Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGA-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 × 10^?11 to 2 × 10^?10 cm²/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.     

Photobleaching recovery studies of membrane events accompanying lectin stimulation of rabbit lymphocytes.

Fluorescence photobleaching recovery methods reveal marked changes in lateral mobilities of rabbit lymphocyte membrane components during the course of stimulation with succinyl concanavalin A (S Con A). The diffusion constant of S Con A receptors on T lymphocytes falls from 1.6×10^?10 cm²/sec to 6.5×10^?11 cm²/sec within 4 hr after stimulation, remains constant for 14 hr, and returns to its former value. The mobility of B cell receptors similarly falls from 1.4×10^?10 cm²/sec to 5.5×10^?11 cm²/sec but regains its unstimulated value much more slowly. In contrast, a fluorescent phospholipid analog shows constant mobilities of 1.9×10^?8 cm²/sec and 1.5×10^?8 cm²/sec in T and B cells, respectively, throughout the experiment.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.     

Translational diffusion in the plasma membrane of sea urchin eggs

Translational diffusion in the plasma membrane of individual egg cells from the sea urchin species Paracentrotus lividus has been studied by fluorescence microphotolysis (FM). In order to probe the lipid phase of the membrane, procedures have been worked out by which the fluorescent analog 3,3′-dioctadecyl-oxatricarbocyanine (C₁₈diO) can be incorporated into the membrane. In the unfertilized egg a fraction R = 0.9 of C₁₈diO was mobile having an apparent diffusion coefficient of D = 6.0 × 10^?9 cm² sec^?1. Fifteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 2.7 × 10^?9 cm² sec^?1, respectively. In order to study diffusion of membrane proteins, procedures have been worked out by which the cell surface can be labeled with fluorescein-isothiocyanate (FITC). FITC binds to both the plasma membrane and the vitelline layer. Together with the vitelline layer two-thirds of the FITC-fluorescence could be removed from the egg surface. Gel electropherograms of isolated egg cortices showed various protein bands; however, only two of the protein bands were labeled with FITC. In the unfertilized egg a fraction R = 0.9 of the FITC-labeled membrane proteins was mobile having an apparent diffusion coefficient of D = 35 × 10^?11 cm² sem^?1. Fiteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 7.0 × 10^?11 cm² sec^?1, respectively. FITC-labeled proteins of the fertilization envelope were immobile. Our studies have shown (i) that the egg surface can be fluorescently labeled without blocking fertilization and early development, (ii) that the plasma membrane of unfertilized eggs is a fluid environment permitting a rapid movement of lipids and proteins, and (iii) that after fertilization a substantial degree of lipid and protein mobility is maintained.     

Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity become extreme upon fertilization

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (～50%) has a fivefold smaller lateral diffusion coefficient (D = 1.5 × 10^?8 cm²/sec) in the animal than in the vegetal plasma membrane (D = 7.6 × 10^?8 cm²/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (>100×) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements (D ? 10^?10 cm²/sec), whereas at the vegetal side D is only slightly reduced (D = 4.4 × 10^?8 cm²/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.     

The concentration dependence of the hemoglobin mutual diffusion coefficient

Laser correlation Spectroscopy was used to measure the mutual diffusion coefficient, D, of human cyanomethemoglobin (Fe⁺⁺⁺:CN) at varying protein concentrations. These measurements were male at 20°C in a 0.1 M phosphate buffer solution at pH 7.0. For low protein concentrations we find D = (6.43 ± 0.26) × 10^?7 cm²/S and that there is a near linear decrease from this value at higher concentrations. The linear relation between the diffusion coefficient and protein concentration allows us to deduce the value of the linear frictional volume fraction coefficient, K_f= 7.75. and to extrapolate to hemoglobin concentrations equivalent to that in the red blood cell where we estimate D = 4.25 × 10^?7 cm²/s Various theoretical predictions of the dependence of the mutual diffusion coefficient on concentration are tested; we find that the generalized Stokes-Einstein relation can be made to fit our high concentration data if we assume a hard-sphere model and if we include a term involving a hydrodynamic interaction integral.     

Translational diffusion coefficient of α-chymotrypsinogen A by laser-light-scattering spectroscopy

The translational diffusion coefficient of a pure sample of α-chymotrypsinogen A is measured by laser light scattering to give a value of D_20,w⁰ = (8.40 ± 0.15) × 10^?7 cm²/sec.     

Physical properties of type i collagen in solution: Structure of α-Chains and β- and γ-components and two-component mixtures

The structure of thermally denatured Type I collagen has been studied using laser light scattering. The results indicate that the diffusion coefficients of α-chains and β- and γ-components are 1.550 ± 0.08 × 10^?7, 1.000 ± 0.05 × 10^?7, and 0.835 ± 0.04 × 10^?7 cm²/sec, respectively, at temperatures between 20 and 40°C. It is concluded from diffusion data that these species have hydrodynamic radii of about 13.8 nm (α-chain), 21.5 nm (β-component), and 25.7 nm (γ-component), consistent with previous studies of thermal denaturation by light scattering. It is also concluded, based on volume calculations, that a large volume increase occurs when the triple helix unfolds. Homodyne correlation functions for two component mixtures of α-chains and β-and γ-components appeared to decay exponentially. In all but one case discussed the correlation function could be fitted with a single component having a translational diffusion coefficient which was an intensity weighted average of the diffusion coefficient of each component present.     

Hydrodynamics,size, and shape of bacteriophage T4D tails and baseplates

We have used translational diffusion coefficient measurements and subunit hydrodynamic theory to determine the dimensions and shape of bacterioophage T4D baseplates and tails. The diffusion coefficient of the baseplate, measured by quasielastic laser light scattering (QLS), was determined previously by Wagenknecht and Bloomfield to be D = 8.56 × 10^?8 cm²/s. For the tail, we found D = 5.88 × 10^?8 cm²/s by QLS, and D = 6.02 × 10^?8 cm²/s by combining sedimentation coefficient and molecular weight in the Svedberg equation. These values, which have an uncertainty of ±2.7%, when combined with subunit hydrodynamic theory, enabled us to refine estimates of dimensions obtained by electron microscopy. For the hexagonal baseplate, the vertex-to-vertex distance is about 480 Å, the thickness is 160 Å, and there are six extended short fibers 320-Å long and 40 Å in diameter. When a baseplate of these dimensions is attached to a tail tube-sheath-connector complex 1050-Å long and 240 Å in diameter, the calculated D is 5.93 × 10^?8 cm²/s, within 1% of experiment. This combined use of electron microscopy and hydrodynamics, using the former to ascertain shape, and the latter to obtain solution dimensions, is a powerful approach to the structure of biomolecular complexes.     

Antigen-specific mouse lymphocyte stimulation by DNP-conjugated T-independent antigens studied by photobleaching recovery

Fluorescence photobleaching recovery techniques have allowed us to measure the lateral mobility of T-independent antigens bound to antigen-specific mouse B cells. The in vitro immunogenicity or tolerogenicity of antigens we have examined, DNP-polymerized flagellin (DNP-POL), and DNP-linear dextran (DNP-DEX), depend upon the antigen dose and epitope density. These factors also determine the mobility of antigen bound to B cell surfaces. For DNP-POL bound to DNP-specific cells, the observed diffusion constants D decrease monotonically with increasing antigen dose and epitope density. Values of D range from 10.4 × 10^?11 cm² sec^?1 for DNP_0.4-POL at 0.15 μg/ml to 0.8 × 10^?11cm² sec^?1for DNP_3.5-POL at 30 μg/ml. For receptor-bound DNP-DEX, D depends strongly on antigen epitope density but not observably on antigen concentration. For epitope densities of 1.2 or less, D is close to the value of 21 × 10^?11cm²sec^?1 observed for single slg receptors. By an epitope density of 4.8, D has fallen to 2.1 × 10^?11cm²sec^?1. Peak immunogenicities for DNP-POL and DNP-DEX arc observed when antigen- receptor aggregates have mobilities 14-fold and 3-fold lower, respectively, than a single slg molecule.     

Lateral diffusion and patch formation of H-2Kk antigens on mouse spleen lymphocytes

We have studied the diffusion and aggregation of H-2K^k antigens labeled with a fluorescent anti-H-2K^k monoclonal antibody (IgG) on mouse splenic lymphocytes, employing fluorescence photobleaching recovery and fluorescence microscopy. The H-2K^k antigens were initially distributed homogeneously on all lymphocytes. Upon antibody binding, sub-micron patches were formed on 50–60% of the cells. A lateral diffusion coefficient, D, of 7.1·10^?10 cm²/s and a mobile fraction of 0.73 were found for H-2K^k antigens on diffusely-labeled cells, while these antigens were immobile (D?5·10^?12 cm²/s) on patched cells. The patched and nonpatched sub-populations did not correspond to B- and T-lymphocytes. Subjection to low temperature or treatment with NaN₃ or cytoskeleton-disrupting drugs did not affect the diffusion or patching of H-2K^k, indicating no involvement of metabolic energy or drug-sensitive cytoskeletal components. These findings could be related to the interactions of H-2 antigens on the cell surface, and to the different susceptibilities of various cells to lysis by cytotoxic T-cells.     

An aminopeptidase from Agave americana,isolation and physical characterization

A new aminopeptidase was isolated from Agave americana by fractionation, chromatography and gel filtration. The mw of the enzyme, determined by different procedures was 86000 ± 1500; the enzyme had a sedimentation coefficient of 4.96 S, a diffusion coefficient of 5.2 × 10^?7 cm²/sec, a Stokes radius of 3.8 nm, a partial specific volume of 0.733 cm³/g, a frictional ratio of 1.40, a molecular absorbancy index at 280 nm of 8.36 × 10⁴, an isoelectric point of 4.53 and contained 1.25% carbohydrate. The amino acid composition of the enzyme was determined and the aminoterminal residue was identified as lysine whilst the carboxylterminal residue was either leucine or isoleucine. No subunit structure was observed for the enzyme.     

Rates of Shrinkage and Growth of Air Bubbles Entrained in Wheat Flour Dough

The rates of shrinkage at constant temperature, and growth under a temperature rise below 100°C, of bubbles entrained in wheat flour dough were analyzed and compared with those of a bubble in water. The rate of shrinkage of bubbles in flour dough was controlled by the diffusion of dissolved air from the surface of bubbles to the bulk of flour dough. The apparent diffusion coefficient of the dissolved air in wheat flour dough with the water fraction of 0.49 calculated from the shrinkage of bubbles, was (3.2 ± 1.5) × 10^1?1 m²/sec (19°C), and (6.4 ± 2.0) × 10^?11 m²/sec (42°C). However, the growth behavior of bubbles in flour dough under a temperature rise was very different from that predicted from the diffusion theory. The critical radius of bubbles to grow was larger than that estimated from the diffusion theory. The mechanism of growth of bubbles in wheat flour dough, which was different from that of a bubble in water, is a subject that needs to be clarified.     

Studies on Sugar-isomerizing Enzyme Purification,Crystallization and Some Properties of Glucose Isomerase from Streptomyces sp.

Glucose isomerase was purified by means of acetone fractionation, DEAE-cellulose column chromatography, DEAE-Sephadex column chromatography and crystallization. The purified enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis. The sedimentation coefficient, s_20,w, the diffusion coefficient, D_20,w, and partial specific volume of the enzyme were 8.0S, 4 × 10^?7cm²/sec and 0.69 ml/g, respectively. The molecular weight of the enzyme was estimated to be 157,000 from the sedimentation and diffusion measurements. The crystalline glucose isomerase contained cobalt and magnesium ions. The properties of the enzyme were also studied.     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998     

Physical and Chemical Properties of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Some physical and chemical properties of the extracellular lipase from the thermophilic fungus, Humico la lanuginosa S–38, were investigated. The results were as follows: Sedimentation coefficient was 2.4 × 10^?13 (cm-g/sec-dyne); diffusion coefficient was 8.8 × 10^?7 (cm²/sec); and frictional coefficient was 1.22. Molecular weight was 27,500±500 and α-helix content was 18.9%. The number of amino acid residues contained in 1 mole of protein of Humicola lipase was 224. Sugar and lipid were not detected. The effect of calcium ion and denaturing reagents, such as urea, sodium dodecyl sulfate and dithiothreitol, on the thermostability of Humicola lipase was examined. It was concluded that the thermostability of Humicola lipase was not influenced by protective cofactors but was attributable to the enzyme itself. Some properties of enzyme structure which were concerned with the thermostability of Humicola lipase are also discussed.     

The two-cross immunodiffusion technique: diffusion coefficients and precipitating titers of IgG in human serum and rabbit serum antibodies.

The two-cross technique, a new two-dimensional double-diffusion technique in gelplates, has been applied for simultaneous determination of precipitating titers and diffusion coeffients of antigen and antibody in body fluids. The advantage of this technique is that it works without using any standard solution and ensures conditions of “time-invariant sink”. The theory of the technique has been verified by experimental results on the precipitating system human serum-rabbit anti-human IgG in phosphate-buffered saline solution at pH 7.4. The results obtained using several modes of calculations from experimental parameters have been compared and found satisfactory. The accuracy and reproducibility of the results have been confirmed. It has been found that at 20°C the diffusion coefficient of human IgG in 10-times-diluted serum is (4.4 ± 0.2) × 10^?7 cm² s^?1, while the diffusion coefficient of rabbit anti-human IgG in a purified preparation is (2.9 ± 0.2) × 10^?7 cm² s^?1. The critical precipitating concentration of human IgG against rabbit anti-human IgG is invariable to concentration and amounts to 0.174 ± 0.03 mg/100 ml at pH 7.4.