Temperature dependence of horse heart cytochromec oxidation by membrane preparations ofAnacystis nidulans: Characterization of two distinct arrhenius discontinuities

The oxidation of reduced horse heart cytochromec by membranes isolated from the cyanobacteriumAnacystis nidulans after growth at different temperatures was studied between 4°C and 41°C in the light and the dark using both spectrophotometric and polarographic techniques. Arrhenius plots of the temperature dependence of cytochromec photooxidation showed a single discontinuity at 25°C, 15°C, and 12°C in membranes derived from cells grown at 40°C, 30°C, and 25°C, respectively. By contrast. Arrhenius plots of the temperature dependence of dark respiratory cytochromec oxidation always displayed two distinct breaks at 25 and 18°C, 15 and 8.5°C, and 12 and 5.5°C in membranes isolated from cells grown at 40°C, 30°C, and 25°C, respectively. The results are discussed in terms of the thermotropic lipid-phase transitions known to take place in the membranes ofA. nidulans. Special reference will be made to possibly distinct localizations of the membrane-bound cytochromec oxidase complexes in respiration and photosynthesis.     

Structural and functional aspects of the respiratory chain of synaptic and nonsynaptic mitochondria derived from selected brain regions

Studies on brain mitochondria are complicated by the regional, cellular, and subcellular heterogeneity of the central nervous system. This study was performed using synaptic and nonsynaptic mitochondria obtained from cortex, hippocampus, and striatum of male Sprague-Dawley rats (3 months old). Ubiquinone content, detected by HPLC analysis, was about 1.5 nmol/mg protein with an approximate CoQ₉/CoQ₁₀ molecular ratio of 2:1. The activities of several respiratory chain complexes were also studied (succinate-cyt.c reductase, NADH-cyt.c reductase, succinate-DCIP, ubiquinol₂-cyt.c reductase, and cytochrome oxidase), and generally found to be higher in mitochondria from cortex than from other regions. Study of the activities of some of these enzymes vs. 1/T (Arrhenius plots) showed a straight line with an activation energy between 7 and 10 kcal/mol in all the three areas considered. Only CoQ₂H₂-cyt.c reductase activity revealed a biphasic temperature dependence. Also anisotropy (as fluorescence polarization) of the hydrophobic probe DPH showed a deviation from linearity; the break points for both enzymatic activity and anisotropy were found at about 23–24°C.     

Effect of temperature on ethylene biosynthesis in carnation petals

Carnation petals, at a stage in which they are already producing ethylene, show a sigmoidal dependency of ethylene production on temperature within the range of 0 to 30°C. An Arrhenius plot of these data show a break atca. 22°C in the straight lines connecting the points. The activity of the ethylene-forming enzyme (EFE), measured bothin vitro, using isolated membranes, andin vivo, using petals pretreated with 1-aminocyclopropane-1-carboxylic acid (ACC), shows an exponential dependency on temperature within the same range. Arrhenius plots of EFE activity fail to show any discontinuity.In contrast, ACC synthase activity measuredin vitro shows the same sigmoidal dependency on temperature as that of the intact petals. We suggest, therefore, that ACC synthase activity is the rate-limiting step mediating the influence of temperature on ethylene biosynthesis by carnation petals over the range studied.     

Effects of Temperature on Electron Transport in Arum maculatum Mitochondria

The effects of temperature upon the respiratory pathways of Arum maculatum mitochondria have been studied. The alternate oxidase sustained a greater proportion of the total respiration at low temperatures than at higher temperatures. Arrhenius plots of respiratory activities show two discontinuities, one at 14°C and one at 21°C. The lower temperature discontinuity was associated with electron transport from succinate dehydrogenase to the alternative oxidase, enzymes that face the inner side of the membrane while the higher temperature discontinuity was associated with electron transport from the external NADH dehydrogenase to cytochrome c oxidase, which face the outer side of the membrane. Both discontinuities resulted in a decrease in the activation energy for electron transport on one side of the membrane. Arrhenius plots of transmembrane electron transport showed discontinuities at both 14° and 21°C but the upper discontinuity resulted in an increase in the activation energy. Activation energies determined for the respiratory activities show that above 21°C the exogenous NADH-cytochrome pathway and the succinate-alternative oxidase pathway were lower than those for the NADH-alternative pathway or the succinate cytochrome pathway.     

CYTOCHEMICAL STUDIES OF MITOCHONDRIA : II. ENZYMES ASSOCIATED WITH A MITOCHONDRIAL MEMBRANE FRACTION

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ~ 12 per cent of the protein N and ~ 35 per cent of the phospholipide of the whole mitochondria and accounted for ~ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.     

Effect of phospholipids from Saccharomyces cerevisiae at different stages of development on restoration of succinooxidase activity in lipid-depleted mitochondria

Phospholipids extracted fromSaccharomyces cerevisiae at different stages of development after glucose repression contain three major fatty acids: palmitic, palmitoleic and oleic. The ratio palmitic: palmitoleic strongly decreases beginning at the 6th hour of growth.To test the effect of fatty acid composition and in particular of unsaturation on succinoxidase activity, all these phospholipids, phospholipids from commercial yeast, and Asolectin were incubated with lipid-depleted yeast mitochondria. The amount of P bound was not much different for the various phospholipids; succinoxidase activity was restored best by Asolectin; the least effective reactivation was given by phospholipids from yeast at the middle stages of growth. There are not great differences between the various phospholipids and there is no correlation with unsaturation. If we compare the pattern of appearance of respiration during morphogenesis of yeast mitochondria with the pattern of the capability of the phospholipids from cells at different stages of mitochondrial morphogenesis to restore activity of lipid-depleted yeast mitochondria, we find no correlation. The results of this investigation are consistent with the idea that changes in phospholipids and changes in enzyme activities are not linked by a causal relation.     

Temperature dependence of membrane-bound enzymes of the energy metabolism in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides

Rhodospirillum rubrum grown either chemotrophically or phototrophically at 14°C and 30°C, was employed to study the effect of temperature on fatty acid composition as well as on several membrane bound functions involved in energy metabolism. Upon growth at both temperatures the fatty acid composition of membranes showed differences, which could be attributed to an incomplete formation of photosynthetically active membranes rather than specifically to the growth temperature. Activities of NADH dependent respiration and light induced proton extrusion by cells did not show discontinuities in Arrhenius plots down to temperatures of 15°C and 5°C, respectively. In contrast, coupling factor Mg²⁺- and Ca²⁺-ATPase as well as succinate cytochrome c oxidoreductase showed significant breaks at 20°C and 18°C, respectively. Similarly, in Rhodopseudomonas sphaeroides. NADH dependent respiration and light induced proton extrusion by cells was continuous over the entire range of temperatures applied. ATPase as well as succinate cytochrome c oxidoreductase, on the other hand, featured discontinuities in Arrhenius plots at 20°C and 19°C. The implication of the data on growth rates and membrane structure are discussed.Abbreviation Bchl baceteriochlorophyll     

Succinoxidase and Cytochrome Oxidase in Mitochondria from the Spadix of Arum

1. Mitochondria prepared from young Arum spadix have enoughcytochrome c oxidase to account for the rate at which succinateis oxidized, but succinoxidase activity increases markedly asthe plants mature so that in old material cytochrome oxidaseactivity is only 10 per cent, of succinoxidase. 2. Disintegration of the mitochondria by vibration with ballotini,treatment with digitonin or incubation in the warm reveals anintra-mitochondrial cytochrome c oxidase probably active enoughto account for the fastest rates of succinate oxidation. 3. Succinic dehydrogenase activity is demonstrated and experimentswith p-chloromercuribenzoate indicate that it plays a part inthe oxidation of succinate. 4. Cyanide completely inhibits both external and internal cytochromeoxidase but even at the earliest stages it only reduces succinoxidaseby about 50 per cent. Antimycin A also inhibits succinoxidaseby about 50 per cent.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Summary Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa ₃ have been isolated. When grown at 41°C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa ₃ and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23°C, they are virtually indistinguishable from wild-type strains.The mutants were selected on the basis of their slow growth at 41°C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41°C. The selection technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa ₃ may be altered in the mutants at the restrictive temperature.Supported by National Research Council of Canada Grant Number A-6351Recipient of a National Research Council of Canada Postgraduate Scholarship     

The effect of temperature on the velocity of exogenous auxin transport in intact chilling-sensitive and chilling-resistant plants

The velocity of exogenous indol-3yl-acetic acid ([1-¹⁴C]IAA) transport from the apical buds of intact pea, sunflower and cotton plants was determined from 0.5° C to 47° C. The minimum temperature at which transport occurred varied from 2° C (pea and sunflower) to 7° C (cotton). Above these temperatures the velocity of transport increased steadily to maxima near 44° C in all three species. Further increase in temperature resulted in a complete cessation of transport, suggesting a sudden high-temperature breakdown of the auxin transport system. Temperature coefficients (Q₁₀) for transport velocity calculated from Arrhenius plots were low (1.36 to 1.41 between 15° C and 30° C).Arrhenius plots for the chilling-sensitive cotton and sunflower plants exhibited abrupt discontinuities at 14.6° C and 8.7° C respectively. An Arrhenius plot for the chilling-resistant pea exhibited no such discontinuity over the whole temperature range at which transport occurred.Abbreviation IAA indol-3yl-acetic acid     

Resolution and Reconstitution of a Mammalian Membrane

The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F ₁, Mg⁺⁺, phospholipids, and F_c. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c ₁, cytochrome c, cytochrome oxidase, phospholipids and Q ₁₀. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin.     

Mechanism of Chilling Injury in Sweet Potato: X. Change in Lipid-Protein Interaction in Mitochondria from Cold-stored Tissue

Seventy per cent of the phospholipid in mitochondria from sweet potato roots was removed by aqueous acetone treatment. The amount of phospholipid that could be rebound to these lipid-depleted mitochondria roughly corresponded to the amount of phospholipid in untreated mitochondria. The activities of NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, cytochrome oxidase, and succinoxidase in lipid-depleted mitochondria were restored by addition of mitochondrial phospholipid to about 60, 50, 15, and 35%, respectively, in comparison to untreated mitochondria. The capacity of lipid-depleted mitochondria from 14-day cold-stored tissue to bind mitochondrial phospholipid from healthy tissue was lower than that from healthy tissue. However, there was no large difference in activities of NADH-cytochrome c oxidoreductase and succinate-cytochrome c oxidoreductase between both phospholipid rebound lipid-depleted mitochondria from healthy and 14-day cold-stored tissues. On the other hand, activity of succinoxidase in phospholipid rebound lipid-depleted mitochondria from 14-day cold-stored tissue was decreased by about 50% of that from healthy tissue. Furthermore, the capacity of lipid-depleted mitochondria from 2-day cold-stored tissue to bind mitochondrial phospholipid from healthy tissue was higher than that from healthy tissue.     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

Thermally induced changes in reconstituted and membranous cytochrome c oxidase.

The Arrhenius plots of electron transport activity in cytochrome c oxidase reconstituted with well-defined phospholipids have been shown to display a change in slope at 20--25 degrees C regardless of the chemical nature of the incorporated lipid. In native membranous cytochrome c oxidase, the discontinuity in Arrhenius activity plot occurred at 16--18 degrees C. These temperature breaks were found to correlate with changes in spin-label mobilities but not with the bulk lipid transition observed by differential scanning calorimetry. Temperature-dependent reciprocal equilibrium between the immobilized and fluid pools is demonstrated. It is suggested that the changes in kinetic and spin-label spectral characteristics in cytochrome c oxidase membranes are related very likely to a lipid-protein interaction prompted by a thermally induced change in the physical state of the lipids that does not involve a gel to liquid crystalline transition.     

Differential effect of temperature on mitrochondrial activity in shoots from freshly harvested and moderately aged kernels of maize (Zea mays L.)

This paper reports on a study of mitochondrial activity in etiolated shoots of freshly harvested and moderately aged kernels of maize. Activity was investigated after incubation at a favourable temperature (25°C), sub-optimal temperature (13°C) and after a heat shock (46°C for 2h). Although impaired mitochondrial activity in shoots from moderately aged maize kernels was not detected at 25°C, deficiencies became evident under low temperature stress (13°C). State 3 oxygen uptake, cyanide-insensitive oxygen uptake and cytochrome oxidase activity were lower in mitochondria from these shoots at 13°C than in mitochondria from shoots of freshly harvested kernels at this temperature. After a heat shock, cyanide-insensitive oxygen uptake was higher, and cytochrome oxidase activity lower, in shoots of aged kernels than in shoots of fresh kernels. No significant differences in ADP: O ratio or succinate dehydrogenase activity occurred between mitochondria from shoots of the two seed lots in any of the temperature treatments.     

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K _m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C.The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.     

Thermal stability of catalases active in dormant saffron (Crocus sativus L.) corms

Catalase activity was detected in crude extract prepared from dormant saffron (Crocus sativus L.) corms. The activity was independent of pH in the range 6.0 – 11.0. Thermostability studies suggested the presence of three isoenzymes with transition temperatures of 30°C, 45°C and 60°C, respectively, as given by Arrhenius plots. When stained for catalase activity gel electropherograms of extract revealed 3 distinct bands with apparent molecular weight of 323,000, 295,000 and 268,000, respectively. Thus it appeared that at least three isoenzymes of catalase were present in dormant saffron corms.     

Comparative growth of the thermal alga Cyanidium caldarium on nitrate and ammonia at different temperatures

Summary The growth of Cyanidium caldarium on nitrate and ammonia as nitrogen sources was studied at different temperatures from 21 to 54°C.Algal growth occurred at temperatures of 24° C or above when ammonia was the nitrogen source, whereas with nitrate, growth occurred at 30° C or above. The optimum and the maximum growth temperatures were 45 and 54° C respectively on both substrates.Arrhenius plots show that the logarithm of the growth rate is not linear with the reciprocal of absolute temperature, but exhibit sharply defined breaks at 30° C on ammonia and at 40° C on nitrate.It is assumed that below 40° C, when Cyanidium grows on nitrate, the utilization of this substrate represents the master reaction which controls the growth rate of the alga.     

Role of proteins and lipids in non-linear Arrhenius plots of Drosophila mitochondrial succinate-cytochrome c reductase studied by rebinding experiments

Abrupt changes in the Arrhenius activation energy of membrane-bound enzymes have often been correlated with changes in the physical state of membrane phospholipids. Similar changes in activation energy have also been found in soluble enzymes. The possibility exists, therefore, that in some of the membrane-bound enzymes the changes might reflect intrinsic changes of the proteins independent of changes in the membrane phospholipids. This hypothesis was investigated using Drosophila mitochondria isolated from wild type and the mutant Ocd^ts-1. In this mutant it has been shown that succinate-cytochrome $\text{c}$ reductase exhibits a change in Arrhenius activation energy at 18°C which is not found in the wild type (Sondergaard, L., Nielsen, N.C. and Smillie, R.M. (1975) FEBS lett. 50, 126–129). A quantitative thin-layer chromatographic analysis of mitochondrial phospholipids showed sphingomyelin to be more abundant in the wild type than in the mutant (5.2% and 4.3% of the total phospholipids, respectively). Since it was shown that the succinate-cytochrome $\text{c}$ reductase had a lipid requirement for full activity, reciprocal rebinding experiments were done. These experiments showed that the reconstituted membranes exhibited the change in activation energy at 18°C only when the protein moiety came from mutant mitochondria, that is, the change was independent of the source of the phospholipids used.