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W Hoppe

Publications and source records attributed to W Hoppe.

At least 37 records · Page 2Linked to original sources
PubMed1986

Negatively stained 50 S ribosomal subunits of Escherichia coli.

Ribosomes are large nucleoproteins of approximately 3 X 10(6) Mr. In contrast to helical or spherical nucleoproteins (viruses) of similar size (which consist of several hundred small asymmetric units reproduced by symmetry), ribosomes are completely asymmetric; therefore, the amount of structural information (defined by the number of independent image elements) necessarily increases from about 10 to 20 to about 1000 to 2000 (at resolutions of the order of 2 nm). With present techniques, only stained single particles can be studied in the electron microscope. Our published work on the 30 S subunit and on the 50 S subunit has demonstrated that three-dimensional reconstructions of stained single particles show a great number of structural details that are reproducible if the particles have the same orientation. One of the main results of this paper is the final proof of this reproducibility from detailed comparisons of individual 50 S subunits and of independent averages over a few (3 to 5) particles in the kidney or crown orientation; in the latter case, even after a chemical modification. The 50 S subunit is non-uniformly stained along the optical axis. It displays a complicated, stain-filled channel-like structure, within which is approximately the partial volume expected for the RNA. The particle shows an irregular but reproducible boundary surface against the stain. At several sites, the channel structure protrudes to the surface. Since the secondary structure of the RNA is well known, one might try to locate it in the subunit after chemical identification of its surface contacts (the 3' end of 23 S RNA and the 3' end of the 5 S RNA have been localized). Most interesting is a groove on the surface, which might accommodate the mRNA.

Computer Simulation↗
PubMed1986

Post-translational phosphorylation of proteodermatan sulfate.

In cultured human skin fibroblasts, the core protein of the small proteodermatan sulfate becomes phosphorylated post-translationally but before the glycosaminoglycan chains are synthesized. This phosphorylation can occur when the intracellular transport is inhibited by carbonyl cyanide m-chlorophenylhydrazone or when the attachment of asparagine-linked oligosaccharides is prevented by tunicamycin. Serine and glycosaminoglycan chains were identified as phosphorylation sites of secreted proteodermatan sulfate. Upon alkaline borohydride treatment and degradation by chondroitin ABC lyase, the main phosphorylated product co-chromatographed with an unsulfated 3H-labeled hexasaccharide prepared analogously from [3H]galactose/[35S]sulfate-labeled proteodermatan sulfate.

Cells, Cultured↗
PubMed1986

Biosynthesis and processing of proteodermatan sulphate.

The biosynthesis and processing of the small iduronic acid-rich proteodermatan sulphate (PDS) was studied in cultured human skin fibroblasts and arterial smooth muscle cells (SMC) with the aid of core-directed antibodies and various inhibitors of protein synthesis, intracellular transport, and glycosylation. Components of the linkage region became attached to the core protein most likely in a pre-Golgi compartment. Phosphorylation of PDS precursors also occurred in the endoplasmic reticulum with a minor contribution by the Golgi complex. Serine residues and the linkage region were identified as phosphorylated species in secreted PDS. Blockade of transport by monensin did not affect 6-sulphation but affected uronic acid epimerization and 4-sulphation. On relief from the monensin block, additional sulphation along the glycosaminoglycan chain was possible, whereas chain elongation was as in the continuous presence of the drug. Asparagine-bound oligosaccharides or glycosaminoglycan chains were not required for secretion of PDS or core protein. PDS from fibroblast and SMC secretions differed markedly in the composition of the glycosaminoglycan chains. No significant difference, however, was found on isoelectric focusing of core protein and after limited proteolysis of chondroitin ABC lyase-treated core protein. Tryptic and chymotryptic peptide maps of iodinated core proteins were similar.

Arteries↗
PubMed1986

Criteria for the comparison of electron microscopical 3D reconstructions of individual particles.

Similarity criteria for electron microscopical 3D reconstructions can be defined in different ways. It is shown that the choice of the zero level in 3D reconstructions plays an important role, (a) for the discrimination of stained versus non-stained structural parts ("discrimination level"), and (b) for the physical definition of the signal ("zero potential level"). Of special interest are further structural features (artifacts) introduced by the reconstruction method chosen (single axis tilting in our papers). A crude estimate of the zero potential level is only possible if we neglect electron scattering from light atoms (concept of the "holey stain structure"). We calculate for the reconstructions of stained ribosome particles (W. Hoppe and H. Oettl and H. Tietz, J. Mol. Biol., in press) a signal-to-noise ratio of the order 3:1 (smaller by a factor of ca. 3 than the ratio calculated from electron noise). This explains why structural details can be recognized in single-particle reconstructions and why averages over only a few particles can already lead to consistent results. An analysis of the contrast decrease along the optical axis shows that this result is only true for strongly stained structural parts; averaging over large numbers of particles is required for the structural study of weakly stained parts. Possibilities of pattern recognition are discussed. The structural feature complementarity is explained by means of a model example; it can drastically reduce cross-correlation coefficients.

Microscopy, Electron↗
PubMed1985

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts.

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.

Cells, Cultured↗
PubMed1983

Three-dimensional reconstruction and averaging of 30 S ribosomal subunits of Escherichia coli from electron micrographs.

From the micrographs of a tilt series, several particles of negatively stained 30 S ribosomal subunits of Escherichia coli were three-dimensionally reconstructed. Three of them showing similar orientation with respect to the supporting foil were averaged after alignment by newly developed three-dimensional correlation methods. As a main result we found a stained channel-like structure inside the particle. We tentatively propose that this corresponds, at least partially, to positively stained segments of the 16 S RNA.

Escherichia coli↗
PubMed1982

Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants.

The three-dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes were analyzed by imaging negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer-aided three-dimensional reconstruction. The information on electron micrographs obtained at 6 degrees angular increments from +60 degrees to -60 degrees was digitized by densitometric scanning, Fourier-transformed, corrected for imaging errors, cross-correlated, and synthesized to the three-dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle, indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16-nm height, 2.0-nm wall thickness, and a 3.0-nm torus at the free end with an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane-binding site, and only small pores of up to 2.0-nm in diameter penetrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of approximately 4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest the MAC may mediate increased membrane permeability by protein channel formation in addition to lipid reorientation.

Cell Membrane↗