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PubMed1997

Impairment of respiration and oxidative phosphorylation by redox cyclers 2-nitrosofluorene and menadione.

The present study was designed to investigate the effects of 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2-acetylaminofluorene, on mitochondrial respiration and oxidative phosphorylation. NOF reacts with the NADH:ubiquinone oxidoreductase (complex I) and consumes oxygen in a rotenone-insensitive manner. Unlike menadione, which is able to bypass the rotenone-block and to restore ATP-formation, NOF-induced electron flow was almost completely uncoupled. In normal respiration both redox-cyclers decreased the respiratory control and P/O ratios at low concentrations (2-20 nmol/mg) in NADH-dependent oxidation. With succinate as substrate, only NOF was significantly active. In contrast to NOF, the hydroxamic acid N-hydroxy-2-acetylaminofluorene (N-OH-AAF) impaired mitochondrial energy conversion only at much higher concentrations (80 nmol/mg). At concentrations > 10 nmol/mg, NOF inhibited electron flow through the respiratory chain in NADH- and succinate-dependent oxidation, as determined by dinitrophenolate-uncoupled respiration. The small protective effect of L-cysteine indicates that covalent binding of the nitroso-compound to SH-groups may not explain sufficiently the inhibitory effect of NOF. The results support the notion that redox cyclers impair oxidative phosphorylation by establishing alternative pathways for electron transport in the respiratory chain.

2,4-Dinitrophenol↗
PubMed1997

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria.

Formation of H2O2 has been studied in rat heart mitochondria, pretreated with H2O2 and aminotriazole to lower their antioxidant capacity. It is shown that the rate of H2O2 formation by mitochondria oxidizing 6 mM succinate is inhibited by a protonophorous uncoupler, ADP and phosphate, malonate, rotenone and myxothiazol, and is stimulated by antimycin A. The effect of ADP is abolished by carboxyatractylate and oligomycin. Addition of uncoupler after rotenone induces further inhibition of H2O2 production. Inhibition of H2O2 formation by uncoupler, malonate and ADP+Pi is shown to be proportional to the delta psi decrease by these compounds. A threshold delta psi value is found, above which a very strong increase in H2O2 production takes place. This threshold slightly exceeds the state 3 delta psi level. The data obtained are in line with the concept [Skulachev, V.P., Q. Rev. Biophys. 29 (1996), 169-2021 that a high proton motive force in state 4 is potentially dangerous for the cell due to an increase in the probability of superoxide formation.

Adenosine Diphosphate↗
PubMed1999

Blockade of mitochondrial Ca2+ uptake by mitochondrial inhibitors amplifies the glutamate-induced calcium response in cultured cerebellar granule cells.

The objective of this study was to evaluate the role of mitochondrial Ca2+ uptake (MCU) in modulation (shaping) of the glutamate (Glu)-induced changes in neuronal cytoplasmic Ca2+ ([Ca2+]i). In order to block MCU, nerve cells were treated with mitochondrial inhibitors (MI) inducing collapse of the mitochondrial potential (Delta Psim). Measurements of changes in [Ca2+]i were performed using either the low-affinity (fura-2FF) or high-affinity (fura-2) Ca2+ indicators. Loading of nerve cells with rhodamine 123 made it possible to monitor changes in Delta Psim. In the first series of experiments it was shown that blockade of MCU in fura-2FF-loaded cells with a cocktail of rotenone (2 microM)+oligomycin (2.5 microg/ml) greatly (2.53+/-0.4 times, n=61) increased the [Ca2+]i response to a 1-min Glu (100 microM) pulse. In fura-2-loaded cells, this increase was small (less than 1.3 times) or absent. In the second series of experiments, cocktails of rotenone+oligomycin or FCCP (1 microM)+oligomycin were applied during a prolonged Glu application. This produced strong mitochondrial depolarisation and an additional [Ca2+]i increase. In most cells the latter could be reversed or prevented by a removal of external Ca2+. The MI-induced additional [Ca2+]i increase was especially pronounced in cells loaded with fura-2FF. In some neurones a removal of external Ca2+ did not produce a decrease in [Ca2+]i during combined Glu+MI application, suggesting an impairment of [Ca2+]i extrusion mechanisms of these cells. The conclusion is drawn that MCU makes a considerable contribution to regulation of [Ca2+]i responses caused by Ca2+ influx via Glu-activated ionic channels. The reasons for a quantitative difference between [Ca2+]i responses observed in fura-2- and fura-2FF-loaded neurones are discussed.

Animals↗
PubMed2001

Stimulation of cardiac glucose transport by inhibitors of oxidative phosphorylation.

Previously we showed that hypoxia in heart stimulates glucose transport via translocation of glucose transporters from intracellular membranes to the plasma membrane. We later showed that rotenone, an inhibitor of oxidative phosphorylation, also decreased intracellular transporters. Here, using another membrane fractionation technique, we show that rotenone increases plasma membrane transporters, and that another respiratory chain inhibitor, azide, acts similarly. Thus, they likely activate the same signaling pathway as hypoxia. Genistein, a tyrosine kinase inhibitor, inhibited insulin- and azide-stimulated 3-O-methylglucose transport similarly in cardiac myocytes. It also increased glucose transporters in the plasma membranes of perfused hearts even though it inhibited glucose uptake, suggesting effects on membrane trafficking. Another tyrosine kinase inhibitor, lavendustin A, and the cyclic nucleotide-dependent protein kinase inhibitors H-8 and H-7 had little effect on basal or azide-stimulated transport. Polymyxin B was a weak inhibitor of basal, insulin-stimulated, and azide-stimulated transport. A nitric oxide donor and a nitric oxide synthase inhibitor had no effect on basal and azide-stimulated transport. The results indicate that tyrosine kinases; protein kinases A, G, and C; and nitric oxide are not involved in the hypoxic activation of cardiac glucose transport.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Control of the pyridine nucleotide-linked Ca2+ release from mitochondria by respiratory substrates.

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca2+ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca2+ release from mitochondria when respiration is supported either by the NAD(+)-linked substrate beta-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca2+ much better with succinate than with beta-hydroxybutyrate as respiratory substrate. When Ca2+ release is induced by t-butylhydroperoxide succinate-supported Ca2+ retention is impeded by rotenone. Ca2+ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca2+ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca2+ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.

3-Hydroxybutyric Acid↗
PubMed2002

Mouse model of optic neuropathy caused by mitochondrial complex I dysfunction.

We developed a mouse model of optic neuropathy caused by mitochondrial complex I dysfunction by intravitreal administration of rotenone, a complex I inhibitor, in CBA/J mice. Retinal thickness was measured in sections stained histochemically for complex I enzymatic activity. The retinal ganglion cell layer of eyes injected with rotenone was significantly thinner than that of the control eyes injected with the vehicle dimethyl sulfoxide at 1, 24, and 48-h survival time groups. The largest reduction was 43% at 24-h post-injection. This effect is consistent with the degeneration of retinal ganglion cells in Leber's hereditary optic neuropathy. This is the first animal model of optic neuropathy caused by mitochondrial dysfunction, and it could be used as a quick and convenient way to test new treatments for mitochondrial neurodegenerative diseases.

Animals↗
PubMed2003

Non-synaptic release of [3H]noradrenaline in response to oxidative stress combined with mitochondrial dysfunction in rat hippocampal slices.

Brain ischemia is frequently associated with oxidative stress in the reperfusion period. It is known that noradrenaline (NA) is released in excess under energy deprivation by the sodium-dependent reversal of the monoamine carrier. However, it is not known how oxidative stress affects NA release in the brain alone or in combination with energy deprivation. As a model of oxidative stress, the effect of H(2)O(2) (0.1-1.5 mM) perfusion was investigated in superfused rat hippocampal slices. It elicited a dose-dependent elevation of the release of [(3)H]NA and its tritiated metabolites as well as a simultaneous drop in the tissue energy charge. Mitochondrial inhibitors, i.e. rotenone (10 microM), and oligomycin (10 microM) in combination, also decreased the energy charge, but they had only a mild effect on [(3)H]NA release. However, when H(2)O(2) was added together with oligomycin and rotenone their effect on [(3)H]NA release was greatly exacerbated. H(2)O(2) and mitochondrial inhibitors also induced an increase in [Na(+)](i) in isolated nerve terminals, and their effect was additive. The effect of H(2)O(2) on tritium release was temperature-dependent. It was also attenuated by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM) and (+/-)-2-amino-5-phosphonopentanoic acid (10 microM), by the nitric oxide synthase inhibitors, N omega-nitro-L-arginine methyl ester (100 microM), or 7-nitroindazole (50 microM) and by the vesicular uptake inhibitor tetrabenazine (1 microM). Our results suggest that oxidative stress releases glutamate followed by activation of postsynaptic ionotropic glutamate receptors that trigger nitric oxide production and results in a flood of NA from cytoplasmic stores. The massive elevation of extracellular NA under conditions of oxidative stress combined with mitochondrial dysfunction may provide an additional source of highly reactive free radicals thus initiating a self-amplifying cycle leading to neuronal degeneration.

Animals↗
PubMed1999

Measurements of bovine sperm velocities under true anaerobic and aerobic conditions.

Velocities of bovine spermatozoa in a medium containing glucose were similar under true anaerobic and aerobic conditions. Spermatozoa were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrates that immobilisation was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 microM antimycin A and 4 microM rotenone when glucose was present in the medium. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into the medium which supported respiration but not glycolysis, and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished sperm motility immediately after addition. Bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Available data on bovine sperm energetics support this view.

Animals↗
PubMed1998

Nitric oxide donors protect cultured rat astrocytes from 1-methyl-4-phenylpyridinium-induced toxicity.

MPP+ is thought to mediate MPTP's toxicity on dopamine neurons by inhibiting mitochondrial respiration. However, astrocytic injuries are also observed in MPTP/MPP+-treated rats. Because nitric oxide (NO.) is suggested to be cytoprotective, we examined the effects of nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1) on MPP+-induced toxicity in astrocytes. Incubation of astrocytes with MPP+ for 2 days produced a dose-dependent toxicity, including increase in lactate level and lipid peroxidation, decrease of metabolic activity and cell damage. SNP, SNAP, and SIN-1 all attenuated MPP+-induced toxicity. The same protection was not achieved with N-acetylpenicillamine or ferrocyanide, structural analogues of SNAP or SNP but devoid of NO.. Further, the effect was not attributed to the increased cGMP levels or blockade of MPP+ accumulation in astrocytes. Notably, catalase, dimethyl sulfoxide and ferricyanide, an extracellular electron acceptor, were also effective in inhibiting MPP+ damage. NO. donors and analogues were also tested against damage produced by rotenone, an irreversible complex I inhibitor. Only ferricyanide and SNP effectively protected rotenone's toxicity. These results concluded that (1) NO. may protect astrocytes from MPP+-induced free radical formation, and (2) prevention of energy depletion/free radicals production alleviate MPP+-induced toxicity.

1-Methyl-4-phenylpyridinium↗
PubMed2002

Inhibitors of mitochondrial complex I attenuate the accumulation of hypoxia-inducible factor-1 during hypoxia in Hep3B cells.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that regulates transcriptional activation of several genes that are responsive to oxygen lack, including erythropoietin, vascular endothelial growth factor, various glycolytic enzymes and the GLUT-1 glucose transporter. Because mitochondria have been postulated to be involved in the regulation of HIF-1, we tested the effects of mitochondrial electron transport chain complex I inhibitors, rotenone and 1-methyl-4-phenylpiridinium (MPP(+)), on hypoxic-induced accumulation of HIF-1 alpha, the regulated component of the dimer. We found, consistent with our previous observations in Cath.a and PC12 cells, that rotenone and MPP(+) attenuated the HIF-1 alpha hypoxic response. Thus, it can be concluded that an intact, functional mitochondrial respiratory chain is required for HIF-1 alpha accumulation.

1-Methyl-4-phenylpyridinium↗
PubMed1992

Identification of the ubiquinone-binding site of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

In order to localize the ubiquinone-binding site of complex I (NADH:ubiquinone oxidoreductase), a novel photoreactive ubiquinone analogue (Q0C7ArN3) has been synthesized. It is shown that the direct chemical precursor of this analogue (Q0C7ArNO2) and the analogue itself are accepted as substrates in an enzyme assay utilizing ubiquinone-depleted mitochondrial membranes of Neurospora crassa. The activity of the enzyme applying these derivatives is inhibited by 50% at a concentration of 9 and 20 microM rotenone. Photoaffinity labeling experiments were performed with both isolated complex I and whole mitochondrial membranes of N. crassa under various conditions. In each of these experiments a protein subunit with an apparent molecular mass of about 9.5 kDa was labeled with high specificity. Radioactive labeling was totally prevented by the addition of ubiquinone-2 at concentrations higher than 500 microM but was not affected by comparable concentrations of rotenone or other hydrophobic substances. In the labeling experiments using whole membranes, the labeling signal was dramatically increased in the presence of 1.5 mM NADH. These results strongly suggest that the ubiquinone analogue interacts specifically with the enzyme.

Affinity Labels↗
PubMed2000

Chronic systemic pesticide exposure reproduces features of Parkinson's disease.

The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity. Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

Animals↗
PubMed1998

Inhibition of mitochondrial respiratory chain complex I by TNF results in cytochrome c release, membrane permeability transition, and apoptosis.

Mitochondria have been shown to play a key role in apoptosis induction. However, the sequence of changes that occur in the mitochondria in the initial step of apoptosis has not been clearly elucidated. Here, we showed that mitochondrial respiratory chain (MRC) complex I was inhibited during the early phase of TNF- or serum withdrawal apoptosis. The importance of complex I inhibition in apoptosis is also supported by the observation that rotenone, an inhibitor of complex I but not that of other complexes, could induce apoptosis in a manner comparable to TNF. We hypothesized that inhibition of complex I could affect electron flow through other complexes leading to cytochrome c release by an antioxidant-sensitive pathway and caspase 3 activation followed by the induction of membrane permeability transition (MPT). This hypothesis is supported by the following observations: (1) TNF and rotenone induced MPT and cytochrome c release; (2) TNF-induced complex I inhibition was observed prior to cytochrome c release and MPT induction; (3) MPT induction was inhibited by a caspase 3 inhibitor, z-DEVD-CH2F, and an antioxidant pyrrolidine dithiocarbamate (PDTC), whereas cytochrome c release was only inhibited by PDTC. Thus, these results suggest that MRC complex I plays a key role in apoptosis signalings.

Antioxidants↗
PubMed1968

The redox state of the nicotinamide-adenine dinucleotides in rat liver homogenates.

1. The redox state of the NAD couple of rat liver mitochondria, as measured by the [beta-hydroxybutyrate]/[acetoacetate] ratio, rapidly changed in the direction of oxidation during the preparation of homogenates in a saline medium. The value of the [beta-hydroxybutyrate]/[acetoacetate] ratio fell from 2.3 to 0.15 in 10min. EDTA diminished the fall and succinate prevented it. 2. The redox state of the rat liver cytoplasm, as measured by the [lactate]/[pyruvate] ratio, changed slightly in the direction of reduction during the preparation of homogenate. This was prevented by succinate. 3. In unsupplemented homogenates the differences in the redox states of mitochondria and cytoplasm decreased. Succinate and EDTA together maintained the differences within the physiological range. A measure of the ability of the mitochondria to maintain different redox states in mitochondria and cytoplasm is the value of the expression [lactate][acetoacetate]/[pyruvate][beta-hydroxybutyrate]. If there are no differences in the redox states of the NAD in the two cell compartments the value of the expression is 444 at 37 degrees . The value in the intact rat liver is between 4.7 and 21. 4. alpha-Oxoglutarate or glutamate were still more effective than succinate in maintaining high [beta-hydroxybutyrate]/[acetoacetate] ratios in the homogenates because these substrates supply a reducing agent of NAD(+) and, through succinate, an inhibitor of the oxidation of NADH. 5. When supplemented with alpha-oxoglutarate and EDTA, homogenates readily adjust the redox state of the beta-hydroxybutyrate dehydrogenase system after it has been upset by the addition of either acetoacetate or beta-hydroxybutyrate. 6. Amytal and rotenone raised the value of the [beta-hydroxybutyrate]/[acetoacetate] ratio. This is taken to indicate that the reduction of acetoacetate in the homogenates was not an energy-linked process. 7. 2,4-Dinitrophenol shifted the [beta-hydroxybutyrate]/[acetoacetate] ratio in the presence of succinate in favour of oxidation because it inhibited the oxidation of succinate and accelerated the oxidation of NADH. 8. Rotenone increased the rate of ketone-body formation of liver homogenates, though it decreased the rate of oxygen uptake.

Acetoacetates↗
PubMed1971

Non-haem iron and the dissociation of piericidin A sensitivity from site 1 energy conservation in mitochondria from Torulopsis utilis.

1. The aerobic incubation of iron-deficient Torulopsis utilis cells for 12h under non-growing conditions results in the recovery by mitochondria of the previously absent site 1 energy conservation and sensitivity to piericidin A. 2. The recovery of piericidin A sensitivity but not site 1 is prevented by the presence of cycloheximide (100mug/ml) in the medium used for aerobic incubation of the cells. Rotenone sensitivity behaved similarly. 3. Chloramphenicol, erythromycin and tetracycline were without effect on the recovery of site 1 and piericidin A sensitivity. 4. Inclusion of (59)Fe in the growth medium can be used as the basis for a highly sensitive assay for non-haem iron. 5. Iron-limited growth of T. utilis lowers the concentration of both non-haem iron and acid-labile sulphide of submitochondrial particles by over 20-fold compared with the ;normal' situation with iron-supplemented glycerol-limited growth. 6. Increases in the non-haem iron and acid-labile sulphide concentrations of submitochondrial particles occur when site 1 and piericidin A sensitivity are recovered. The increase is approximately halved by the presence of cycloheximide. 7. The non-haem iron of T. utilis submitochondrial particles does not exchange with added iron. 8. Continuous culture of T. utilis at the transition between glycerol- and iron-limitation results in cells where mitochondria possess site 1 energy conservation but lack piericidin A sensitivity. 8. It is concluded, in contrast with widely held views to the opposite, that energy conservation at site 1 does not require electron flow to proceed through a piericidin A- or rotenone-sensitive route. 9. Restriction of the iron supplied to growing T. utilis to a concentration just above that required for growth limitation demonstrates that a 10- to 20-fold decrease of the ;normal' non-haem iron concentration of both cells and mitochondria is without effect on the growth yield per unit of carbon source. Submitochondrial particles prepared from such iron-restricted but otherwise functionally normal cells have a non-haem iron concentration of about 0.5-0.8ng-atom/mg of protein. It is concluded that the concentration of iron-sulphur protein required for normal function by the respiratory chain is close to the concentrations of cytochromes and flavoproteins.

Chloramphenicol↗
PubMed1973

The electron-transport system of mitochondria from the slime mould Physarum polycephalum.

A method is described for the preparation of mitochondria from the slime mould Physarum polycephalum; the mitochondria were not coupled. P. polycephalum mitochondria oxidized added NADH via a rotenone-insensitive pathway, but the oxidation of malate plus glutamate was rotenone sensitive; both of these substrates reduced much less cytochrome b than did succinate, in both aerobic and anaerobic steady states. Spectroscopy at 77 degrees K separated three absorption maxima in the alpha-band region, at 560nm, 553nm and one at 547nm due to cytochrome c. The absorption at 553nm was increased in the aerobic steady state by the addition of 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that it was due to a b-type cytochrome. All three absorption maxima appeared in the aerobic steady state after the addition of a range of substrates. The respiratory activity with different substrates and the response to inhibitors of respiration were similar to those previously described for fungus mitochondria (Weiss et al., 1970; Erickson & Ashworth, 1969). When grown under conditions of haem limitation the mitochondria contained a lower concentration of cytochromes than normal.

Adenosine Triphosphate↗
PubMed1976

Transport of calcium ions by Ehrlich ascites-tumour cells.

Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.

Adenosine Triphosphate↗
PubMed1977

Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase from bovine heart mitochondria.

1. Incubation of NADH-ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH-K(3)Fe(CN)(6) oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH-cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH-K(3)Fe(CN)(6) oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.

Animals↗