Cooperative Substrate Oxidation by Mitochondria from Castor Bean Hypocotyls

Cooperative oxidation of succinate and exogenous NADH was followed in the mitochondria from five- to six-day-old castor bean (Ricinus communisL.) seedlings. Although succinate was oxidized at a much higher rate than NADH, the former inconsiderably (less than 15%) inhibited the oxidation of the latter substrate in state 4, while, in state 3 (in the presence of ATP), the two substrates did not compete and were jointly oxidized. When two substrates were oxidized by the mitochondria with the alternative CN-resistant oxidase (AO) inhibited with salicylhydroxamic acid, the rate of NADH oxidation in state 4 dropped by over 40% as compared to the initial rate. Meanwhile, the rate of succinate oxidation was not considerably affected by AO inhibition. We believe that one of the AO functions in the mitochondria is to provide for noncompeting oxidation of two (or more) substrates by employing two (or several) dehydrogenases of the respiratory chain.     

Alternative Oxidase Activity and the Ubiquinone Redox Level in Soybean Cotyledon and Arum Spadix Mitochondria during NADH and Succinate Oxidation

In Arum and soybean (Glycine max L.) mitochondria, the dependence of the alternative oxidase activity on the redox level of ubiquinone, with NADH and succinate as substrates, was studied, using a voltametric procedure to measure the ubiquinone redox poise in the mitochondrial membrane. The results showed that when the enzyme was activated by pyruvate the relationship between the alternative oxidase rate and the redox state of the ubiquinone pool was the same for both NADH and succinate oxidations. In the absence of pyruvate the alternative oxidase had an apparent lower affinity for ubiquinol. This was more marked with NADH than with succinate and was possibly due to pyruvate production during succinate oxidation or to an activation of the alternative oxidase by succinate itself. In Arum spadix (unlike soybean cotyledon) mitochondria, succinate oxidation via the alternative oxidase maintained the ubiquinone pool in a partially reduced state (60%), whereas NADH oxidation kept it almost completely reduced. Previous data comparing mitochondria from thermogenic and nonthermogenic tissues have not examined the full range of ubiquinone redox levels in both tissues, leading to the suggestion that the activity of alternative oxidase for Arum was different from nonthermogenic tissues. When the complete range of redox states of ubiquinone is used and the oxidase is fully activated, the alternative oxidase from thermogenic tissue (Arum) behaves similarly to that of nonthermogenic tissue (soybean).     

Effects of flavone on the oxidative properties of intact plant mitochondria

The effects of flavone on the oxidative and phosphorylative properties of plant mitochondria from potato tubers and etiolated mung bean hypocotyls were investigated. Flavone inhibited the state 3 oxidation rates of malate, NADH and, to a lesser extent, succinate but was without effect on the ascorbate-TMPD oxidation rate. The inhibition was the same whether the mitochondria were in state 3 or in an uncoupled state 3. When 100 μM flavone was added during the state 4, the tight coupling of succinate or NADH oxidation was not released. In the electron transfer chain, flavone inhibition appeared to be located in the flavoprotein region. All forms of NADH dehydrogenases seemed to be affected but the greatest inhibition appeared when exogenous NADH was used.     

The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.     

Oxidation processes and ubiquinone localization in the branched respiratory system of mi-1 mutant of Neurospora crassa.

1. Stimulation of succinate oxidation in mi-1 mitochondria by Mg2+ and Pi is abolished on uncoupling, which points to the energy-linked activation of succinate oxidation. 2. Mitochondria exhibited respiratory control with succinate and NADH when the cyanide-insensitive oxidation was inhibited by salicylhydroxamic acid (SHAM). SHAM lowered the oxidation rate with NADH and succinate to the same level, though the NADH oxidation rate was 2.5 times as high as with succinate. 3. Despite the high stimulation of succinate oxidation via the SHAM-sensitive pathway in the active and controlled state of mitochondria, the redox state of UQ in all metabolic states remains unchanged. On inhibition of the cyanide-insensitive pathway, UQ reduction is greatly increased only in the controlled and active state. With NADH as a substrate, UQ does not respond to the metabolic states of mitochondria. 4. The redox changes of cytochrome c parallel those of UQ. 5. Branching of the respiratory chain in mi-1 mitochondria is discussed.     

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD⁺-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD⁺-linked dehydrogenases and ubiquinone.     

The inhibition of plant mitochondrial respiration by the synthetic analog of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT)

The quinone analog, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), has been shown to inhibit cyanide-sensitive and cyanide-insensitive respiration in higher plant mitochondria. The inhibition is dependent upon the concentration of mitochondrial protein. The low concentrations of UHDBT required to inhibit the cyanide-sensitive pathway (microM) and the cyanide-insensitive pathway (nM) indicate that UHDBT is acting as a tight-binding inhibitor of ubiquinol oxidation. Inhibition of both pathways was dependent upon pH. It is shown that UHDBT appears to be a less potent inhibitor of cyanide-sensitive NADH oxidation than of cyanide-sensitive succinate oxidation, and that the pH dependence of inhibition of these two pathways differs. The inhibition of NADH and succinate oxidation by the cyanide-insensitive pathway shows similar pH dependences although at a given pH NADH oxidation is more susceptible to inhibition than succinate oxidation.     

Chlortetracycline and the transmembrane potential of the inner membrane of plant mitochondria.

The oxidation of NADH or succinate by Jerusalem-artichoke (Helianthus tuberosus L.) mitochondria in the presence of chlortetracycline induced an increase in chlortetracycline fluorescence. Any treatment that prevented the formation of a transmembrane potential (as monitored by changes in safranine absorbance, A511-A533), e.g. uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, inhibition of dehydrogenase activity or electron transport, anaerobiosis or depletion of substrate, prevented the increase in chlortetracycline fluorescence or caused it to disappear. Changes in chlortetracycline fluorescence were always slower than changes in the safranine absorbance. The increase in chlortetracycline fluorescence caused by succinate oxidation had an excitation maximum at 393 nm, indicating that a Ca2+-chlortetracycline complex was involved. The increase in fluorescence was observed even in the presence of EDTA, which removes all external bivalent cations, indicating that internal Ca2+ is mobilized. Although NADH and succinate oxidations gave the same membrane potential and qualitatively had the same effect on chlortetracycline fluorescence, NADH oxidation caused a much larger (over 3-fold) increase in chlortetracycline fluorescence than did succinate oxidation. It is possible that this is connected with the Ca2+-dependence of NADH oxidation. In the presence of 2 mM external Ca2+, chlortetracycline collapsed the transmembrane potential and uncoupled succinate and duroquinone oxidation.     

Effects of kaempferol on the oxidative properties of intact plant mitochondria

The effects of kaempferol on the oxidative and phosphorylative properties of plant mitochondria from potato tubers and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls were investigated. Kaempferol inhibited the state 3 oxidation rate of malate, NADH, and succinate, but was without effect on the ascorbate-tetramethyl p-phenylenediamine oxidation rate. The inhibition was almost the same whether the mitochondria were in state 3 or in an uncoupled state 3. When 180 micromolar kaempferol was added during state 4, the tight coupling of succinate or NADH oxidation was not released. The results obtained indicate that kaempferol inhibits the mitochondrial electron flow at, or just after, the flavoprotein site.     

Glyceollin: a site-specific inhibitor of electron transport in isolated soybean mitochondria

The glyceollin inhibition of electron transport by isolated soybean and corn mitochondria was similar to that of rotenone, acting at site I between the internal NADH dehydrogenase and coenzyme Q. Coupled state 3 malate oxidation was inhibited by glyceollin and rotenone with apparent K_i values of about 15 and 5 micromolar, respectively. Carbonylcyanide m-chlorophenyl hydrazone uncoupled state 4 malate oxidation was also inhibited by glyceollin and rotenone, but uncoupled succinate and exogenous NADH state 4 oxidation was only slightly inhibited by both compounds. Glyceollin also inhibited ferricyanide reduction with malate as the electron donor, with an apparent K_i of 5.4 micromolar, but failed to inhibit such reduction with succinate or externally added NADH as electron donors. Glyceollin did not inhibit state 4 oxidation of malate, succinate, or exogenous NADH. Glyceollin did not act as a classical uncoupler or as an inhibitor of oxidative phosphorylation.     

Respiratory Mechanisms in the Flexibacteriaceae: Terminal Oxidase Systems of Saprospira grandis and Vitreoscilla Species

Particles from both Saprospira grandis and Vitreoscilla species, obtained by high-pressure extrusion and sonic treatment, respectively, actively catalyze the oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate with O(2). These activities are inhibited by cyanide but not by antimycin; Saprospira is also amytal- and rotenone-insensitive. Vitreoscilla preparations were unable to oxidize mammalian ferrocytochrome c and reduced tetramethyl-p-phenylenediamine, whereas the Saprospira preparations did so actively. Low-temperature (77 K) difference spectroscopy of Vitreoscilla cells and particles indicates the presence of three maxima in the cytochrome alpha-region at 554, 558, and 562 nm. All three cytochromes are active in NADH and succinate oxidation, but none is ascorbate reducible. Cytochrome o is the only CO-binding pigment present and is probably the terminal oxidase; it has properties similar to the cytochrome o isolated in solubilized form from this organism. Saprospira cells and membranes exhibit four cytochrome absorption bands whose maxima are at 550, 554, 558, and 603 nm at 77 K. The latter component has not been noted previously. NADH and succinate reduce all four cytochromes, but ascorbate reduces only the 550- and 603-nm pigments. CO spectra indicate the presence of cytochrome a,a(3) which is probably the oxidase. A second CO-binding pigment is present which is not a peroxidase but may be a cytochrome.     

Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola

Beffa, T., Pezet, R. and Turian, G. 1987. Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola. Mitochondria from young dormant α spores of Phomopsis viticola Sacc. (ATCC 44940) were isolated by grinding and differential centrifugation. They presented a good integrity of their inner and outer membranes as measured by biochemical assays. Electron microscopic analysis revealed an homogenous population. The highest respiratory activities were observed with NADH and ascorbate + tetra-methyl-p-phenylenediamine (TMPD). Malate stimulated the oxidation of pyruvate, citrate or α-ketoglutarate. The coupling of respiration to oxidative phosphorylation appeared at the time of spore germination. The respiratory activities of mitochondria isolated from young dormant α spores of P. viticola were strongly inhibited by S°. The sensitivity of mitochondrial oxidation of different substrates (NADH, pyruvate + malate, succinate and ascorbate + TMPD) to S° was heterogenous and indicated multiple-site action. Thus preincubation of mitochondria with 30 μM S° before addition of substrates fully prevented NADH oxidation (>98%), and strongly inhibited oxidation of pyruvate + malate (85%), succinate (60%) and ascorbate + TMPD (74%). S° inhibited more rapidly the oxidation of succinate than that of other substrates. In the presence of dithiothreitol (DTT), S°-inhibited oxidation of all substrates (except ascorbate + TMPD) could only be transiently and weakly reestablished. The inhibitory action of S° on the oxidation of NADH, pyruvate + malate and succinate was higher than that observed with sulfhydryl group reagents such as mersalyl, Hg-acetate or p - chloromercuribenzoate. In contrast to S° these SH-group reagents could not inhibit oxidation of ascorbate + TMPD. S°, by its dual capacity to oxidize the SH-groups and to self-reduce, probably at the level of cytochrome c oxidase, could produce a modification of the oxidation state of the respiratory complexes thereby disturbing the electron flux.     

Energy transduction in photosynthetic bacteria VI. Respiratory sites of energy conservation in membranes from dark-grown cells of Rhodopseudomonas capsulata

Membranes prepared from Rhodopseudomonas capsulata grown heterotrophically in the dark perform phosphorylation linked to oxidation of NADH and succinate, with P/2e⁻ ratios of about 0.5 and 0.15, respectively. The localization of the sites of energy conservation was investigated by observing the respiration-induced quenching of the fluorescence of atebrine.

Energization of the membrane can be demonstrated when NADH is oxidized by O₂, ferricyanide or Q₁, when succinate is oxidized by O₂ or by oxidized diaminodurene, and during the oxidation of reduced diaminodurene.

Antimycin A completely inhibits energization between succinate and O₂ or succinate and diaminodurene; however, it only inhibits partially NADH or succinate oxidases and energization between NADH and O₂. KCN inhibits NADH oxidase in a biphasic way: the first level of inhibition is observed at concentrations which block the oxidation of exogenous cytochrome c or of diaminodurene and energization between succinate or ascorbate-diaminodurene and O₂. The second level corresponds to the inhibition of the antimycin-insensitive oxidase.

The results are interpreted as evidence of the presence in these bacteria of a respiratory chain branching after the dehydrogenase system, one arm of the chain being sensitive to antimycin A and low concentrations of KCN and capable of energy conservation, the other being represented by a completely uncoupled system.     

Some Reactions of Isolated Corn Mitochondria Influenced by Juglone

The effects of juglone on the uptake of O₂ by excised corn roots (Zea mays L., Wf9 cms- T × M14) and isolated corn mitochondria arc reported. The O₂ uptake by excised corn roots, as measured by an O₂ electrode, was inhibited more than 90% after a one-hour treatment of 500 μM juglone. Lesser inhibitions were observed with 50 μM and 250 μM juglone. In a KC1 reaction medium in the absence of inorganic phosphate (Pi), juglone stimulated the rate of O₂ uptake by isolated mitochondria oxidizing NADH, succinate, or malate + pyruvate. In the presence of Pi, juglone concentrations of 3 μM and greater inhibited the state 3 oxidation rates of succinate and malate + pyruvate, lowered respiratory control and ADP/O ratios obtained from the oxidation of NADH, malate + pyruvate, or succinate, and reduced the coupled deposition of calcium phosphate within isolated mitochondria driven, by the oxidation of malate + pyruvate. The inhibition of state 3 O₂ uptake by isolated mitochondria, an oxidative state in which electron transfer is coupled to ATP production, is seen to correlate with the inhibition affected by juglone when applied to tissues in vivo.     

Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

Eupafolin: Effect on mitochondrial energetic metabolism

This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3',4'-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25-200microM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated approximately 30% by eupafolin (100microM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA-Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.     

The Inhibition of the Activity of Apple Mitochondria by Oxaloacetate

The inhibitory effect of oxaloacetate (OAA) on the activityof mitochondria isolated from the peel of Cox's Orange Pippinapples has been investigated. A given concentration of OAA causesa longer inhibition of succinate than of malate oxidation andthe rate of disappearance of OAA is faster in the presence ofmalate than in that of succinate. Mg⁺⁺⁺, Al⁺⁺⁺, ATP, and glutamateaccelerate the disappearance of inhibition by OAA; Ca⁺⁺ reinforcesthe inhibition. It is established, by estimation of the oxoacids in the reaction mixtures, that the relief from inhibitionis directly due to removal of OAA. The fall in rate of O₂ uptakewith time, using succinate or malate as substrate, is accompaniedby an accumulation of OAA, and the inhibition of succinate oxidationby malate is due to the increased OAA production when malateis oxidized. Some OAA is broken down non-enzymically to formpyruvate and the rate of breakdown is enhanced by Mg⁺⁺; someis metabolized via the Krebs cycle; some disappear in a coupledreaction between pyruvic and malic dehydrogenases to form citrateand malate, and some can be removed by transamination. It issuggested that all these may be factors in a regulatory actionof OAA on the operation of the Krebs cycle. It is relevant inthis connexion that very small amounts of OAA inhibit the activityof the Krebs cycle when they are produced at the active siteswithin the mitochondrion.     

Calcium uptake by bovine epididymal spermatozoa is regulated by the redox state of the mitochondrial pyridine nucleotides

Immature caput epididymal sperm accumulate calcium from exogenous sources at a rate 2- to 4-fold greater than mature caudal sperm. Calcium accumulation by these cells, however, is maximal in the presence of lactate as external substrate. This stimulation of calcium uptake by optimum levels of lactate (0.8-1.0 mM) is about 5-fold in caput and 2-fold in caudal sperm compared to values observed with glucose as substrate. Calcium accumulation by intact sperm is almost entirely mitochondrial as evidenced by the inhibition of uptake by rotenone, antimycin, and ruthenium red. The differences in the ability of the various substrates in sustaining calcium uptake appeared to be related to their ability to generate NADH (nicotinamide adenine dinucleotide). Previous reports have documented that mitochondrial calcium accumulation in several somatic cells is regulated by the oxidation state of mitochondrial NADH. A similar situation obtains for bovine epididymal sperm since calcium uptake sustained by site III oxidation of ascorbate in the presence of tetramethyl phenylenediamine and rotenone was also stimulated by NADH-producing substrates, including lactate, and inhibited by substrates generating NAD+ (nicotinamide adenine dinucleotide, oxidized form). Further, calcium uptake by digitonin-permeabilized sperm in the presence of succinate was stimulated when NADH oxidation was inhibited by rotenone. The compounds alpha-keto butyric, valeric, and caproic acids, which generate NAD+, inhibited the maximal calcium uptake observed in the presence of succinate and rotenone, and the hydroxy acids lactate and beta-hydroxybutyrate reversed this inhibition. These results document the regulation of sperm calcium accumulation by the physiological substrate lactate, emphasize the importance of mitochondria in the accumulation of calcium by bovine epididymal sperm, and suggest that the mitochondrial location of the isozyme LDH-X in mammalian sperm may be involved in the regulation of calcium accumulation.     

Iron-sulphur centres in mitochondria from Arum maculatum spadix with very high rates of cyanide-resistant respiration.

X-band electron-paramagnetic-resonance spectroscopy at 4.2--77K combined with measurements of oxidation-reduction potential was used to identify iron--sulphur centres in Arum maculatum (cuckoo-pint) mitochondria. In the oxidized state a signal with a derivative maximum at g = 2.02 was assigned to succinate dehydrogenase centre S-3. Unreduced particles showed additional signals at g = 2.04 and 1.98 (at 9.2 GHz), which may be due to a spin-spin interaction. In the reduced state a prominent signal at g = 1.93 and 2.02 was resolved into at least three components that could be assigned to centres S-1 and S-2 of succinate dehydrogenase (midpoint potentials -7 and -240 mV respectively at pH 7.2) and a small amount of centre N-1b (e'o= -240 mV) of NADH-ubiquinone reductase. In addition, changes in line shape around -10 mV indicated the presence of a fourth component in this signal. The latter was more readily reduced by NADH than by succinate, suggesting that it might be associated with the external NADH dehydrogenase. The iron-sulphur centres of NADH-ubiquinone reductase were present in an unusually low concentration, indicating that the alternative, non-phosphorylating, NADH dehydrogenase containing a low number of iron-sulphur centres may be responsible for most of the high rate of oxidation of NADH.