Search

This study investigates the physical basis of color effects in the detection of proteins in polyacrylamide gels by silver staining. Specifically, the hypothesis that different colors may correlate with the development of silver grains of characteristic sizes was investigated by electron microscopy. Protein bands that stained brown, yellow, and blue were excised from stained gels and prepared for electron microscopy by thin-sectioning. In each case, the size distributions of globular silver grains were determined directly from the electron micrographs. We found that blue bands have larger silver grains (with diameters of 40-100 nm) than yellow (21-39 nm) or brown bands (17-35 nm). On the basis of these and other observations, a general mechanism is proposed whereby chemical specificity of electrophoretically separated proteins is expressed in color-specific silver staining.

We investigated the cholesterol content of highly purified populations of coated vesicles from rat liver by biochemical quantitation and by cytochemical electron microscopy using the polyene antibiotic filipin. Failure of this reagent to elicit its typical response for a cholesterol-containing membrane, i.e., a characteristically corrugated or rippled appearance by thin section analysis, had led to the hypothesis (Montesano, R., A. Perrelet, P. Vassalli, and L. Orci, 1979, Proc. Natl. Acad. Sci. USA., 76:6391-6395) that cholesterol is specifically excluded from the plasma membrane domains associated with coated pit regions. The present electron microscopic results showed that although the response of coated vesicle membranes to filipin was also negative, uncoated vesicles whose clathrin coats had been removed in vitro exhibited a strong filipin-positive response. Quantitated biochemically, the cholesterol-to-phospholipid ratio of the coated vesicles was found to be indistinguishable from that of control preparations of plasma membranes isolated from rat liver. Taken together, the results indicate that the filipin-negative response of coated vesicles (and probably also that of coated pits) is due not to abnormally low cholesterol content, but rather to the stabilizing influence of their enveloping clathrin coats which inhibit the characteristic structural expression of the filipin-cholesterol complexes.

Cells of Bordetella pertussis BP353, a nonfimbriated Eldering serotype 1.3 strain, were used as an immunogen to produce three monoclonal antibodies, BPE3, BPD8, and BPE8, that agglutinated the immunizing cells, as well as certain other nonfimbriated and fimbriated serotype 3-containing B. pertussis strains. The antibodies did not agglutinate serotype 1 or nontypable B. pertussis cells. These monoclonal antibodies specifically detected a 69-kilodalton (kDa) band on Western blots (immunoblots) containing whole B. pertussis cell lysates of Eldering agglutinogen serotypes 1.3, 1.3.6, 1.2.3.4, and 1.2.3.4.6. This 69-kDa antigen was released from the bacteria by cell incubation for 60 min at 60 degrees C, and it was purified by affinity chromatography with a BPE3-agarose affinity matrix. Purified material was used to produce a polyclonal antiserum that agglutinated all nonfimbriated and fimbriated B. pertussis cells containing serotype 3 agglutinogen. Immunogold electron microscopy and indirect immunofluorescence studies demonstrated that it is an outer membrane constituent but nonfimbrial in appearance. BPE3 did not detect purified fimbriae on Western blots, and antibodies to these fimbriae did not bind to the 69-kDa component. Although B. bronchiseptica and B. parapertussis cells were not agglutinated by the monoclonal antibodies, antigenically similar proteins were detected in extracts of the bacteria. These results identify the 69-kDa protein as a nonfimbrial agglutinogen present on all virulent strains of B. pertussis. The monoclonal antibodies described here should be useful for further studies on the structure and function of this protein.

The structures of the hexavalent capsomers of herpes simplex virus type 2 were analyzed by negative staining electron microscopy of capsomer patches derived from partially disrupted nucleocapsids. Optimally computer-averaged images were formed for each of the three classes of capsomer distinguished by their respective positions on the surface of the icosahedral capsid with a triangulation number of 16; in projection, each capsomer exhibited unequivocal sixfold symmetry. According to correspondence analysis of our set of capsomer images, no significant structural differences were detected among the three classes of capsomers, as visualized under these conditions. Taking into account information from images of freeze-dried, platinum-shadowed nucleocapsid fragments, it was established that each hexavalent capsomer is a hexamer of the 155-kilodalton major capsid protein. The capsomer has the form of a sixfold hollow cone approximately 12 nm in diameter and approximately 15 nm in depth, whose axial channel tapers in width from the outside towards the inner capsid surface.

The helical structures of Bordetella pertussis fimbriae of serotypes 2 and 6 were determined by optical diffraction analysis of electron micrographs of negatively stained paracrystalline bundles of purified fimbriae. The fimbrial structure is based on an axial repeat of 13 nm that contains five repeating units in two complete turns of a single-start helix. This structure was confirmed by direct measurements of mass per unit length for individual fimbriae performed by dark-field scanning transmission electron microscopy of unstained specimens. These data further established that the helically repeating unit is a monomer of fimbrial protein (Mr congruent to 22,000 for type 2 and Mr congruent to 21,500 for type 6). Radial density profiles calculated from the scanning transmission electron micrographs showed that the fimbria has peak density at its center, i.e., no axial channel, consistent with the results of conventional negative-staining electron microscopy. The radial profile gives an outermost diameter of approximately 7.5 nm, although the peripheral density is, on average, diffuse, allowing sufficient intercalation between adjacent fimbriae to give a center-to-center spacing of approximately 5.5 nm in the paracrystals. Despite serological and biochemical differences between type 2 and type 6 fimbriae, the packing arrangements of their fimbrial subunits are identical. From this observation, we infer that the respective subunits may have in common conserved regions whose packing dictates the helical geometry of the fimbria. It is plausible that a similar mechanism may underlie the phenomenon of phase variations in other systems of bacterial fimbriae.