Electromobile surface charge alters membrane potential changes induced by applied electric fields.

The relation between extracellular electric fields and changes in membrane potential that such fields directly induce has previously been described both theoretically and experimentally. It is clearly established that extracellular electric-field-induced membrane potential changes are well described by Poisson's equation of electrostatics. A modification of this simple theory to include effects of the electric-field-induced redistribution of charged cell surface components is introduced and is shown to produce major alterations in calculated membrane potential changes over times of the order of minutes to hours. Implications for biological systems which respond to extracellular electric fields are discussed.     

Scanning tunneling microscopy of human erythrocyte membranes.

Images of surfaces of human erythrocyte ghosts, lecithin liposomes, spectrin, erythrocyte membrane skeleton, concanavalin A and concanavalin A--decorated erythrocyte ghosts were obtained by scanning tunneling microscopy. The dimensions and surface topography of some membrane structures are described and discussed.     

Hydrophobic thickness of fluid planar monooleylglycerol membranes maximally thinned by inversed micellisation

A procedure of making membranes of amphiphilic materials at the bottom of a U-shaped flexible plastic tube within an aqueous medium is described. The membranes were made sufficiently large in order for the annulus area to be neglected. Consequently the hydrophobic thickness of the membrane could be measured by a capacitance technique assuming the relative permittivity of the hydrophobic part of the bilayer. Introduction of an AC microvolt technique allowed manufacture of stable thick membranes by quenching the electroconstriction observed when DC electrical potentials in the millivolt range are used. By continuously monitoring the hydrophobic thickness and by use of the AC microvolt technique the membrane-thinning process by chemical means could be studied in isolation because the electroconstriction was quenched. The maximally thinned hydrophobic thickness of a monooleylglycerol membrane measured at 38 degrees C was found to be 25+/-1.2 A. Criteria and argumentation for maximal thinning of the membrane are put forward. A distinction between genuine and modified cholesterol was demonstrated to be possible by the described method.     

ISOLATION OF THE PLASMA MEMBRANE OF THE LUMINAL SURFACE OF RAT BLADDER EPITHELIUM, AND THE OCCURRENCE OF A HEXAGONAL LATTICE OF SUBUNITS BOTH IN NEGATIVELY STAINED WHOLE MOUNTS AND IN SECTIONED MEMBRANES

A method of isolating the thick luminal membrane from homogenates of bladder epithelium is described, which entails pretreatment of the epithelium with fluorescein mercuric acetate and centrifugation of the homogenate on sucrose density gradients. A hexagonal array of hexamers is illustrated by negative contrast staining in whole mounts of the isolated thick membrane. Subunits are also shown in tangential sections of this thick membrane, in fixed, embedded bladder epithelium. The significance of the subunits is discussed in the context of membrane structure and permeability.     

Surface-induced lamellar orientation of multilayer membrane arrays. Theoretical analysis and a new method with application to purple membrane fragments.

The orientation of membrane fragments into a lamellar array by a flat surface is analyzed. This analysis includes processes such as centrifugation and drying and physical effects due to membrane fragment steric interactions, finite size, elasticity, and thermal fluctuations. Several model calculations of optimal orientational order in multilayer membrane arrays are presented. The predictions of a smectic A model agree quantitatively with the measured spatial dependence of the fluctuations in layer orientation in a multilamellar arrays. A new technique, based in part on this analysis, for the preparation of well-oriented multilamellar arrays of natural and artificial membranes, isopotential spin-dry centrifugation, is described. The method involves the use of specially designed inserts for the buckets of a standard vacuum ultracentrifuge. The membrane fragments to be oriented are sedimented from solution or suspension onto a substrate of a convenient material which forms a gravitational isopotential surface at high g. Sedimentation is accompanied by removal of the suspending medium at high g to produce oriented films with a selected degree of solvation. In addition, a method is described whereby small solute molecules can be maintained in constant concentration with the membrane fragments during this process. Initial application of the method to the orientation of purple membrane fragments is described. The degree of orientation obtained in this system is evaluated using freeze-fracture and scanning electron microscopy, optical birefringence, linear dichroism, and microscopy.     

An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins

A new method for the measurement of rapid isotopic release from a membrane compartment is described. Membrane vesicles loaded with isotope, or broken membranes with bound radioactive ligand, are filtered onto the surface of a cellulose ester filter; the rate of the loss of isotope from the membrane compartment is followed continuously by collecting fluid which is passed through the filters under high pressure. A change in release rate is initiated by changing the solution or by delivering a flash of light to a photosensitive sample. The approach has been used to study rapid 22Na efflux from membrane vesicles rich in the ouabain-sensitive Na pump, and to examine dissociation of 32P and 86Rb from membrane-bound Na,K-ATPase. Since the rate of efflux is measured, and not the total counts remaining on the filter, the technique has high sensitivity. A complete time course is obtained using only a few micrograms of membrane protein. The apparatus described is simple, inexpensive, and easily constructed; with the present device, time resolution is approximately 10 ms.     

Galactosyltransferase: multiple forms in serum of normal and hepatoma Mc-29 bearing chickens and from liver and hepatoma microsomal and plasma membrane preparations

The multiple forms of galactosyltransferase in chicken serum and in microsomal and plasma membrane preparations from liver and viral induced hepatoma Mc-29 have been studied by isoelectric focussing. An elevation of the hepatoma plasma membrane enzyme activity was described and in the pattern of the multiple forms of the enzyme two forms were found (pI-5.34 and 8.22) which were similar to those described in the serum of hepatoma bearing chickens (pI-5.36 and 8.24). A conclusion is drawn that these enzyme forms are apparently present to a greater extent in the hepatoma plasma membrane enriched fractions than in liver membranes and are probably shed into the serum of the tumor bearing animals.     

Preparative capillary electrophoresis based on adsorption of the solutes (proteins) onto a moving blotting membrane as they migrate out of the capillary.

A micropreparative capillary electrophoresis apparatus equipped with a new type of fraction collection device is described: solutes, such as proteins, are adsorbed onto a moving blotting membrane (for instance a polyvinylidene difluoride membrane) as they migrate electrophoretically out of the capillary. The adsorbed proteins are visualized by conventional protein staining methods or by fluorescent labeling. Specific identification of separated components by an immunological technique is demonstrated. The method also offers the potential to analyze proteins and peptides collected on the membrane by gas phase sequencing and mass spectrometry.     

Uptake across the cell envelope and insertion into the inner membrane of ion channel-forming colicins in E coli.

Pore-forming colicins exert their lethal effect on E coli through formation of a voltage-dependent channel in the inner (cytoplasmic-membrane) thus destroying the energy potential of sensitive cells. Their mode of action appears to involve 3 steps: i) binding to a specific receptor located in the outer membrane; ii) translocation across this membrane; iii) insertion into the inner membrane. Colicin A has been used as a prototype of pore-forming colicins. In this review, the 3 functional domains of colicin A respectively involved in receptor binding, translocation and pore formation, are defined. The components of sensitive cells implicated in colicin uptake and their interactions with the various colicin A domains are described. The 3-dimensional structure of the pore-forming domain of colicin A has been determined recently. This structure suggests a model of insertion into the cytoplasmic membrane which is supported by model membrane studies. The role of the membrane potential in channel functioning is also discussed.     

The formation of basal body domains in the membrane skeleton of Tetrahymena

Differentiated regions within the membrane skeleton are described around basal bodies in the ciliary rows of Tetrahymena. These domains, approximately 1 micron in diameter, are characterized by a relatively dense ultrastructure, the presence of a family of proteins called K antigens (Mr 39-44 x 10(3)) that are recognized by mAb 424A8, and the apparent exclusion of major membrane skeleton proteins that are present in most other regions of the cell (Mr 135, 125 x 10(3]. Mature basal body domains are asymmetric, reflecting the polarity of the cell as a whole. A similar differentiation of the membrane skeleton occurs in the oral apparatus, except here the K antigens surround four clusters of basal bodies (from which this cell takes its name) rather than the individual basal bodies. The development of new basal body domains in the cell cycle is described, with similarities and differences noted between somatic and oral regions of the cell. It is concluded that the capacity of this cell for precise topographic regulation of molecular events in the membrane skeleton makes it a useful model for the study of cortical differentiation in cells generally.     

Isolation and characterization of Neurospora crassa plasma membranes.

The isolation and characterization of plasma membranes from a cell wall-less mutant of Neurospora crassa are described. The plasma membranes are stabilized against fragmentation and vesiculation by treatment of intact cells with concanavalin A just prior to lysis. After lysis, the concanavalin A-stabilized plasma membrane ghosts are isolated by low speed centrifugation techniques and the purified ghosts subsequently converted to vesicles by removal of the bulk of the concanavalin A. The yield of ghosts is about 50% whereas the yield of vesicles is about 20%. The isolated plasma membrane vesicles have a characteristically high sterol to phospholipid ratio, Mg2+-dependent ATPase activity and (Na+ plus K+)-stimulated Mg2+ATPase activity. Only traces of succinate dehydrogenase and 5'-nucleotidase are present in the plasma membrane preparations.     

In vitro effects of vitamin D3 on the phospholipids of isolated renal brush border membranes

A model system is described in which cholecalciferol (vitamin D3) is incorporated into phosphatidylcholine liposomes and then the liposomes are incubated in vitro with isolated renal brush border membrane vesicles. The incubation results in an alteration of the phospholipid composition, the fluidity, and the transport properties of the membrane. The findings provide evidence consistent with the hypothesis that vitamin D3 and metabolites modify membrane structure and function by "liponomic regulation."     

STUDIES ON THE OXIDATIVE METABOLISM OF SACCHAROMYCES CEREVISIAE : I. Observations on the Fine Structure of the Yeast Cell

Vegetative cells of Saccharomyces cerevisiae were fixed with potassium permanganate followed by uranyl nitrate, embedded in methacrylate, and studied in electron micrographs of thin sections. Details of the structure of the cell wall, cytoplasmic membrane, nucleus, vacuole, and mitochondria are described. Cell membranes, about 70 to 80 A thick, have been resolved into two dense layers, 20 to 25 A thick, separated by a light layer of the same dimensions, which correspond in thickness and appearance to the components of the "unit membrane" as described by Robertson (15). The cell wall is made up of zones of different electron opacity. Underlying the cell wall is the cytoplasmic membrane, a sinuous structure with numerous invaginations. The nucleoplasm, often of uneven electron opacity, is enclosed in a pair of unit membranes in which nuclear pores are apparent. The vacuole, limited by a single unit membrane, is usually irregular in outline and contains some dense material. Rod-shaped mitochondria, 0.4 to 0.6 µ in length and 0.2 to 0.3 µ in diameter, are smaller in size, but similar in structure to some of those described in plant and animal cells. Attempts to use osmium tetroxide as fixative were unsuccessful, a result similar to that obtained by other workers. It is suggested that yeast cells are impermeable to osmium tetroxide, except when grown under specific conditions.     

pH-Dependent membrane interactions of diphtheria toxin: A genetic approach

A genetic approach is described for exploring the mechanism by which diphtheria toxin undergoes pH-dependent membrane insertion and transfer of its enzymic A fragment into the cytoplasm of mammalian cells. The cloned toxin expressed inEscherichia coli is secreted to the periplasmic space, where it is processed normally and folds into a native structure. When bacteria synthesizing the toxin are exposed to pH 5, they die rapidly. The toxin undergoes a conformational change that is believed to allow it to be inserted into the bacterial inner membrane and form channels, which proves lethal for the cell. The membrane insertion event mimics the process by which the toxin inserts into the endosomal membrane of mammalian cells, leading to release of the enzymic A fragment into the cytoplasm. The observation of pH-dependent bacterial lethality provides the basis for a positive genetic selection method for mutant forms of the toxin that are altered in ability to undergo membrane insertion or pore formation.     

The use of polyvinylidenedifluoride membranes as a general blotting matrix

A fast chemical staining of protein by Coomassie blue on a stable nonshrinking membrane of polyvinylidenedifluoride is described. The procedure is especially useful for detection of nonlabeled marker proteins in comparison to specifically detected proteins. The membrane is highly suitable for other chemical stains like silver, gold, or india ink for immunostaining, lectin binding, and binding of radioactive ligands like 45Ca. Blocking of the background against nonspecific binding to proteins was studied using different reagents. A multiple slit plate is described for volume-saving simultaneous application of multiple reactants on one protein-loaded membrane.     

5'-Nucleotidase activity of lymphocyte plasma membranes. Effect of concanavalin A]

The 5'-nucleotidase properties of isolated lymphocyte plasma membranes from young pig mesenteric nodes are described; nucleosides-5'-monophosphates are the substrates of this specific enzyme. Concanavalin A inhibits this enzyme; on the same membranes this mitogen does not affect alkaline phosphatase and activates the membrane bound (Ca2+) ATPase. The 5'-nucleotidase inhibition is due to a specific interaction of Con A with carbohydrate groups of the membrane; its high positive cooperativity suggests that the lectin promotes reorganization of the membrane bound 5'-nucleotidase. Solubilization of the 5'-nucleotidase does not prevent the effect of Con A and the solubilized enzyme is firmly bound by Con A-Sepharose 4B; these results suggest that Con A inhibits the enzyme by a direct interaction and that 5'-nucleotidase can be considered as an eventual receptor for the lectin.     

A Rapid and Reproducible Hcmogenization Procedure for the Isolation of Plasma Membranes from Rat Liver

A simplified modification of the Neville procedure for the isolation of plasma membranes from rat liver is described in which cells are broken by low-shear homogenizetion with a Polytron homogenizer. Plasma membranes are recovered from the homogenates by differential and discontinuous sucrose gradient centrifugetions. The procedure provides plasma membrane fractions enriched 25-fold for AMPase, a marker enzyme for the plasma membrane of rat liver, with a combined contamination from endoplasmic reticulum, Golgi apparatus and mitochondria of less than 10% The procedure is uncomplicated, reproducible, and yields enzymatically active plasma membrane fractions of high purity.     

Selective Mycobacteriophage Isolation with Membrane Filters

A simple technique is described whereby mycobacteriophages are selectively isolated by adsorption on mycobacteria layered within membrane filter sandwiches before standard enrichment procedures.     

Partial purification of an oxygen scavenging cell membrane fraction for use in anaerobic biochemical reactions

Anaerobic conditions are necessary to prevent the autoxidation of tetrahydrobiopterin. An enzymatic method for achieving such an anaerobic condition is shown to be obtained by a lactate oxidase activity in the membrane of Escherichia coli when the enzyme concentration exceeds 0.05 unit/ml. A procedure is described for partial purification of the membrane fragment that involves salt precipitation and gel sieve chromatography on Bio-Gel A-50m. The rate of removal of oxygen is used to define the enzyme activity, to determine stability during storage, and to define conditions for stabilization of tetrahydrobiopterin. A second assay procedure that measures the rate of reduction of resazurin is described and is useful when the enzyme concentration is low.     

HELA CELL PLASMA MEMBRANES : I. 5'-Nucleotidase and Ouabain-Sensitive ATPase as Markers for Plasma Membranes

A method for the preparation of HeLa cell plasma membrane ghosts is described. The purity of the plasma membrane fraction was examined by phase contrast and electron microscopy, by chemical analysis, and by assay of marker enzymes. Data on the composition of the plasma membrane fraction are given. It was observed that the distribution pattern of 5'-nucleotidase activity among the subcellular fractions differed from that of ouabain-sensitive ATPase. In addition, the specific activity of 5'-nucleotidase did not follow the distribution of the membrane ghosts. Thus, this enzyme would seem unsuitable as a plasma membrane marker. A complete balance sheet for marker enzyme activities during the fractionation is necessary for the calculation of increase in specific activity because the activities of both 5'-nucleotidase and ouabain-sensitive ATPase might change during the fractionation procedures.