Hydrodynamic properties of rigid macromolecules composed of ellipsoidal and cylindrical subunits.

A procedure is devised for the calculation of hydrodynamic properties of rigid macromolecules composed subunits that are modeled as ellipsoids of revolution and cylinders. Owing to the axial symmetry of these shapes, smooth shell models can be constructured for the subunit structure. The bead shell model so constructed is employed for the calculation of the properties. A computer program, HYDROSUB, has been written implementing both the model building and the hydrodynamic calculation. A detailed example of the use of this methodology is presented for the case of the solution properties of the human antibody molecule immunoglobulin G3 (IgG3). Finally, hints are given on other uses and applications of the procedure.     

Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment

The transmembrane potential on a cell exposed to an electric field is a critical parameter for successful cell permeabilization. In this study, the effect of cell shape and orientation on the induced transmembrane potential was analyzed. The transmembrane potential was calculated on prolate and oblate spheroidal cells for various orientations with respect to the electric field direction, both numerically and analytically. Changing the orientation of the cells decreases the induced transmembrane potential from its maximum value when the longest axis of the cell is parallel to the electric field, to its minimum value when the longest axis of the cell is perpendicular to the electric field. The dependency on orientation is more pronounced for elongated cells while it is negligible for spherical cells. The part of the cell membrane where a threshold transmembrane potential is exceeded represents the area of electropermeabilization, i.e. the membrane area through which the transport of molecules is established. Therefore the surface exposed to the transmembrane potential above the threshold value was calculated. The biological relevance of these theoretical results was confirmed with experimental results of the electropermeabilization of plated Chinese hamster ovary cells, which are elongated. Theoretical and experimental results show that permeabilization is not only a function of electric field intensity and cell size but also of cell shape and orientation.     

Microscopic description of voltage effects on ion-driven cotransport systems

A microscopic model for the analysis of voltage effects on ion-driven cotransport systems is described. The model is based on the notion that the voltage dependence of a given rate constant is directly related to the amount of charge which is translocated in the corresponding reaction step. Charge translocation may result from the movement of an ion along the transport pathway, from the displacement of charged ligand groups of the ion-binding site, or from reorientation of polar residues of the protein in the course of a conformational transition. The voltage dependence of overall transport rate is described by a set of dimensionless coefficients reflecting the dielectric distances over which charge is displaced in the elementary reaction steps. The dielectric coefficients may be evaluated from the shape of the experimental flux-voltage curve if sufficient information on the rate constants of the reaction cycle is available. Examples of flux-voltage curves which are obtained by numerical simulation of the transport model are given for a number of limiting cases.     

Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.     

On the mechanics of viscous bodies and elongation in ellipsoidal cells

After deriving some auxiliary equations for the average elongation of a viscous body under the action of forces derived from a potential, the diffusion problem for an ellipsoidal cell with a constant rate of reaction is solved for the case of an infinite permeability. The equation of elongation of such a cell under the influence of diffusion forces is derived, and compared with the, approximate expression obtained by N. Rashevsky for any kind of oblong cell. The two equations are in fair agreement. Effects of constant and variable surface tension are studied.     

ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage.

ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts.     

Stereological analysis of the guinea pig pancreas. I. Analytical model and quantitative description of nonstimulated pancreatic exocrine cells

A stereological model which provides detailed quantitative information on the structure of the fasted, nonstimulated gland has been developed for the guinea pig pancreas. The model consists of morphologically defined space and membrane compartments which were used to describe the general composition of the tissue and the specific components of exocrine cells. The results are presented, where appropriate, relative to a cubic centimeter of pancreas, a cubic centimeter of exocrine cell cytoplasm, and to the volume of an average exocrine cell. The exocrine cells, accounting for 82% of the pancreas volume, consisted of 54% cytoplasmic matrix, 22% rough-surfaced endoplasmic reticulum (RER), 8.3% nuclei, 8.1% mitochondria, 6.4% zymogen granules, and 0.7% condensing vacuoles. Their total membrane surface area was distributed as follows: 60% RER, 21% mitochondria, 9.9% Golgi apparatus, 4.8% plasma membranes, 2.6% zymogen granules, 1.8% plasma membrane vesicles, and 0.4% condensing vacuoles. The application of this model to the study of membrane movements associated with the secretory process is discussed within the framework of an analytical approach.     

Formation of the transmembrane electrochemical potential on Staphylococcus cells and membrane vesicles

The generation of transmembrane difference of electrochemical potentials was registered on the intact cells and ultrasonication-obtained membrane vesicles of Staphylococcus aureus with the application of transmembrane electrophoresis of permeant anions, potassium transport in the presence of valinomycin and 8-anilinonaphthalene-1-sulphonate fluorescence. The membrane potential is formed when the chain of electron transfer or H+-ATPase functions or when the pH gradient varies (the nonenzymic pathway). M-chlorinecarbonylcyanidephenylhydrazonium, a protonophore uncoupler potassium cyanide, an inhibitor of the respiratory chain, N',N-dicyclohexylcarbodiimide, an inhibitor of ATPase, cause the membrane potential dissipation. The orientation of the transmembrane electric field is as follows: "minus" inside cells and "plus" inside membrane vesicles.     

The transmembrane domain of surfactant protein C precursor determines the morphology of the induced membrane compartment in CHO cells

Surfactant protein C (SP-C) is a small lipopeptide of which the main part consists of a typical valyl-rich transmembrane domain. The protein is expressed as a propeptide (proSP-C) which is processed and sorted via the regulated secretory pathway to the lamellar body, where mature SP-C is stored before secretion into the alveolar space. In this study we investigated the identity of the compartment to which proSP-C is sorted in cells that do not have a regulated secretory pathway, such as CHO cells. By electron microscopy we determined that proSP-C was localized in an uncommon membrane compartment with very regular morphology, which was not present in control cells. This membrane compartment is not influenced by the palmitoylation of proSP-C and is probably derived from the endoplasmic reticulum. However, proSP-C chimeras with artificial transmembrane domains induced a membrane compartment with a different morphology. Therefore we propose that the typical amino acid sequence of the transmembrane domain of proSP-C plays a role in membrane formation and morphology, which may be relevant under physiological conditions.     

Autonomous transmembrane segment S4 of the voltage sensor domain partitions into the lipid membrane

The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2±0.2kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9?. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence.     

Use of an oriented transmembrane protein to probe the assembly of a supported phospholipid bilayer.

Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer.     

The relationship between sensitivity to 60-Hz electric fields and induced transmembrane potentials in plants root cells

Summary Growth rates and cell diameters were determined from 12 species of plant roots exposed to a 60-Hertz (Hz) electric field of 360 Volts per meter (V/m) in an aqueous inorganic nutrient medium [conductivity: 0.07–0.09 Siemens per meter (S/m)]. The degree of growth depression ranged from zero to nearly 100 percent of control. Cell diameters ranged from 13.5 to 31.8 µm as an averaged value for procambial, cortical, and meristem cells. Sensitivity to the electric field as determined by root growth rate reduction increased with increasing cell size. Sensitivity also increased with increase in 60 Hz induced transmembrane potentials; the transmembrane potential threshold for growth reduction was about 6.0 mV and the potential for near-complete cessation of growth was about 10–11 mV.Two different hypothetical mechanisms of action by which applied electric fields induce biological effects at the cellular level were tested. The two mechanisms pertain to different possible modes of action of applied electric fields: one mechanism postulates the involvement of the transmembrane field, the other mechanism postulates the tangential electric field as the important factor for inducing biological effects. The data support the transmembrane and not the tangential field mechanism. It is concluded that the effects observed are consistent with a membrane related mechanism and that there is a narrow range (a few mV) between threshold and debilitating induced membrane potentials.