Biomedical subjects
Publications and source records attributed to L Bachmann.
The nucleotide sequences of nine clones, pKA191/1-4 from Drosophila kitumensis and pMR190/1-5 from D. microlabis, were determined. They represent a tandemly arranged and highly repetitive satellite DNA family, KM190, which is specific for the two species.
The proteasome or multicatalytic proteinase from the archaebacterium Thermoplasma acidophilum is a 700 kDa multisubunit protein complex. Unlike proteasomes from eukaryotic cells which are composed of 10-20 different subunits, the Thermoplasma proteasome is made of only two types of subunit, alpha and beta, which have molecular weights of 25.8 and 22.3 kDa, respectively. In this communication we present a three-dimensional stoichiometric model of the archaebacterial proteasome deduced from electron microscopic investigations. The techniques which we have used include image analysis of negatively stained single particles, image analysis of metal decorated small three-dimensional crystals after freeze-etching and STEM mass measurements of freeze-dried particles. The archaebacterial and eukaryotic proteasomes are almost identical in size and shape; the subunits are arranged in four rings which are stacked together such that they collectively form a barrel-shaped complex. According to a previous immunoelectron microscopic investigation, the alpha-subunits form the two outer rings of the stack, while the two rings composed of beta-subunits, which are supposed to carry the active sites, are sandwiched between them. Each of the alpha- and beta-rings contains seven subunits; hence the stoichiometry of the whole proteasome is alpha 14 beta 14 and the symmetry is 7-fold. Image simulation experiments indicate that the alpha- and beta-subunits are not in register along the cylinder axis; rather it appears that the beta-rings are rotated with respect to the alpha-rings by approximately 25 degrees. In contrast to some previous reports we have not been able to find stoichiometric amounts of RNA associated with highly purified proteolytically active proteasome preparations.
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Pyruvate dehydrogenase complex (pyruvate : lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1), from pig heart, was studied by spray freeze etching and negative staining. From freeze etching experiments an average particle weight of 7-10(6) was estimated. Negative staining after glutaraldehyde fixation and freeze etching of unfixed and prefixed enzyme solutions yielded no significant difference in particle dimensions: the majority of the isometric complex molecules measured approximately 400 A in diameter. Tantalum tungsten shadowed freeze etch replicas indicated that the surface of the complex is built up of globular units. The relative positions of these units are in good agreement with the model still under discussion.
In paramecia the membranes of alveoli and trichocysts are permanently connected to the cell membrane by membrane-junctions, which consist of membrane-intercalated particles in a regular geometrical arrangement. Trichocysts contain secretory material discharged by exocytosis. In unfixed or fixed cells these two compartments were impermeable to the following tracers: To "microperoxidases", i.e. a cytochrome c-derived heme-nonapeptide and a heme-undecapeptide (WM approximately 1650, 1900) applied in vivo, as well as to lanthanum and cytochrome c used during (La) or after (cytochrome c) fixation. The heme-nonapeptide was prepared by TPCK trypsin digestion of cytochrome c and subsequent purification by Sephadex gel chromatography--a simple and inexpensive new procedure resulting in preparations of high yield and purity. Tracers entered alveoli only when the plasmalemma and the alveolar membranes ruptured upon glutardialdehyde fixation. In no case were transmembraneous channels detectable in regions containing membrane-intercalated particles; this holds true for all tracers used and for freeze-fracture replicas obtained by tantalum-tungsten evaporation. With regard to attachment sites over trichocysts our results do not support the assumptions by others according to which exocytosis would be driven by an osmotic shift via transmembraneous channels (which would be analogous to inter-cellular coupling phenomena mediated by gap-junctions), unless such channels would be assumed to operate as carriers rather than via diffusion. Tracers did not penetrate trichocysts before exocytosis occurred. The functional role of membrane-intercalated particles on trichocyst attachments remains unclear. Despite some resemblance with gap-junctions all types of intra-cellular membrane-junctions investigated are functionally "tight" at the level of "resolution" obtained with tantalumtungsten-shadowing and with the tracers used.
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