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Biomedical subjects

M Schindler

Publications and source records attributed to M Schindler.

At least 145 records · Page 8Linked to original sources
PubMed1985

Lateral diffusion in nuclear membranes.

Chemical modification of rat liver nuclei with citraconic anhydride selectively removed outer nuclear membrane. This conclusion was based on (a) transmission electron microscopy, (b) lipid analysis, (c) lamin B as an inner membrane-associated marker, and (d) the demonstration of phospholipid lateral mobility on outer membrane-depleted nuclei as a criteria for inner membrane integrity. Addition of urea or N-ethylmaleimide resulted in the additional disruption of inner membrane. Fluorescence photobleaching was used to determine the long range (greater than 4 microns) lateral transport of lectin receptors and a phospholipid analog in both membranes. The diffusion coefficient for wheat germ agglutinin on whole nuclei was 3.9 X 10(-10) cm2/s whereas the diffusion coefficient for wheat germ agglutinin in outer membrane-depleted nuclei was less than or equal to 10(-12) cm2/s. Phospholipid mobilities were the same in whole and outer membrane-depleted nuclei (3.8 X 10(-9) cm2/s). The protein diffusion differences observed between whole and outer membrane-depleted nuclei may be interpreted in the context of two functionally different membrane systems that compose the double bilayer of the nucleus.

Animals↗
PubMed1984

Structural transitions of porin, a transmembrane protein.

Conformational transitions of porin were monitored using 3 independent criteria: (i) oligomeric state as observed by SDS-polyacrylamide gel electrophoresis; (ii) spectroscopic titrations (ultraviolet and circular dichroism) and (iii) chemical modifications. Four pH-dependent transitions were observed with half-maximal changes occurring at pH values of 1.6, 3.5, 11.2 and 12.4. Two of these pH values differ significantly from intrinsic pK values of the constituent amino acids of this membrane protein. Since porin is very polar despite its location predominantly within the outer membranes, this may be due to ion pair formation in the hydrophobic environment of the membrane.

Bacterial Proteins↗
PubMed1984

Alterations in nuclear anatomy by chemical modification of proteins in isolated rat liver nuclei.

Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, approximately 2-4s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while approx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA [less than or equal to 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli, and ribonucleoprotein (predominantly hnRNA group A and B). These nucleic acid-deficient structures have been termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants, or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA treatment), but missing the double bilayer and a 51K polypeptide that is a major component of the other structures. The use of chemical modification on the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve two functions: (a) as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; (b) offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.

Animals↗
PubMed1983

Lectin receptors on the plasma membrane of soybean cells. Binding and lateral diffusion of lectins.

Protoplasts prepared from suspension cultures of root cells of Glycine max (SB-1 cell line) bound soybean agglutinin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA). Binding studies carried out with 125I-labeled SBA, Con A, and WGA showed that these interactions were saturable and specific. Fluorescence microscopy demonstrated uniform membrane labeling. The mobility of the lectin-receptor complexes was measured by fluorescence redistribution after photobleaching. The diffusion constants (D) for SBA and Con A were 5 X 10(-11) and 7 X 10(-11) cm2/s, respectively. In contrast, WGA yielded a diffusion constant of 3 X 10(-10) cm2/s. Pretreatment of the protoplasts with either SBA or Con A resulted in a 6-fold reduction in the mobility of WGA (D congruent to 5 X 10(-11) cm2/s). The results suggest that the binding of SBA or Con A may lead to alterations of the soybean plasma membrane which, in turn, may restrict the mobility of other receptors.

Cell Membrane↗
PubMed1982

Lateral mobility of cytochrome c on intact mitochondrial membranes as determined by fluorescence redistribution after photobleaching.

Lateral mobility of an active fluorescent derivative of cytochrome c on the membranes of giant mitochondria was measured by fluorescence redistribution after photobleaching. A diffusion coefficient of 1.6 X 10(-10) cm2/sec was determined for the labeled cytochrome c on inner mitochondrial membranes under conditions where succinate oxidase activity was demonstrated. This relatively low rate of diffusion, together with results of other investigators, is explained in terms of a model involving a dynamic equilibrium between freely diffusing and associated forms of electron-transfer components.

Animals↗
PubMed1982

Chemical modification of matrix porin from Escherichia coli: probing the pore topology of a transmembrane protein.

Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta-octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with increasing pH or upon denaturation. Citraconic anhydride modified approximately 12-14 residues in native porin and 15-16 of the total of 19 amino groups in the denatured state. Fluorescamine, another amine-specific reagent of intermediate size, derivatized 3 and 16 residues in the native and denatured states, respectively. These results indicate that reactive probes of various sizes may serve as indicators for the surface accessibility of reactive residues in matrix porin. The increased derivatization of lysyl residues at high pH (or in phosphate buffer) suggests the method's sensitivity to different conformational states of the protein. The extent of tyrosine modification (1-2 residues in the native, and approximately 22 in the denatured porin) depended on the state of protein folding, even with reagents of small size. The approach of using various probes with differing properties and specificities thus appears useful for the determination of membrane protein asymmetry, pore topology, and conformational states of transmembrane proteins.

Bacterial Proteins↗
PubMed1981

Matrix control of protein diffusion in biological membranes.

Lateral diffusion coefficients of fluorescently labeled lipids and integral membrane proteins were determined in the membranes of normal and spectrin-deficient spherocytic mouse erythrocytes by the technique of fluorescence redistribution after photobleaching. The results were used to generate a mathematical description of a matrix-control model of membrane protein diffusion. In the spherocytic cells, which lack the principal components of the cytoskeletal matrix of normal cells, the diffusion coefficients of lipid (1.5 +/- 0.5 X 10(-8) cm2/s) and protein (2.5 +/- 0.6 X 10(-9) cm2/s) differ only by a factor of 6, close to the difference predicted on the basis of size by the two-dimensional bilayer continuum model of Saffman and Delbrück [Saffman, P. G. l& Delbrück, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111-3113]. In contrast, the membranes of normal cells show a lipid diffusion coefficient (1.4 +/- 0.5 X 10(-8) cm2/s) that is some 300-fold greater than that of the membrane proteins (4.5 +/- 0.8 X 10(-11) cm2/s). Analysis of these results, based on the hypothesis that protein diffusion in normal membranes is sterically hindered by a labile matrix, yields an effective matrix surface viscosity consistent with the viscoelastic mechanical properties of the membranes. Thus, a relationship is established between the deformation characteristics of the membrane and the lateral mobility of proteins suspended in the membrane.

Animals↗
PubMed1980

Lateral mobility of integral membrane proteins is increased in spherocytic erythrocytes.

Alterations of glycoprotein distribution and lateral mobility in cell membranes can provide transmembrane signals for several membrane-related phenomena. Control of the transmembranous events has been ascribed to interaction between submembranous protein matrices (or 'cytoskeletons') and membrane glycoproteins. A consequence of such interaction would be differential inhibition of protein lateral diffusion in biological membranes. Measurements of the lateral diffusion coefficients of membrane proteins, in fact, have generally yielded values much less than were predicted for unhindered diffusion in a fluid bilayer. The mouse spherocytic erythrocyte, which lacks the major components of the normal erythrocyte membrane matrix (composed of spectrin, actin, bands 4.1 and 4.9 (ref. 16), in the nomenclature of Fairbanks et al.), provides a unique system for a direct evaluation of the effect of the matrix on protein lateral mobility. After using a modification of the technique of fluorescence redistribution after photobleaching (FRAP), we report here that membrane proteins diffuse about 50 times faster in spherocytic than in normal mouse erythrocytes.

Animals↗
PubMed1980

Lateral diffusion of lipopolysaccharide in the outer membrane of Salmonella typhimurium.

Gram-negative enteric bacteria are enveloped by two membrane systems. The inner or cytoplasmic membrane is responsible for the major metabolic functions including biosynthetic activities, while the major known functions of the outer membrane are primarily physical: it contains receptors for bacteriophages and bacteriocins; it contributes to the maintenance of cell shape; and it controls access of nutrient solutes and agents such as antibiotics and detergents to the cytoplasmic membrane. Several investigations have indicated that mobility of membrane components, particularly lipopolysaccharide, is essential for biogenesis of the outer membrane, and is a primary event in phage infection. To define more accurately the fluid dynamic properties of the outer membrane as related to function, we have now developed the capability to measure lateral diffusion coefficients in vivo of rhodaminated G30 lipopolysaccharide fused into Salmonella typhimurium G30A filamentous bacteria. The method used extends the FRAP procedure (fluorescence redistribution after photobleaching) to bacteria and the results demonstrate rapid diffusion of lipopolysaccharide (D = 2.0 +/- 0.9 x 10(-10) cm2s-1) over micrometre distances.

Cell Membrane↗
PubMed1980

Lateral mobility in reconstituted membranes--comparisons with diffusion in polymers.

The diffusion coefficients (D) of lipopolysaccharide, phospholipid, and Escherichia coli matrix protein were determined in reconstituted multibilayer membranes. Over a range of protein concentration of 0--60% by weight, D for lipopolysaccharide decreased 10-fold, whereas D for phospholipid remained essentially constant. The diffusion coefficient of matrix protein at a concentration of 50% was less than or equal to 10(-12) cm2 s-1. These results are discussed in terms of a model for diffusion in polymeric networks.

Bacterial Proteins↗
PubMed1980

Lateral diffusion in biological membranes. A normal-mode analysis of diffusion on a spherical surface.

A new approach is described for the analysis of lateral diffusion in biological membranes. It is shown that a suitably defined first moment of the concentration distribution on a spherical surface decays as a single exponential with a relaxation rate proportional to the diffusion coefficient and inversely proportional to the square of the radius of the sphere. The approach is illustrated with an example of fluorescence redistribution after photobleaching of membrane proteins in a spectrin-deficient spherocytic mouse erythrocyte membrane.

Diffusion↗
PubMed1980

Modulation of membrane protein lateral mobility by polyphosphates and polyamines.

The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of "fluorescence redistribution after fusion." Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.

Adenosine Triphosphate↗
PubMed1978

Bacteriophage P22 is not a likely probe for zones of adhesion between the inner and outer membranes of Salmonella typhimurium.

Thin-section electron micrographs of plasmolyzed Salmonella typhimurium infected with bacteriophage P22 demonstrated that phage adsorbed to cells over sites of inner- and outer-membrane contact. Efforts were made to isolate such adsorption sites by infection of cells with 35S- and 32P-labeled phage and by separation of the membranes on sucrose gradients. At 37 degrees C, about 75% of the 35S radioactivity could be recovered in a region of intermediate density between the inner and outer membranes. This region (phi band) did not contain 32P. The gradient profile was independent of the multiplicity of infection (between 0.2 and 50) and of the presence or absence of chloramphenicol, dinitrophenol, or cyanide. However, ethylenediaminetetraacetate, when present during the infection step, prevented the formation of phi band. The density of phi band was at least 1.30 g/cm3, as demonstrated by prolonged centrifugation on a D2O-sucrose gradient. phi Band was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy to contain empty phage heads and contaminating cellular debris. In purified preparations, phage heads were the only structures, visible by negative staining, and very little cellular phospholipid or protein was associated with the phage proteins (less than 2% and 30% by weight, respectively, as determined by using [3H]glycerol or [3H]leucine). The residual cellular protein included all of the major outer-membrane proteins rather than any one specific protein. These results are interpreted as indicating that phi band probably does not contain adhesion site material stably associated with phage heads.

Adsorption↗