Effect of growth at low temperature on the alternative pathway respiration in Acanthamoeba castellanii mitochondria

Mitochondria of amoeba Acanthamoeba castellanii in addition to the conventional cytochrome pathway possess, like plant mitochondria, a cyanide-resistant alternative quinol oxidase. In mitochondria isolated from amoeba batch culture grown temporarily at low temperature (6 degrees C), higher respiration was accompanied by lower coupling parameters as compared to control culture (grown at 28 degrees C). In the presence of benzohydroxamate, respiratory rates and coupling parameters were similar in both types of mitochondria indicating that growth in cold conditions did not disturb the cytochrome pathway. Increased contribution of alternative oxidase in total mitochondrial respiration in low-temperature-grown amoeba cells was confirmed by calculation of its contribution using ADP/O measurements. Furthermore, in mitochondria from low-temperature- grown cells the content of the alternative oxidase was increased and correlated with the increase in the unstimulated and GMP-stimulated cyanide-resistant respiratory activity. A possible physiological role of higher activity of alternative oxidase as response to growth at a low temperature in unicellular organisms, such as amoeba, is discussed.     

The contribution of uncoupling protein and ATP synthase to state 3 respiration in Acanthamoeba castellanii mitochondria

Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical gradient. We show that AcUCP activity diverts energy from ATP synthesis during state 3 mitochondrial respiration in a fatty acid-dependent way. The efficiency of AcUCP in mitochondrial uncoupling increases when the state 3 respiratory rate decreases as the AcUCP contribution is constant at a given linoleic acid concentration while the ATP synthase contribution decreases with respiratory rate. Respiration sustained by this energy-dissipating process remains constant at a given linoleic acid concentration until more than 60% inhibition of state 3 respiration by n-butyl malonate is achieved. The present study supports the validity of the ADP/O method to determine the actual contributions of AcUCP (activated with various linoleic acid concentrations) and ATP synthase in state 3 respiration of A.castellanii mitochondria fully depleted of free fatty acid-activated and describes how the two contributions vary when the rate of succinate dehydrogenase is decreased by succinate uptake limitation.     

The Effects of Low Growth and Measurement Temperature on the Respiratory Properties of Five Temperate Species

Five temperate ruderal species, Epilobium glandulosum, Matricariamatricariodes, Melilotus alba, Sonchus asper and Taraxacum officinale,were grown under controlled conditions at both 10 °C and20 °C. Respiration was measured at 10 °C and 20 °C.Total dark respiration rates of plants grown at 10 °C wereconsistently higher than the rates of plants grown at 20 °C.This was due to greater respiratory activity along both thecytochrome (KCN-sensitive) and alternative (SHAM-sensitive)pathways in the cold grown plants. The capacity to use the alternativepathway was also greater in the 10 °C grown plants. Theincrease in the relative contribution of each pathway was speciesspecific. At a given growth temperature the activity of thealternative pathway varied little between measurements at 10°C and 20 °C. In contrast, the activity of the cytochromepathway was greater when measured at 20 °C than when itwas measured at 10 °C. The capacity of the alternative pathwaywas lower when measured at 10 °C. These results suggestthat the cytochrome pathway is more sensitive to temperaturestress than is the alternative pathway. Alternative pathway, cyanide resistance, cytochrome pathway, respiration, temperature     

Activation of alternative oxidase and uncoupling protein lowers hydrogen peroxide formation in amoeba Acanthamoeba castellanii mitochondria

Mitochondria of amoeba Acanthamoeba castellanii were used to determine the role of two energy-dissipating systems, i.e., a free fatty acid (FFA)-activated, purine nucleotide-inhibited uncoupling protein (AcUCP) and a FFA-insensitive, purine nucleotide-activated ubiquinol alternative oxidase (AcAOX), in decreasing reactive oxygen species production in unicellular organisms. It is shown that the activation of AcUCP by externally added FFA resulted in a strong decrease in H2O2 production, whilst the inhibition of the FFA acid-induced AcUCP activity by GDP or addition of bovine serum albumin (BSA) enhanced production of H2O2. Similarly, the activation of antimycin-resistant AcAOX-mediated respiration by GMP significantly lowered H2O2 production, while inhibition of the oxidase by benzohydroxamate cancelled the GMP-induced effect on H2O2 production. When active together, both energy-dissipating systems revealed a cumulative effect on decreasing H2O2 formation. The results suggest that protection against mitochondrial oxidative stress may be a physiological role of AOX and UCP in unicellulars, such as A. castellanii.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

Homeoviscous and functional adaptations of mitochondrial membranes to growth temperature in soybean seedlings

The present study investigated whether the cold‐sensitive character of soybean is reflected at the level of mitochondrial membranes. When exposed to an increase of temperature (from 25 to 35 °C), mitochondrial membranes were characterized by a higher phosphatidylcholine : phosphatidylethanolamine ratio and a lower content in 18 : 3 fatty acid. After a reduction of temperature (from 25 to 18 °C) the opposite changes were found. Lipid lateral diffusion and local microviscosity appeared to be comparable in mitochondria from plantlets grown at 25 or 35 °C when assayed at the respective growth temperatures. Some functional aspects (cytochrome c oxidase activity or membrane conductance) tended to this behaviour whereas others (respiration rate or maximum membrane potential) did not. On the other hand, membranes from plants grown at 18 °C were more rigid. Moreover, as illustrated by cytochrome c oxidase activity or respiration rate, functional measurements suggested that these membranes were less active at this temperature. Thus the dynamic characteristics and functional properties measured in mitochondrial membranes were in favour of an adaptive trend at 35 °C, but not at 18 °C despite changes in lipid composition, in accordance with the cold‐sensitive character of the plant.     

Thermal inactivation of electron-transport functions and F0F1-ATPase activities

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of temperature on arginine incorporation by ribosomeless extracts of cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

The effect of transformation of normal rat kidney cells by a temperature-sensitive mutant of the Prague strain of Rous sarcoma virus (ts LA 24 PR-A) on the post-translational addition of arginine to the NH2-terminus of preformed acceptor molecules has been studied. Cells maintained at the permissive (35 degrees C) temperature show a high arginine-incorporating activity in ribosome free extracts compared to that found in extracts of cells grown at the non-permissive (40 degrees C) temperature. Temperature shift experiments as well as studies with cells transformed by wild type Rous sarcoma virus suggest that the decreased activity in cells grown at 40 degrees C is not due to a high temperature per se. The lower arginine incorporation in the 40 degrees C cell extracts is partially due to a decrease in the activity of arginyl transferase which catalyses the transfer of arginine from arginyl tRNA to the acceptor protein. Polyacrylamide gel electrophoresis of the radioactive product shows that the acceptor molecules present in extracts of cells grown at 40 degrees C are larger and qualitatively different from those found in extracts of cells grown at 35 degrees C.     

Identification and characterization of a protozoan uncoupling protein in Acanthamoeba castellanii.

An uncoupling protein (UCP) has been identified in mitochondria from Acanthamoeba castellanii, a nonphotosynthetic soil amoeboid protozoon that, in molecular phylogenesis, appears on a branch basal to the divergence points of plants, animals, and fungi. The existence of UCP in A. castellanii (AcUCP) has been revealed using antibodies raised against plant UCP. Its molecular mass (32,000 Da) was similar to those of plant and mammalian UCPs. The activity of AcUCP has been investigated in mitochondria depleted of free fatty acids. Additions of linoleic acid stimulated state 4 respiration and decreased transmembrane electrical potential (DeltaPsi) in a manner expected from fatty acid cycling-linked H(+) reuptake. The half-maximal stimulation by linoleic acid was reached at 8.1 +/- 0.4 microM. Bovine serum albumin (fatty acid-free), which adsorbs linoleic acid, reversed the respiratory stimulation and correspondingly restored DeltaPsi. AcUCP was only weakly inhibited by purine nucleotides like UCP in plants. A single force-flow relationship has been observed for state 4 respiration with increasing concentration of linoleic acid or of an uncoupler and for state 3 respiration with increasing concentration of oligomycin, indicating that linoleic acid has a pure protonophoric effect. The activity of AcUCP in state 3 has been evidenced by ADP/oxygen atom determination. The discovery of AcUCP indicates that UCPs emerged, as specialized proteins for H(+) cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world.     

Cold stress induces switchover of respiratory pathway to lactate glycolysis in psychrotrophicRhizobium strains

Two psychrotrophic strains of Rhizobium, DDSS69, a non-cold acclimated strain, and ATR1, a cold acclimated strain, were subjected to cold stress. A 4-fold increase in the specific activity of lactate dehydrogenase (LDH) was characteristic for cold stressed cells of DDSS69, whereas ATR1 showed a higher LDH activity in general, which increased 1.5-fold under cold stress. Cold sensitive mutants of DDSS69 which could not grow below 15 degrees C, in contrast to the wild type which could grow at 5 degrees C, were isolated using Tn5-tagged mutagenesis. These mutants showed a 40% lower LDH activity than the wild type grown at 5 degrees C that was comparable to the wild type grown at 15 degrees C. High specific activity of succinic dehydrogenase (SDH) at 28 degrees C in both strains and mutants indicated that aerobic respiration via the citrate cycle is the normal mode of saccharide utilization. Shifts to lower temperatures decreased the specific activity of SDH. However, alcohol dehydrogenase (ADH) activity remained very low in both the strains and the mutants at low temperatures indicating that a shift from aerobic saccharide metabolism to anaerobic one under cold stress involves lactate glycolysis rather than alcohol fermentation. There was an increase in membrane-bound ATPase activity under cold stress which is correlated to higher LDH activity. These data show that, in psychrotrophic Rhizobium strains, cold stress induces a switchover of respiratory metabolism from aerobic to anaerobic pathway, especially lactate glycolysis.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Influence of salicylic acid on biosynthesis of nucleic acids in Polyscias filicifolia cell culture under the action of unfavorable temperatures

Amounts of DNA and RNA was increased (from 20 to 50%) in the presence of salicylic acid in cells of Polyscias filicifolia tissue culture grown in Murachige-Skoog modified medium. Treatment of the tissue culture with salicylic acid resulted in a significant increase of intracellular protein and decrease of proteolytic activity. In cells treated with salicylic acid, the amounts of DNA and RNA was higher in conditions of heat (3 h, 45 degrees C) and cold (24 h, 7 degrees C) stress in comparison with cells exposed to unfavorable temperatures without the initial treatment with salicylic acid.     

The Alternative Respiratory Pathway of Euglena Mitochondria

Mitochondria, isolated from heterotrophic Euglena gracilis , have cyanide-resistant alternative oxidase (AOX) in their respiratory chain. Cells cultured under a variety of oxidative stress conditions (exposure to cyanide, cold, or H2O2) increased the AOX capacity in mitochondria and cells, although it was significant only under cold stress; AOX sensitivity to inhibitors was also increased by cold and cyanide stress. The value of AOX maximal activity reached 50% of total respiration below 20 degrees C, whereas AOX full activity was only 10-30% of total respiration above 20 degrees C. The optimum pH for AOX activity was 6.5 and for the cytochrome pathway was 7.3. GMP, AMP, pyruvate, or DTT did not alter AOX activity. The reduction level of the quinone pool was higher in mitochondria from cold-stressed than from control cells; furthermore, the content of reduced glutathione was lower in cold-stressed cells. Growth in the presence of an AOX inhibitor was not affected in control cells, whereas in cold-stressed cells, growth was diminished by 50%. Cyanide diminished growth in control cells by 50%, but in cold-stressed cells this inhibitor was ineffective. The data suggest that AOX activity is part of the cellular response to oxidative stress in Euglena .     

Effect of different carbon sources and cold shock on protein synthesis by a psychrotrophic Acinetobacter sp

The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80.     

Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria

Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.     

Growth temperature-dependent stearoyl coenzyme A desaturase activity of Fusarium oxysporum microsomes.

The characteristics of the microsomal stearoyl CoA desaturase (EC 1.14.99.5) of vegetative Fusarium oxysporum cells grown at different temperatures were studied. The enzyme had an unusual preference for NADPH (Km = 38 micrometers) over NADH (Km = 89 micrometers) as electron donor, and a relatively high optimum pH of 8.3. Enzyme activity was highest in microsomes from cells grown at 37 degrees C and lowest in cells grown at 15 degrees C. This result correlated well with the observed changes in oleic acid content of the microsomal lipids. Both NADPH-linked reductase activities and hemoprotein content were lowest in cells grown at 37 degrees C. Spectrophotometric analysis of the microsomal hemoproteins indicated the absence of cytochrome b5 and the presence of a b-type heme with a pyridine hemochrome alpha band absorption maximum at 565 nm. Labile sulfide analysis and inhibitor studies with thenoyltrifluoroacetone suggested a role for an iron-sulfur protein in the electron transfer system associated with the desaturase.     

Effect of cold shock on lipid A biosynthesis in Escherichia coli. Induction At 12 degrees C of an acyltransferase specific for palmitoleoyl-acyl carrier protein

Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 degrees C or higher, but it comprises approximately 11% of the fatty acyl chains of lipid A in cells grown at 12 degrees C. The appearance of palmitoleate at 12 degrees C is accompanied by a decline in laurate from approximately 18% to approximately 5.5%. We now report that wild-type E. coli shifted from 30 degrees C to 12 degrees C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA. The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 degrees C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 degrees C.     

The molecular and biochemical basis of nonshivering thermogenesis in an African endemic mammal, Elephantulus myurus

Uncoupling protein 1 (UCP1) mediated nonshivering thermogenesis (NST) in brown adipose tissue (BAT) is an important avenue of thermoregulatory heat production in many mammalian species. Until recently, UCP1 was thought to occur exclusively in eutherians. In the light of the recent finding that UCP1 is already present in fish, it is of interest to investigate when UCP1 gained a thermogenic function in the vertebrate lineage. We elucidated the basis of NST in the rock elephant shrew, Elephantulus myurus (Afrotheria: Macroscelidea). We sequenced Ucp1 and detected Ucp1 mRNA and protein restricted to brown fat deposits. We found that cytochrome c oxidase activity was highest in these deposits when compared with liver and skeletal muscle. Consistent with a thermogenic function of UCP1 isolated BAT mitochondria showed increased state 4 respiration in the cold, as well as palmitate-induced, GDP-sensitive proton conductance, which was absent in liver mitochondria. On the whole animal level, evidence of thermogenic function was further corroborated by an increased metabolic response to norepinephrine (NE) injection. Cold acclimation (18 degrees C) led to an increased basal metabolic rate relative to warm acclimation (28 degrees C) in E. myurus, but there was no evidence of additional recruitment of NE-induced NST capacity in response to cold acclimation. In summary, we showed that BAT and functional UCP1 are already present in a member of the Afrotheria, but the seasonal regulation and adaptive value of NST in Afrotherians remain to be elucidated.     

Proton re-uptake partitioning between uncoupling protein and ATP synthase during benzohydroxamic acid-resistant state 3 respiration in tomato fruit mitochondria

The yield of oxidative phosphorylation in isolated tomato fruit mitochondria depleted of free fatty acids remains constant when respiratory rates are decreased by a factor of 3 by the addition of n-butyl malonate. This constancy makes the determination of the contribution of the linoleic acid-induced energy-dissipating pathway by the ADP/O method possible. No decrease in membrane potential is observed in state 3 respiration with increasing concentration of n-butyl malonate, indicating that the rate of ATP synthesis is steeply dependent on membrane potential. Linoleic acid decreases the yield of oxidative phosphorylation in a concentration-dependent manner by a pure protonophoric process like that in the presence of FCCP. ADP/O measurements allow calculation of the part of respiration leading to ATP synthesis and the part of respiration sustained by the dissipative H(+) re-uptake induced by linoleic acid. Respiration sustained by this energy-dissipating process remains constant at a given LA concentration until more than 50% inhibition of state 3 respiration by n-butyl malonate is achieved. The energy dissipative contribution to oxygen consumption is proposed to be equal to the protonophoric activity of plant uncoupling protein divided by the intrinsic H(+)/O of the cytochrome pathway. It increases with linoleic acid concentration, taking place at the expense of ADP phosphorylation without an increase in the respiration.     

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