or equal to 1 micronm radius) on the cell surface. Transport rates are measured from the rates at which the measured fluorescence intensity changes due to entrance or departure of fluorophores from the region. One method, "Fluorescence Photobleach Recovery" (FPR), uses a brief intense pulse of light to create an initial concentration gradient over the spot by irreversible photochemical destruction of fluorophores. The rate of fluorescence recovery due to transport of unbleached fluorophores into the observation region is the primary experimental datum. The other method, "Fluorescence Correlation Spectroscopy" (FCS), is based on a statistical analysis of spontaneous fluctuations of numbers of fluorophores in the observation region and does not require a perturbation to generate an initial concentration gradient. FCS is mainly useful to measure relatively fast processes (D greater than or equal to 10(-9) cm 2/sec) in stable systems; FPR can be used to measure both slow and fast transport in less stable systems. Using both FCS and FPR, the diffusion coefficient of a fluorescent lipid probe in rat myoblast plasma membranes was measured to be D = (9 +/- 4) X 10(-9) cm2/sec over a range of at least 4 micronm. FPR was used to measure the lateral mobility of the fluorescent labeled lectin concanavalin A complexed to myoblast plasma membrane "receptors." A fraction of the complexes were immobile on the time scale of the experiment (D less than 8 x 10(-12) cm2/sec). The remainder of the complexes had effective diffusion coefficients far smaller than expected from the measurements on the lipid probe (8 x 10(-12) less than or equal to D less than or equal to 3.3 x 10(-11) cm2/sec). Moreover the mobility depended on the valence, dose, and time of occupancy of the lectin on the membrane, suggesting that an aggregation of complexes was occurring during the experiment. Cytochalasin B decreased the mobility of complexes, suggesting an influence of microfilaments on the transport process. Neither azide nor colchine affected measured transport rates. These results indicate the operation of constraints on the mobility of the lectin-receptor complexes beyond that exerted by the viscous resistance of the lipid bilayer membrane matrix. The two types of interactions revealed by our current experiments - interactions of the complexes with microfilaments and with each other (aggregation) - seem insufficient to account entirely for the low observed mobility.

Animals↗
PubMed1972

Properties of the refolding and unfolding reactions of ribonuclease A.

Both the refolding kinetics and unfolding kinetics of ribonuclease A have been measured at the same final conditions, as a function of temperature at pH 3.9, by stopped-flow (pH-jump) experiments; absorbance changes at 240 and 286.5 nm were measured. Refolding follows first-order kinetics in the upper two-thirds of the thermal transition zone. Under the same conditions, the unfolding kinetics are biphasic; the terminal phase has the same rate constant as refolding. The biphasic kinetics of unfolding demonstrate the presence of intermediate states. Since both the refolding and unfolding kinetics are consistent with a simple sequential model, the intermediates satisfy kinetic criteria for being on the direct pathway of unfolding. At temperatures just above the transition zone, the fast phase of unfolding becomes the major kinetic phase. The rate of the slow unfolding reaction increases rapidly with temperature, and approaches the average rate of the fast phase at temperatures just above the transition zone. The entire set of kinetic results can be reproduced semiquantitatively by assignment of values to four parameters in a cooperative sequential model. However, reasons are given for the belief that this simple model will have to be generalized before it can give a realistic description of the kinetics of the unfolding reaction.

Animals↗
PubMed1971

The sequential unfolding of ribonuclease A: detection of a fast initial phase in the kinetics of unfolding.

Temperature-jump studies have been used to detect a rapid reaction in the thermal unfolding of ribonuclease A (RNase A). The fast reaction occurs over a wide range of pH, and the results of a detailed study at pH 1.3 are reported here. Although its amplitude is small, the reaction is easily measurable over the entire temperature range of thermal unfolding. It occurs in the millisecond time range, and is faster by 3-4 orders of magnitude than the slow unfolding reaction studied previously. Unfolding is measured here by the change in absorbance at 287 nm, which reflects the exposure to solvent of buried tyrosine groups. Since the fast reaction has a detectable amplitude only in the temperature range of unfolding, it apparently detects the presence of intermediate, partly-folded states. Previous equilibrium studies of the unfolding of RNase A in the pH range 1-2 have indicated that it is essentially a 2-state reaction, without detectable intermediates. The existence of a rapid transient phase in the unfolding of RNase A had been predicted previously from a model for this unfolding reaction, based on nucleation-dependent sequential folding. The model served to reconcile kinetic and equilibrium studies of the thermal unfolding reaction of RNase A at neutral pH. Kinetic studies had shown that the slow unfolding reaction, measured at 287 nm, could be represented as a single exponential process, as expected for a 2-state reaction. However, earlier equilibrium measurements, especially the calorimetric studies of Sturtevant and coworkers, had revealed significant deviations from the 2-state behavior at neutral pH. These conflicting observations are explained by the model, which satisfies closely many criteria for a 2-state unfolding, even when appreciable concentrations of partly folded molecules are present. In particular, it predicts that the final, and major, portion of the kinetic reaction will occur as a single process characterized by an exponential time course.

Chemical Phenomena↗