Characterization of Neurospora crassa Mitochondria Prepared with a Grind-Mill
TL;DR: Mitochondria can be isolated rapidly from Neurospora crassa with high yields by disrupting the hyphae suspension between the two grinding wheels of a simple mill and can be looked upon in analogy to mitochondria from yeast, particularly from Torulopsis utilis.
Abstract: Mitochondria can be isolated rapidly from Neurospora crassa with high yields by disrupting the hyphae suspension between the two grinding wheels of a simple mill.
The isolated mitochondria respire with pyruvate + malate, succinate, and with NADH and NADPH in high rates and well coupled to oxidative phosphorylation.
Three phosphorylation steps are involved in the oxidation of pyruvate and two in the oxidation of succinate, NADH and NADPH.
Rotenone, antimycin and KCN inhibit the electron flow at the known steps.
The cytochrome composition pattern is characterized by a particularly high content of cytochrome c.
Evidence is given for the existence of two NADH-dehydrogenases for the oxidation of exogenous and endogenous NADH with different localizations on the inner mitochondrial membrane.
On the basis of these properties, mitochondria from Neurospora crassa can be looked upon in analogy to mitochondria from yeast, particularly from Torulopsis utilis.
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TL;DR: It is concluded that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation.
Abstract: Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation
273 citations
TL;DR: The effect of ten naturally occurring and two synthetic inhibitors of NADH:ubiquinone oxidoreductase (complex I) of bovine heart, Neurospora crassa and Escherichia coli and fructose dehydrogenase of Gluconobacter oxidans was investigated.
Abstract: The effect of ten naturally occurring and two synthetic inhibitors of NADH:ubiquinone oxidoreductase (complex I) of bovine heart, Neurospora crassa and Escherichia coli and glucose:ubiquinone oxidoreductase (glucose dehydrogenase) of Gluconobacter oxidans was investigated. These inhibitors could be divided into two classes with regard to their specificity and mode of action. Class I inhibitors, including the naturally occurring piericidin A, annonin VI, phenalamid A2, aurachins A and B, thiangazole and the synthetic fenpyroximate, inhibit complex I from all three species in a partially competitive manner and glucose dehydrogenase in a competitive manner, both with regard to ubiquinone. Class II inhibitors including the naturally occurring rotenone, phenoxan, aureothin and the synthetic benzimidazole inhibit complex I from all species in an non-competitive manner, but have no effect on the glucose dehydrogenase. Myxalamid PI could not be classified as above because it inhibits only the mitochondrial complex I and in a competitive manner. All inhibitors affect the electron-transfer step from the high-potential iron-sulphur cluster to ubiquinone. Class I inhibitors appear to act directly at the ubiquinone-catalytic site which is related in complex I and glucose dehydrogenase.
244 citations
TL;DR: The knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms, which showed that most organisms contain genes that potentially encode NDH-2.
Abstract: Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the
two-electron transfer from NAD(P)H to quinones, without any
energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H,
regenerating the NAD(P) + pool, and may contribute to
the generation of a membrane potential through complexes III and IV.
These enzymes are usually constituted by a nontransmembrane polypeptide
chain of ∼50 kDa, containing a flavin moiety. There are a few
compounds that can prevent their activity, but so far no general
specific inhibitor has been assigned to these enzymes. However, they
have the common feature of being resistant to the complex I classical
inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular
relevance in yeasts like Saccharomyces cerevisiae and in
several prokaryotes, whose respiratory chains are devoid of complex I,
in which NDH-2 keep the
[NADH]/[NAD + ] balance and are
the main entry point of electrons into the respiratory chains. Our
knowledge of these proteins has expanded in the past decade, as a
result of contributions at the biochemical level and the sequencing of
the genomes from several organisms. The latter showed that most
organisms contain genes that potentially encode NDH-2. An overview of
this development is presented, with special emphasis on microbial
enzymes and on the identification of three subfamilies of
NDH-2.
241 citations
Cites background from "Characterization of Neurospora cras..."
...eukaryotic organisms from the fungal (16, 89) and plant (55)...
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TL;DR: After isolation, the bound Triton X-100 could be replaced by other nonionic detergents such as: alkylphenyl polyoxyethylene ethers,Alkyl polyoxy ethylene ethers and acyl poly Oxyethylene sorbitan esters.
Abstract: The electron transfer complexes, succinate: ubiquinone reductase, ubiquinone: cytochrome c reductase, and cytochrome c:O2 oxidase were isolated from the mitochondrial membranes of Neurospora crassa by the following steps. Modification of the contents of the complexes in mitochondria by growing cells on chloramphenicol; solubilisation of the complexes by Triton X-100; affinity chromatography on immobilized cytochrome c and ion exchange and gel chromatography. Ubiquinone reductase was obtained in a monomeric form (Mr∼ 130000) consisting of a flavin subunit (Mr 72000) an iron-sulfur subunit (Mr 28000) and a cytochrome b subunit (Mr probably 14000). Cytochrome c reductase was obtained in a dimeric form (Mr∼ 550000), the monomeric unit comprising two cytochromes b (Mr each 30000), a cytochrome c1 (Mr 31000), an iron-sulfur subunit (Mr 25000), and six subunits without known prosthetic groups (Mr 9000, 11000, 14000, 45000, 45000, and 52000). Cytochrome c oxidase was also isolated in a dimeric form (Mr∼ 320000) comprising two copies each of seven subunits (Mr 9000, 12000, 14000, 18000, 21000, 29000, and 40000). The complexes were essentially free of phospholipid. Each bound one micelle of Triton X-100 (Mr∼ 90000). After isolation, the bound Triton X-100 could be replaced by other non-ionic detergents such as: alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers and acyl polyoxyethylene sorbitan esters.
187 citations
TL;DR: The mechanism of inter-complex electron transfer mediated by ubiquinone is reviewed and the kinetic consequences of the supramolecular organization of the respiratory complexes (randomly dispersed vs. super-complexes) in terms of Coenzyme Q pool behavior vs. metabolic channeling are discussed.
184 citations
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TL;DR: In this article, a methodische Teil enthält neben technischen Einzelheiten für die Aufarbeitung im größeren Maßstab eine neue Ableitung zur Errechnung der Salzzugabe für bestimmte Ammonsulfatkonzentrationen and Vorschriften für the Ammonulfat-and Eiweißbestimmung im Routinebetrieb.
Abstract: Extrakt von Kaninchenmuskulatur wird durch steigende Sättigung mit Ammonsulfat in amorphe Fraktionen aufgeteilt, aus denen sich ohne weitere Reinigungsschritte die obengenannten Fermente kristallisieren lassen. Die Reinigung der Rohkristallisate erfolgt durch Waschungen mit Ammonsulfatlösungen und Umkristallisationen. Jedes Ferment wird an Hand seines Absorptionsspektrums, der Umsatzzahl, Kristallform und spezifischen Eigenschaft beschrieben und den entsprechenden reinsten Präparaten anderer Autoren gegenübergestellt. Der methodische Teil enthält neben technischen Einzelheiten für die Aufarbeitung im größeren Maßstab eine neue Ableitung zur Errechnung der Salzzugabe für bestimmte Ammonsulfatkonzentrationen und Vorschriften für die Ammonsulfat- und Eiweißbestimmung im Routinebetrieb.
1,018 citations
TL;DR: Results showed that well preserved mitochondria isolated from Saccharomyces carlsbergensis could be stored for a long period of time without appreciable loss of phosphorylation activity and were completely insensitive to Amytal and rotenone.
281 citations
TL;DR: With mitochondria from Saccharomyces carlsbergensis the functional relationship of the exogenous and endogenous NAD system has been investigated with respect to the membrane barrier, the localisation of NADH dehydrogenase and other NAD-and flavine-linked dehydrogenases and the existence of a mitochondrial alcohol dehydrogensase is established.
Abstract: With mitochondria from Saccharomyces carlsbergensis the functional relationship of the exogenous and endogenous NAD system has been investigated with respect to the membrane barrier, the localisation of NADH dehydrogenase and other NAD-and flavine-linked dehydrogenases.
1
Added NAD and NADH do not permeate through the inner membrane into the matrix space of mitochondria from S. carlsbergensis despite the high rate of oxidation. There is no exchange between exogenous and endogenous NADH and NAD.
2
A transhydrogenation between external and internal NADH and NAD across the inner membrane is not observed.
3
By applying the “ferricyanide method” two separate dehydrogenases can be identified: one for the oxidation of exogenous NADH, located towards the outer surface of the inner mitochondrial membrane; and a second for the oxidation of endogenous NADH directed towards the inner surface. Both dehydrogenases are connected to the cytochrome chain through the ubiquinone pool.
4
About 50% of the total NADH dehydrogenase activity of yeast mitochondria is solubilized by sonication. The solubilized NADH dehydrogenase is tentatively identified with the external enzyme. This agrees with the finding that on opening the membrane by sonication the total activity of NADH-ferricyanide reduction increases by 65%.
5
Applying both [4B-3H]NADH and [4A-3H]NADH the oxidation of both exogenous and endogenous NADH by ferricyanide and by oxygen are found to be B-specific.
6
By application of the “ferricyanide method” to other substrates it is concluded that the dehydrogenases for glycerolphosphate and possibly lactate are localized before the barrier for ferricyanide while the dehydrogenases for ethanol, isocitrate and succinate are localized behind it. This barrier is identical with the inner mitochondrial membrane.
7
The existence of a mitochondrial alcohol dehydrogenase, accounting for about 6% of the total cellular activity and located in the matrix space, is established. The dual localization of alcohol dehydrogenase is considered to facilitate the equilibration between the intra- and extra-mitochondrial NAD systems via an ethanol-acetaldehyde shuttle.
221 citations
TL;DR: The data suggest that the mitochondrial mass is increased by a continuous process of addition of new lecithin units to the already existing mitochondrial framework.
Abstract: Cells of a choline-requiring mutant of Neurospora crassa, labeled with radioactive choline, were transferred to unlabeled medium. At various times during their subsequent logarithmic growth, a highly purified mitochondrial fraction was prepared by sucrose density gradient centrifugation, and the distribution of label among individual mitochondria was determined by quantitative autoradiography. Preliminary experiments indicated that, under the conditions of this "washout" experiment, choline served as a stable mitochondrial label. Radioautographic analysis showed that, in fully labeled mycelia and for three mass doubling cycles in the unlabeled medium, radioactivity was randomly distributed among all mitochondria; i.e., the distribution of autographic grains among individual mitochondria followed a Poisson distribution. In experiments in which pulse labeling for 10 minutes was used, the label was randomly distributed among all mitochondria. The data suggest that the mitochondrial mass is increased by a continuous process of addition of new lecithin units to the already existing mitochondrial framework.
178 citations
145 citations