Search PubMed⌕ Search

Biomedical subjects

R E Webster

Publications and source records attributed to R E Webster.

At least 73 records · Page 4Linked to original sources
PubMed1978

Association of the major coat protein of fd bacteriophage with phospholipid vesicles.

The association of the major coat protein of fd bacteriophage with a phospholipid bilayer was investigated by analyzing the protein's susceptibility to proteolysis and its circular dichroism spectrum when incorporated into single-walled phospholipid vesicles. In the limits tested, this association appeared to be independent of the mass ratio of protein to lipid and of vesicle size, phospholipid composition, and method of preparation. The circular dichroism data are consistent with a similar "membrane-bound" conformation for all cases of vesicle-associated coat protein and for deoxycholate micelle-associated coat protein. Proteolysis of coat protein associated with deoxycholate micelles and with phospholipid vesicles defined the central hydrophobic core presumed to represent that portion of the protein which associates with membrane bilayers in vivo. The isolated core, which assumed a predominantly beta-type conformation in detergent solution, maintained a beta conformation when associated with a vesicle phospholipid bilayer.

Amino Acid Sequence↗
PubMed1978

Three-dimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin- and tubulin-containing structures.

Cytoskeletons prepared by Triton X-100 treatment of tissue culture cells appear in stereo electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. Microfilament bundles and also tonofilament-like bundles are readily discerned when present in the cell type. In addition thinner fibers, some of which branch (smallest diameter 30--40 A), as well as fibers of larger diameter, some of which correspond to microtubules, can be seen. Since such cytoskeletons are an open, membrane-free system, individual fibrous organizations can be identified by specific antibodies. An indirect immunoferritin procedure using antibodies to tubulin or actin visualizes microtubules or actin-containing structures. Stereo electron microscopy of cytoskeletons decorated with actin antibody reveals, in addition to the F-actin-containing microfilament bundles, an extended fine actin lattice. This actin net is displayed throughout the cytoplasm not only between the microfilament bundles but also in those regions of the cytoskeleton that in the intact cell correspond to the submembraneous regions. Thus all actin-containing fibrous cytoplasmic structures may be interconnected in the living cell.

Actins↗
PubMed1978

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell.

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

Animals↗
PubMed1978

Effect of membrane-associated f1 bacteriophage coat protein upon the activity of Escherichia coli phosphatidylserine synthetase.

The effects of insertion of the major coat protein of f1 bacteriophage into Escherichia coli membranes were investigated under conditions allowing in vivo analysis of phosphatidylserine synthesis. An E. coli strain possessing a temperature-sensitive phosphatidylserine decarboxylase was utilized under conditions in which the decarboxylase activity was reduced but nonlethal. The presence of the coat protein in the host membranes inhibits the activity of the phosphatidylserine synthetase and perhaps affects the activity of the phosphatidylserine decarboxylase.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
PubMed1977

Adsorption protein of bacteriophage fl: solubilization in deoxycholate and localization in the fl virion.

A complex containing the minor coat protein or adsorptionprotein (A protein) of bacteriophage fl has been solubilized from the fl virion, using the detergent deoxycholate. This complex was resolved from the fl DNA and from the fl major coat protein, or B protein, by gel filtration in the presence of deoxycholate. The A protein complex migrated as a single band on sodium dodecyl sulfate-urea-polyacrylamide gels corresponding to a molecular weight of 60 000. Analysis of the amino acid composition and amino terminal residues of this preparation indicates that the preparation contains a 20% contamination of additional protein species. Antibody against purified fd A protein is cross-reactive with deoxycholate-purified fl A protein and with fl phage. Electron microscopic observation of negatively stained complexes of fl phage with this anti-fd A protein antibody and ferritin conjugated goat anti-rabbit IgG antibody revealed phages with ferritin particles at their termini or complexes of two or more phages joined together at one end by ferritin, indicating that the complex of A protein molecules is located at one end of the filamentous fl virion.

Adsorption↗
PubMed1977

Effect of cessation of phospholipid synthesis on the synthesis of a specific membrane-associated bacteriophage protein in Escherichia coli.

The major coat protein of the bacteriophage f1 is synthesized during infection of Escherichia coli and becomes tightly associated with the host membrane. This synthesis was studied in conjunction with the strain BB26-36, a mutant defective in phospholipid synthesis, to investigate basic questions concerning membrane protein and phospholipid synthesis. Coat protein synthesis is decreased in the absence of net phospholipid synthesis. The coat protein produced under these conditions is still found tightly associated with the membrane. Resumption of phospholipid synthesis leads to an increase in the synthesis and accumulation of the coat protein. Therefore, a correlation between coat protein and phospholipid synthesis seems to exist. However, the packaging of phage deoxyribonucleic acid into phage particles proceeds in the absence of phospholipid synthesis, and the number of phage particles produced appears to depend only on the amount of coat protein in the membrane.

Bacterial Proteins↗
PubMed1976

Lipid-protein interactions in Escherichia coli. Membrane-associated f1 bacteriophage coat protein and phospholipid metabolism.

The effects of insertion of the major coat protein of f1 bacteriophage into Escherichia coli membranes were investigated. The relative level of phosphatidylethanolamine decreased due to the failure to accumulate phosphatidylethanolamine when the cellular levels of phosphatidylglycerol and cardiolipin were increasing. This decreased accumulation was correlated with a 4-fold reduction in phosphatidylethanolamine synthesis. A 10- to 20-fold increase in cardiolipin content resulted from both a 3-fold increase in cardiolipin synthesis and a decrease in cardiolipin turnover. As long as cell division and protein synthesis continued, the number of cardiolipin molecules per coat protein molecules in the bacterium attained a constant value. The coat protein had little effect of phosphatidylglycerol synthesis. This data suggests that the coat protein froms a specific association with cardiolipin in the host membranes. Additional evidence suggests that cardiolipin also may facilitate the entry of coat protein into membranes.

Binding Sites↗
PubMed1975

The in vitro translation of a terminating signal by a single Escherichia coli ribosome. The fate of the subunits.

A complex was isolated containing one 70 S ribosome bound to the RNA of the f2 bacteriophage in the region of the coat gene. Either the f2 RNA and 70 S ribosome or both ribosomal subunits in this complex were differentially labeled with tritiated lysine or phosphorous 32 RNA. Incubation of this complex in a ribosome-free protein-synthesizing system lacking initiating factor and appreciable nuclease activities allowed the synthesis of coat protein and subsequent termination. Using this techique, it was found that termination resulted in the release of ribosome as subunits. Intermediate in this process is a transient 46 S complex composed of the f2 RNA and 30 S ribosomal subunit, presumably the result of the initial release of the 50 S ribosomal subunit. This 46 S complex subsequently breaks down to a free f2 RNA and 30 S ribosomal subunit. The rate of this latter process is faster in the presence of crude initiating factors. These results are discussed in terms of the mechanism of termination and reading of the intercistronic region.

Bacterial Proteins↗
PubMed1975

Interaction of deoxycholate and of detergents with the coat protein of bacteriophage f1.

The major coat protein of bacteriophage f1, which is localized in the host membrane during phage maturation, has a hydrophobic binding site capable of binding deoxycholate and a variety of detergents to form a soluble particle, and in that respect, resembles many membrane proteins. The soluble particle has properties that suggest it is formed by simple insertion of protein into a deoxycholate or detergent micelle, but molecular weight measurements show that the protein is present as a dimer, even in sodium dodecyl sulfate, indicating the existence of unusually strong forces for self-association. A by-product of the investigation has been to show that detergents can be very helpful in the fractionation of the constituent molecules of the virus: deoxycholate-solubilized virus is readily fractionated by gel chromatography into DNA, A protein, and B protein, with virtually no cross-contamination.

Binding Sites↗
PubMed1975

Proteolytic digestion of the micellar complex of f1 coat protein and deoxycholate.

The major coat protein of bacteriophage f1 radioactively labeled with specific amino acids was solubilized with deoxycholate and digested with trypsin or alpha-chymotrypsin. The degree of proteolysis of the coat protein was assayed by gel filtration chromatography of the digest in the presence of deoxycholate. Hydrolysis occurred at residues in the hydrophilic termini of the coat, releasing peptides containing proline, lysine, and phenylalanine. No cleavage occurred at the tyrosine or methionine residues in the hydrophobic core. However, chymotrypsin could cleave somewhat at these residues in the absence of deoxycholate. A model for the topography of the micellar complex of coat protein and deoxycholate is presented in which the hydrophobic sequence of the coat is bound to deoxycholate within a micelle, while the hydrophilic termini of the coat project from the micelle.

Amino Acid Sequence↗