Search PubMed⌕ Search

Biomedical subjects

H Tiedge

Publications and source records attributed to H Tiedge.

30 records · Page 2Linked to original sources
PubMed1992

Immunoelectron microscopy of enzymes, multienzyme complexes, and selected other oligomeric proteins.

The collective term "immunoelectron microscopy" subsumes a number of techniques in which the biological material is decorated with specific antibodies, prior to being visualized in the electron microscope. In this article, we have reviewed literature on immunoelectron microscopy that focusses on the analysis of the molecular architecture of proteins, in particular of enzymes and of multienzyme complexes. Molecular immunoelectron microscopy has been remarkably successful with multi-subunit enzymes of complex quaternary structures, and in many cases the data have been the basis for the eventual development of detailed three-dimensional molecular models. The elucidation of subunit composition and juxtaposition of a given enzyme, an important accomplishment in itself, has in turn stimulated and guided discussions on the catalytic mechanism; illustrative examples include F1 ATPase and citrate lyase, among others. Here we have chosen a variety of enzymes, multienzyme complexes, and non-enzymatic proteins to demonstrate the versatility of immunoelectron microscopy, to illustrate methodological prerequisites and limitations, and to discuss significance and implications of individual immunoelectron microscopy studies.

Animals↗
PubMed1991

Dendritic location of neural BC1 RNA.

In nerve cells, a specialized protein synthetic machinery is thought to operate in local compartments of dendrites, in particular beneath synaptic junctions, and thereby to facilitate swift adjustments of the postsynaptic protein repertoire in situ. This notion has been supported by the identification of polyribosomes and selected mRNAs in those compartments. In this study, we report the discovery of a specific RNA polymerase III transcript in dendrites. This RNA, a noncoding, 152-nucleotide-long, single-gene transcript known as BC1 RNA, is expressed almost exclusively in the nervous system. In adult rats as well as in immature rats in late developmental stages, BC1 RNA has been located in the dendrites and somata of a subset of neurons in the central and peripheral nervous system. The colocalization of BC1 RNA with dendritic mRNAs and polyribosomes may indicate a role--possibly within the functional unit of a high molecular mass ribonucleoprotein particle--in specific pre- or posttranslational processes in postsynaptic compartments of neurons.

Animals↗
PubMed1991

The use of UV light as a cross-linking agent for cells and tissue sections in in situ hybridization.

UV cross-linking is introduced as a novel method to stabilize tissue on microscopic slides and to immobilize target molecules in biological material for in situ hybridization. UV illumination dramatically improves the stability of tissue sections and isolated cells on slides coated with gelatin/poly-lysine. Even during prolonged high-stringency washes, specimens remain firmly attached to the support layer. At the same time, the signal intensity is increased significantly whereas background levels remain as low as without UV illumination. These results indicate that while target RNAs and other molecules in the biological material are covalently cross-linked with their nearest neighbor molecules, tissues nonetheless remain penetrable, and target molecules remain accessible. We expect that this simple and efficient technique will find widespread applications in in situ hybridization methodology.

Animals↗
PubMed1988

Diazepam binding inhibitor gene expression: location in brain and peripheral tissues of rat.

Diazepam binding inhibitor (DBI), an endogenous 10-kDa polypeptide was isolated from rat and human brain by monitoring displacement of radioactive diazepam bound to specific recognition sites in brain synaptic and mitochondrial membranes. The cellular location of DBI mRNA was studied in rat brain and selected peripheral tissues by in situ hybridization histochemistry with a 35S-labeled single-stranded complementary RNA probe. DBI mRNA was heterogeneously distributed in rat brain, with particularly high levels in the area postrema, the cerebellar cortex, and ependyma of the third ventricle. Intermediate levels were found in the olfactory bulb, pontine nuclei, inferior colliculi, arcuate nucleus, and pineal gland. Relatively low but significant levels of silver grains were observed overlying many mesencephalic and telencephalic areas that have previously been shown to contain numerous DBI-immunoreactive neurons and a high density of central benzodiazepine receptors. In situ hybridizations also revealed high levels of DBI mRNA in the posterior lobe of the pituitary gland, liver, and germinal center of the white pulp of spleen, all tissues that are rich in peripheral benzodiazepine binding sites. The tissue-specific pattern of DBI gene expression described here could be exploited to further understand the physiological function of DBI in the brain and periphery.

Animals↗
PubMed1986

Interaction between the soluble F1 ATPase and its naturally occurring inhibitor protein. Studies using hydrophilic high-performance liquid chromatography and immunoelectron microscopy.

Binding of the isolated ATPase (F1) to its naturally occurring inhibitor protein was studied by two novel, independent techniques. High-pressure gel permeation chromatography revealed one tight binding site (Kd = 0.46 microM) for the inhibitor on F1, and a number of weak, non-specific sites. Use of an antibody directed against a non-binding region of the inhibitor protein demonstrated the formation of inhibitor/F1/immunoglobulin G complexes of 1:1:1 and 2:2:1 stoichiometry, but not of the putatively more stable cyclic 4:2:2 complexes. It was concluded that, despite the presence of three beta-subunits, only one site per F1 molecule is available for binding its inhibitor protein.

Animals↗
PubMed1986

Site-site interaction on mitochondrial F1-ATPase. Functional symmetry of the high-affinity nucleotide binding sites.

Interactions between the high affinity binding sites on mitochondrial F1 were analysed by combined use of the nucleotide analogues 3'-O-(1-naphthoyl)-ADP (N-ADP) and 2'-3'-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP). The binding behaviour of F1 with respect to these ligands was studied by measuring the fluorescence of F1 and of TNP-ADP and the fluorescence anisotropy of N-ADP. A total of 3 high affinity binding sites can be occupied by TNP-ADP. By exchange experiments, it could be shown that binding of TNP-ADP to such a site considerably accelerates the dissociation of a ligand bound to a neighbouring site. These results support the notion that the functional behaviour of F1 is symmetric: during the catalytic cycle any individual site can successively assume different affinity states as has been predicted by hypotheses such as the binding change model.

Animals↗
PubMed1985

Subunit stoichiometry and juxtaposition of the photosynthetic coupling factor 1: Immunoelectron microscopy using monoclonal antibodies.

Monoclonal antibodies specific to the alpha subunits of the photosynthetic coupling factor 1 (CF(1)) were used as marker molecules in an electron microscopic analysis of the subunit organization of this enzyme. Immune complexes were obtained by incubation of CF(1) with saturating amounts of anti-alpha-subunit IgG, isolated by gel filtration, and visualized by electron microscopy. The maximum number of antibodies bound to a CF(1) molecule was three, the angle defined by a neighboring pair of antibodies characteristically being 120 degrees . These results are interpreted as direct evidence for the presence of three alpha subunits in the CF(1) complex, the relative orientation of them being described by 3-fold rotary symmetry. Our observations thus favor an overall subunit stoichiometry of alpha(3)beta(3)gammadeltaepsilon.

Journal Article↗
PubMed1983

An electron microscopic approach to the quaternary structure of mitochondrial F1-ATPase.

The three-dimensional structure of F1-ATPase from beef heart mitochondria was investigated by electron microscopic techniques. The presence of high concentrations of nucleotides is essential for preservation of the quaternary structure. When investigated under such conditions, monodisperse F1-ATPase could not be distinguished from the membrane-bound enzyme. At low resolution, the particle shape resembles an oblate ellipsoid of revolution with an axial ratio of about 2:1. From several lines of evidence (including field micrographs at higher magnifications, Markham rotational analysis, and tilting experiments), two conclusions may be drawn concerning the three-dimensional fine structure of F1-ATPase. 1. At the periphery of the molecule, six globular protein masses are orientated in a way similar to the chair conformation of cyclohexane. This array is interpreted to be made up of an alternating sequence of alpha and beta subunits. 2. Part of the central space is occupied by a seventh protein mass, protrusions of which are likely to be in contact with some of the outer subunits. A gamma subunit is supposed to be constituent part of this central protein mass. As a consequence, this model favours a stoichiometry of alpha 3 beta 3 gamma for the large subunits of beef heart F1-ATPase.

Animals↗
PubMed1982

High-affinity binding of ADP and of ADP analogues to mitochondrial F1-ATPase.

Nucleotide-depleted F1-ATPase was prepared from beef heart mitochondria. By use of fluorescence techniques and isotope binding analyses, we investigated the occupation of the high-affinity binding sites on F1 by ADP and the ADP analogues 3'-O-(1-naphthoyl)adenosine diphosphate (N-ADP) and 3'-O-[1-(5-dimethylamino)-naphthoyl]adenosine diphosphate (DMAN-ADP). F1-ATPase was found to exhibit three binding sites for ADP (Kd = 50 nM for one site; Kd = 3 microM for the remaining two sites), two binding sites for N-ADP (Kd = 20 - 50 nM for both of the sites), and three binding sites for DMAN-ADP (Kd = 50 nM for all of the sites). Since the adenine nucleotides under consideration are bound to the same class of sites, the binding data can be explained best on the basis of the hypothesis that the binding process is anticooperative with ADP, whereas the analogues are able to overcome anticooperativity partially (N-ADP) or completely (DMAN-ADP). This binding model is consistent with the view that the exchangeable tight sites are involved directly in the catalytical process of ATP-synthesis in oxidative phosphorylation.

Adenosine Diphosphate↗