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H Weiss

Publications and source records attributed to H Weiss.

At least 19 recordsLinked to original sources
PubMed1992

Accumulation of the pre-assembled membrane arm of NADH:ubiquinone oxidoreductase in mitochondria of manganese-limited grown Neurospora crassa.

The NADH:ubiquinone oxidoreductase (complex I) of mitochondria is constructed from two arms arranged perpendicular to each other. The peripheral arm protruding into the matrix contains the proximal section of the electron pathway, and the membrane arm with all mitochondrially encoded subunits contains the distal section of the electron pathway. When Neurospora crassa is grown under manganese limitation the formation of the peripheral arm is disturbed, but the membrane arm containing the iron-sulfur cluster N-2, is accumulated. An extra-polypeptide, assumed to be a chaperone, is found to be associated with this pre-assembled membrane arm.

Electron Spin Resonance Spectroscopy↗
PubMed1992

Primary structure and mitochondrial import in vitro of the 20.9 kDa subunit of complex I from Neurospora crassa.

The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nuclear-coded component of the hydrophobic arm of the enzyme. We have determined the primary structure of this subunit by sequencing a full-length cDNA and a cleavage product of the isolated polypeptide. The deduced protein sequence is 189 amino acid residues long and contains a putative membrane-spanning domain. Striking similarity over a 60 amino-acid-residue domain with the M (matrix) protein of para-influenza virus was found. No other relationship with already known sequences could be detected, leaving the function of this subunit in complex I still undefined. The biogenetic pathway of this polypeptide was studied using a mitochondrial import system in vitro. The 20.9 kDa subunit synthesized in vitro is efficiently imported into isolated mitochondria, where it obtains distinct features of the endogenous subunit. Our results suggest that the 20.9 kDa polypeptide is made on cytosolic ribosomes lacking a cleavable targeting sequence, interacts with the mitochondrial outer membrane (in a process that does not require an energized inner membrane), and is imported into mitochondria at contact sites. The 20.9 kDa subunit is then inserted into the inner membrane acquiring a topology similar to that of the already assembled subunit.

Amino Acid Sequence↗
PubMed1992

Characterization of assembly intermediates of NADH:ubiquinone oxidoreductase (complex I) accumulated in Neurospora mitochondria by gene disruption.

NADH:ubiquinone oxidoreductase, the respiratory chain complex I of mitochondria, is an assembly of some 25 nuclear-encoded and 7 mitochondrially encoded subunits. The complex has an overall L-shaped structure formed by a peripheral arm and an elongated membrane arm. The peripheral arm containing one FMN and at least three iron-sulphur clusters constitutes the NADH dehydrogenase segment of the electron pathway. The membrane arm with at least one iron-sulphur cluster constitutes the ubiquinone reducing segment. We are studying the assembly of the complex in Neurospora crassa. By disrupting the gene of a nuclear-encoded subunit of the membrane arm a mutant was generated that cannot form complex I. The mutant rather pre-assembles the peripheral arm with all redox groups and the ability to catalyse NADH oxidation by artificial electron acceptors. The final assembly of the membrane arm is blocked in the mutant leading to accumulation of complementary assembly intermediates. One intermediate is associated with a protein that is not present in the fully assembled complex I. The results demonstrate that the two arms of complex I are assembled independently on separate pathways, and gave a first insight into the assembly pathway of the membrane arm. It is also shown for the first time that the obligate aerobic fungus N. crassa can grow and respire without an intact complex I. Gene replacement in this fungus is therefore a tool for investigation of this complex.

Blotting, Southern↗
PubMed1992

De novo synthesis and desaturation of fatty acids at the mitochondrial acyl-carrier protein, a subunit of NADH:ubiquinone oxidoreductase in Neurospora crassa.

We have cultivated the cel mutant of Neurospora crassa defective in cytosolic fatty acid synthesis with [2-14C]malonate and found radioactivity covalently attached to the mitochondrial acyl-carrier protein (ACP), a subunit of the respiratory chain NADH:ubiquinone oxidoreductase. We purified the ACP by reverse-phase HPLC: the bound acyl groups were trans-esterified to methylesters and analyzed by gas chromatography. The saturated C6 to C18 fatty acids and oleic acid were detected. De novo synthesis and desaturation of fatty acids at the ACP subunit of NADH:ubiquinone oxidoreductase and use of the products of this mitochondrial synthetic pathway for cardiolipin synthesis is discussed.

Acyl Carrier Protein↗
PubMed1992

Non-linear inhibition curves for tight-binding inhibitors of dimeric ubiquinol-cytochrome c oxidoreductases. Evidence for rapid inhibitor mobility.

Steady-state electron flow through and electron delivery into isolated dimeric bc1 complex (ubiquinol--cytochrome c oxidoreductase) from Neurospora crassa and beef heart mitochondria were studied in the presence of increasing concentrations of antimycin A, funiculosin and/or myxothiazol. Parabolic or linear inhibition curves were obtained, depending upon the different quinols and inhibitors that were used. Linear curves occur when the inhibitor directly affects the rate-determining step. The most reasonable explanation for the parabolic curves is given by a fast intradimeric exchange of the hydrophobic inhibitors antimycin A, funiculosin (rate less than 500 s-1) and of myxothiazol (rate greater than 1 s-1). Using mitochondria from beef heart, the shape of the inhibition curve with antimycin A is parabolic if the quinol--O2 oxidoreductase turns over at about 300 s-1, but hyperbolic if the rate is 5 times less. The hyperbolic titration curve may be the result of both intradimeric and an additional interdimeric redistribution (rate approximately 100 s-1) of inhibitors between enzymes incorporated in a continuous phospholipid membrane. This explanation is supported by experiments with chromatophores obtained from Rhodobacter capsulatus. As recently described [Fernandez-Velasco, J. & Crofts, A. R. (1992) Biophys. J. 2, A153], cytochrome b becomes fully reoxidized within 1 s after a flash at substoichiometric concentrations of antimycin A. This kinetic of the slow reoxidation can be expressed in terms of the intradimeric and interdimeric redistribution with rate constants of about 10 s-1 and 2 x 10(6) M-1 s-1, respectively. It seems that rapid inhibitor redistribution may be a widespread phenomenon for hydrophobic inhibitors of enzymes incorporated in lipid membranes.

Animals↗
PubMed1992

Effect of calcium channel antagonists on cell membrane potential oscillations and proliferation of cells expressing the ras oncogene.

NIH fibroblasts expressing the Ha-ras oncogene (+ras), unlike otherwise identical cells not expressing the oncogene (-ras), are able to grow in serum-depleted media (0.5% fetal calf serum). Electrophysiological experiments revealed that in +ras fibroblasts but not in -ras fibroblasts, bradykinin leads to sustained, calcium-dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K+ channels, resulting from oscillating intracellular calcium activity. The present study was performed to test for an effect of calcium channel antagonists on these phenomena. Whereas 10 mumol/l verapamil and 10 mumol/l diltiazem did not significantly interfere with either oscillations or proliferation, 10 mumol/l nifedipine completely abolished both the oscillations and the proliferation of +ras fibroblasts. The number of -ras fibroblasts remained virtually constant in both the presence and absence of 10 mumol/l nifedipine. These observations show the antiproliferative action of nifedipine and suggest that the oscillations of cell membrane potential are pertinent for the proliferation of +ras cells in serum-depleted media.

3T3 Cells↗
PubMed1992

Electron transfer complex I defect in idiopathic dystonia.

It has been suggested that dystonia is caused by an autosomal gene with reduced penetrance and a consequent biochemical abnormality affecting cell activity within the basal ganglia. No consistent biochemical disturbance has been identified. In the present study, activities of the mitochondrial electron transfer complexes were measured in platelets of 31 patients with idiopathic dystonia. Enzyme assays of these patients were compared to measurements in 28 control subjects. A significant decrease of complex I activity was observed in the majority of the patients, whereas the activities of other electron transfer complexes were normal. The severity of the complex I defect was more pronounced in patients with the segmental or generalized form than in those with focal dystonia. Complex I activity was not age dependent in the patients or control subjects. Although the electron pathway in complex I is disturbed in patients with idiopathic dystonia, complex I protein content seems to be normal. Whether abnormalities of complex I activity play a role in the pathogenesis of idiopathic dystonia remains to be determined.

Adult↗
PubMed1992

Ion channels activated by swelling of Madin Darby canine kidney (MDCK) cells.

According to previous studies hyposmotic swelling of Madin Darby Canine Kidney (MDCK) cells leads to a marked decrease of cell membrane resistance. The present study has been performed to identify the underlying ion channels using the patch-clamp technique: reduction of extracellular osmolarity to 230 mmol/liter leads to a transient activation of K+ channels and a sustained activation of anion channels. The K+ channels are inwardly rectifying with a single-channel slope conductance of 56 +/- 3 pS at -50 mV (cell negative) and of 29 +/- 2 pS at 0 mV PD across the patch (150 mmol/liter K+ in pipette). The same channels are activated by an increase of intracellular calcium activity, as shown previously. The anion channels display a single-channel slope conductance of 41 +/- 4 pS at -50 mV (cell negative) and of 25 +/- 3 pS at 0 mV PD across the patch (150 mmol/liter Cl- in pipette). The channel is anion selective and conducts both bicarbonate and chloride with a preference for bicarbonate. Its open probability is not affected by changing intracellular calcium from 0.1-10 mumol/liter. The channels observed explain the effects of cell swelling on PD, ion selectivity and resistance of the cell membrane in MDCK cells.

Animals↗
PubMed1992

Altered cell volume regulation in ras oncogene expressing NIH fibroblasts.

Expression of the Ha-ras oncogene has been reported to stimulate the dimethylamiloride sensitive Na+/H+ exchanger and Na+,K+,2Cl- cotransport, both transport systems which are involved in cell volume regulation. The present study has been performed to test for an influence of ras oncogene expression on cell volume regulation in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras). As controls served NIH 3T3 fibroblasts not expressing the ras oncogene (-ras). In isotonic extracellular fluid, the cell volume of + ras cells (2.70 +/- 0.08 pl) is significantly greater than the cell volume of -ras cells (2.04 +/- 0.10 pl). Both, + ras and -ras cells exhibit a regulatory cell volume increase in hypertonic extracellular fluid and a regulatory cell volume decrease in hypotonic extracellular fluid. The regulatory cell volume decrease is inhibited by 1 mmol/l quinidine and barium, the regulatory cell volume increase is inhibited in -ras and +ras cells by dimethyl-amiloride (100 mumol/l) and, only in +ras cells, by furosemide (100 mumol/l) and bumetanide (10 mumol/l). In conclusion, expression of the ras oncogene leads to a shift of the set point for cell volume regulation to greater cell volumes, which may contribute to the activation of the Na+/H+ exchanger and Na+,K+,2Cl- cotransport.

3T3 Cells↗
PubMed1992

[Morphological and numerical studies of the stress compatibility of patella implants].

Failure of total knee arthroplasty is relatively often caused by problems of the patellofemoral replacement. The purpose of this study was to analyze the distribution of stresses within an anatomical patella and the changes in stress distribution after patellar resurfacing with a Miller-Galante I patellar implant using two- and three dimensional finite element models (FEM). To assess validity, FEM results were compared with morphological findings from contact radiographs and densitographs. Internal orientation of bone trabeculae is in good agreement with the orientation of theoretically calculated principal stresses. Almost unchanged principal tensile stresses after implantation, together with the lack of extreme stress peaks within the cancellous bone ensure stress compatibility of the implant. In the case of a firmly seated implant with good bone ingrowth, increased von Mises stresses are found near the fixation peg/plate junction. Their relevance for improved bone ingrowth near this part of the interface is emphasized. At the same time, material failure at the peg/plate junction can be better understood. An analysis of the early postoperative period assuming nonlinear interface conditions failed to demonstrate an uniform distribution of normal and tangential interface forces.

Aged↗
PubMed1991

Effect of BHT 920 on calcium-activated K+ channels in renal epithelioid MDCK cells.

In Madin Darby canine kidney (MDCK) cells, epinephrine has been shown to increase intracellular calcium, activate calcium-dependent K+ channels and hyperpolarize the cell membrane. The present study has been performed to test for the possible involvement of alpha 2-adrenergic receptors. To this end, the effects of alpha 2-adrenoceptor agonist BHT 920 have been studied on cell membrane potential, ion channel activity and intracellular calcium: Similar to epinephrine, BHT 920 hyperpolarizes the cell membrane, increases intracellular calcium and activates inwardly rectifying K+ channels (single channel slope conductances 30-80 pS). Half-maximal hyperpolarization is achieved at concentrations between 10 and 100 nmol/l. The hyperpolarizing effect of BHT 920 is abolished in the presence of alpha 2-adrenoceptor antagonist yohimbine (100 nmol/l) but not in the presence of alpha 1-adrenoceptor antagonist prazosin (100 nmol/l). At extracellular calcium activity below 100 nmol/l BHT 920 still leads to a transient hyperpolarization of the cell membrane but, in contrast to epinephrine, is unable to significantly increase intracellular calcium or significantly activate the calcium-sensitive K+ channels. The observations indicate that stimulation of alpha 2-receptors participates in the epinephrine-induced increase of intracellular calcium, channel activation and hyperpolarization.

Adrenergic beta-Agonists↗
PubMed1991

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.

Crystallography↗
PubMed1991

The acyl-carrier protein in Neurospora crassa mitochondria is a subunit of NADH:ubiquinone reductase (complex I).

We determined the primary structure of a 9.6-kDa subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa mitochondria and found a close relationship between this subunit and the bacterial or chloroplast acyl-carrier protein. The degree of sequence identity amounts to 80% in a region of 19 residues around the serine to which the phosphopantetheine is bound. The N-terminal presequence of the subunit has the characteristic features of a mitochondrial import sequence. We cultivated the auxotroph pan-2 mutant of N. crassa in the presence of [14C]pantothenate and recovered all radioactivity incorporated into mitochondrial protein in the 9.6-kDa subunit of complex I. We cultivated N. crassa in the presence of chloramphenicol to accumulate the nuclear-encoded peripheral arm of complex I. This pre-assembled arm also contains the 9.6-kDa subunit. These results demonstrate that an acyl-carrier protein with pantothenate as prosthetic group is a constituent part of complex I in N. crassa.

Acyl Carrier Protein↗
PubMed1991

Primary structure of the nuclear-encoded 29.9 kDa subunit of NADH: ubiquinone reductase from Neurospora crassa mitochondria.

We isolated and sequenced cDNA for the 29.9 kDa subunit of mitochondrial NADH: ubiquinone reductase (complex I) from a Neurospora crassa library in the lambda gt11 expression vector. The N-terminus of the mature protein was determined by Edman-degradation. The cDNA contains an open reading frame encoding a preprotein of 273 amino acids. The presequence of the transit protein essential for mitochondrial import is eight residues long. Northern-blot analysis shows, that the level of the corresponding mRNA is increased 3-fold if cells are grown in the presence of chloramphenicol.

Amino Acid Sequence↗
PubMed1991

Primary structure of the nuclear-encoded 18.3 kDa subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria.

The primary structure of the nuclear-encoded 18.3 kDa subunit of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA and the N-terminus of the protein. The cDNA contains an open reading frame for a protein of 206 amino acids. The mature protein consists of 173 amino acids and has a molar mass of 18,341 Da. The precursor protein includes a characteristic mitochondrial import sequence with a typical matrix peptidase processing site.

Amino Acid Sequence↗
PubMed1991

The iron-sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.

Animals↗
PubMed1991

cDNA and genomic DNA sequence of the 21.3 kDa subunit of NADH:ubiquinone reductase (complex I) from Neurospora crassa.

The primary structure of a nuclear-encoded subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the protein. The sequence correlates to a protein of 200 amino acids and a molecular mass of 21349 Da. The protein is synthesized without a cleavable presequence. It contains two alpha-helices predicted to traverse the bilayer and is a constituent of the membrane part of complex I.

Amino Acid Sequence↗