High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CII_hmw) that can be resolved by clear native electrophoresis. CII_hmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CII_hmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CII_hmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.     

Type selective inhibition of microbial fatty acid synthases by thiolactomycin

The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g. Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g. Saccharomyces cerevisiae enzyme). We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus. Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin. E. coli synthase, as expected, was strongly inhibited by thiolactomycin. Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E. coli synthase, it is largely insensitive to thiolactomycin.     

Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2-terminal binding sites on a single enzyme

Previous studies have indicated the existence of separate binding sites of ubiquitin-protein ligase, E3, specific for basic (Type I) or bulky hydrophobic (Type II) NH2-terminal amino acid residues of proteins. Another class (Type III) of protein substrates appeared to interact with E3 at regions other than the NH2 terminus (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698). In the present study we have used affinity chromatography on immobilized protein substrates to examine the question of whether the different binding sites belong to one E3 enzyme, or to different E3 species. Another objective was to develop a procedure for the extensive purification of E3. When a crude extract of reticulocytes is applied to Type I or Type II protein substrates linked to Sepharose, E3 becomes strongly bound to the affinity columns and is not eluted with salt at high concentration. However, the enzyme can be specifically eluted by a dipeptide that has an NH2-terminal residue similar to that of matrix-bound protein substrate. A 350-fold purification is obtained in this single step. Preparations of E3 purified on either Type I or Type II protein substrate affinity columns act on both types of protein substrates, indicating that the separate binding sites for basic and hydrophobic NH2-terminal residues belong to one enzyme. Another species of E3 that acts strongly on some Type III protein substrates does not bind to Type I or Type II protein substrate affinity columns.     

Temporal Aspects of Singing Interactions among Territorial Ovenbirds (Seiurus aurocapillus)

The temporal aspects of singing interactions among birds have received relatively little attention. To determine if the song delivery of one individual is affected by that of its territorial neighbor, I recorded singing interactions between territorial ovenbirds (Seiurus aurocapillus). Ovenbirds appeared to adopt one of two roles during singing interactions, Type I or Type II singers. Type II singers placed more of their songs immediately after the song of their neighbor than expected. The singing pattern of Type I singers could not be distinguished from a random pattern with respect to their neighbor's songs. In each observed pair of interacting birds, one individual was a Type I singer and one was a Type II singer. Although there was some intra-individual variation, most birds maintained the same role with each of their recorded territorial neighbors. Variation occurred between the two study sites in the extent that individuals overlapped the songs of their neighbors. Song overlap was common at one site, but occurred as, or less, often than expected at the other site.     

Cyanide-induced transition from endogenous carbohydrate to lipid oxidation as indicated by the carbon-13 content of respiratory CO2

The respiration of thin aerated discs of potato tuber tissue rises sigmoidally through 24 h. Aged disc respiration is ostensibly resistant to concentrations of cyanide which inhibit the respiration of fresh discs. It has been shown that cyanide-resistant respiration does not represent indifference to the inhibitor, but is rather due to the suppression of one respiratory carbon path and the evocation of another. The predominant respiratory carbon path of aged discs in the absence of cyanide comprises glycolysis linked to the tricarboxylic acid cycle. The carbon path mediating the cyanide-induced respiration reflects tricarboxylic acid cycle-independent lipid degradation.

The respiratory substrate at any time was deduced by comparing the ¹³C/¹²C ratio of respired CO₂, collected from discs in the presence or absence of cyanide, with the ¹³C/¹²C ratios characterizing endogenous potential metabolites. The determination of the predominant respiratory substrate in potato discs, which have an endogenous substrate reserve, proved possible because the relative concentrations of the stable carbon isotopes in endogenous compounds such as lipid and starch are widely different.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.     

Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: Effects of growth,respiration and protein biosynthesis

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O₂ consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.Abbreviations DNP dinitrophenol - PG propylgallate - SHAM salicylhydroxamic acid     

Keto acid metabolism in Desulfovibrio.

Four strains of Desulfovibrio each excreted pyruvate to a constant level during growth; it was re-absorbed when the substrate (lactate) was exhausted. Malate, succinate, fumarate and malonate also accumulated during growth. One of the strains (Hildenborough) excreted alpha-ketoglutarate as well as pyruvate when incubated in nitrogen-free medium; the former was re-absorbed on addition of NH4Cl. In a low-lactate nitrogen-free medium, strain Hildenborough rapidly re-absorbed the pyruvate initially excreted, but did not re-absorb the alpha-ketoglutarate. Arsenite (I mM) prevented the accumulation of alpha-ketoglutarate; I mM-malonate did not affect the accumulation of keto acids. Isocitrate dehydrogenase activity (NAD-specific) in all strains was lower than NADP-specific glutamate dehydrogenase activity. Alpha-Ketoglutarate dehydrogenase could not be detected in any strain. NADPH oxidase activity was demonstrated. This and previous work indicate that a tricarboxylic acid pathway from citrate to alpha-ketoglutarate exists in Desulfovibrio spp., and that succinate can be synthesized via malate and fumarate; however, an intact tricarboxylic acid cycle is evidently not present. The findings are compared with observations on biosynthetic pathways in clostridia, obligate lithotrophs, phototrophs, and methylotrophs, and various facultative bacteria.     

Metabolic characteristics of mitochondria isolated from rabbit brain

Abstract— Data on the metabolic characteristics of mitochondria from rabbit brain have been obtained using low concentrations (100–300 μm) of ¹⁴C-labelled substrates of the Krebs tricarboxylic acid cycle. Oxygen consumption was measured polarographically with a Clark-type electrode. Products of the reactions were separated by chromatography on Dowex-1 columns. Data are reported for relative respiratory quotients of various substrates, respiratory control ratios, accumulation of ¹⁴C in various tricarboxylic acid cycle intermediates and the effect of malate and several inhibitors on these parameters. The data have been compared with similar experiments on mitochondria from rabbit heart. Mitochondria from rabbit brain differ substantially from heart mitochondria: those from brain have a very low capacity for oxidation of acetate and other short chain fatty acids; they do not form j3-hydroxybutyrate; they produce substantial amounts of α-ketoglutarate and require added dicarboxylic acid to yield a maximum respiratory quotient. Like those from heart, brain mitochondria have a high capacity for pyruvate oxidation and a low K_m for this substrate. The results have been discussed and compared with work reported by other investigators.     

The distribution in the methylobacteria of some key enzymes concerned with intermediary metabolism

Summary Cell-free extracts of methane-utilizing bacteria (methylobacteria) were examined for the presence of enzymes of the tricarboxylic acid cycle. Representative organisms of the Type II group had a complete set of enzymes for the operation of this cycle. Members of the Type I group lacked -ketoglutarate dehydrogenase. All the methylobacteria examined had an NADH-oxidase which had the properties of a flavoprotein type enzyme. Glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase was NADP-specific and could only be detected in cell extracts of Type I methylobacteria.     

Direct evidence for expression of type II flavoprotein subunit in human complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in aerobic respiration and the tricarboxylic acid cycle. We recently identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual and demonstrated mRNAs of these two isoforms, Type I Fp and Type II Fp, in skeletal muscle, liver, brain, heart, and kidney. Type I Fp was expressed at higher levels than Type II Fp in all cases. In the present study, the biochemical properties of Type II Fp-containing complex II in Raji cells predominantly expressing Type II Fp were investigated. Complex II having Type II Fp was separated from that having Type I Fp by isoelectric focusing in the presence of sucrose monolaurate. Together with the fact that succinate-ubiquinone reductase activity of mitochondria prepared from Raji cell was almost identical to that from human liver, these results clearly indicate the presence of two distinct isoforms of active complex II in human mitochondria.     

Photodynamic effects of hypericin on photosynthetic electron transport and fluorescence of Anacystis nidulans (Synechococcus 6301)

We investigated the photodynamic action of hypericin, a natural naphthodianthrone, on photosynthetic electron transport and fluorescence of the cyanobacterium Anacystis nidulans (Synechococcus 6301). The most drastic effect was the inactivation of photosynthetic oxygen evolution in the presence of the electron acceptor phenyl-p-benzoquinone in aerobic cells which required 1 hypericin/5 chlorophyll a for half-maximal effect. Anaerobic A. nidulans was only partially inactivated and variable chlorophyll a fluorescence remained unperturbed suggesting that photoreaction center II was not a target. Further, hypericin, stimulated photoinduced oxygen uptake in the presence of methylviologen in aerobic cells. This action was less specific than the inactivation of oxygen evolution (1 hypericin/0.5–0.7 chlorophyll a for half-maximal effect). Results point to the involvement of molecular oxygen in two ways. Type I mechanism (Henderson BW and Dougherty TJ (1992) Photochem Photobiol 55: 145–157) in which ground state oxygen reacts with excited substrate triplets appears probable for the inactivation of oxygen evolution. On the other hand, Type II mechanism in which excited oxygen singlets react with ground state substrate molecules appears probable in the stimulation of methylviologen mediated oxygen uptake.Abbreviations Chl chlorophyll - DAD diaminodurene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - Hepes N-[2-hydroxyethyl]-N-[ethanesulfonic acid] - MV methyl viologen - PBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PS I, PS II Photosystems I and II - RC I, RC II reaction centers of PS I and PS II     

Changes in primary metabolism leading to citric acid overflow in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

For citric acid-accumulating Aspergillus niger cells, the enhancement of anaplerotic reactions replenishing tricarboxylic acid cycle intermediates predisposes the cells to form the product. However, there is no increased citrate level in germinating spores and a complex sequence of developmental events is needed to change the metabolism in a way that leads to an increased level of tricarboxylic acid cycle intermediates in mycelia. A review of physiological events that cause such intracellular conditions, with the special emphasis on the discussion of hexose transport into the cells and regulation of primary metabolism, predominantly of glycolytic flux during the process, is presented.     

Indirect evidence of impairment of platelet desaturase enzymes in diabetes mellitus

We measured the platelet total phospholipid fatty acid profiles of 20 insulin treated (Type I) diabetics, 20 non-insulin treated (Type II) diabetics and 20 matched non-diabetic controls to determine the relationship between the omega 6 and omega 3 series of fatty acids in diabetes. A significant inverse correlation between linoleic acid and arachidonic acid occurred in the normal subjects (r = -0.61; P less than 0.001) but was not seen in the Type I diabetics (r = -0.13; P = NS) or in the Type II diabetics (r = -0.27; P = NS). No significant correlation was seen between linolenic acid and eicosapentaenoic acid in the normal controls (r = -0.34; P = NS) or in the Type I diabetics (r = 0.21; P = NS) or in the Type II diabetics (r = -0.20; P = NS). The results suggest that a functional impairment of platelet delta 5 and delta 6 desaturase may occur in diabetes which disrupts the normal equilibrium between linoleic acid and arachidonic acid. However, the level of eicosapentaenoic acid appears to be less dependent on conversion from linolenic acid. Our findings are of importance to studies designed to reduce platelet aggregation in diabetics and non-diabetics by manipulation of the levels of the precursor fatty acids of thromboxane.     

Changes in skeletal muscle in males and females following endurance training

Gender differences in substrate selection have been reported during endurance exercise. To date, no studies have looked at muscle enzyme adaptations following endurance exercise training in both genders. We investigated the effect of a 7-week endurance exercise training program on the activity of beta-oxidation, tricarboxylic acid cycle and electron transport chain enzymes, and fiber type distribution in males and females. Training resulted in an increase in VO2peak, for both males and females of 17% and 22%, respectively (P < 0.001). The following muscle enzyme activities increased similarly in both genders: 3-beta-hydroxyacyl CoA dehydrogenase (38%), citrate synthase (41%), succinate-cytochrome c oxidoreductase (41%), and cytochrome c oxidase (COX; 26%). The increase in COX activity was correlated (R2 = 0.52, P < 0.05) with the increase in VO2peak/fat free mass. Fiber area, size, and % area were not affected by training for either gender, however, males had larger Type II fibers (P < 0.05) and females had a greater Type I fiber % area (P < 0.05). Endurance training resulted in similar increases in skeletal muscle oxidative potential for both males and females. Training did not affect fiber type distribution or size in either gender.     

METABOLISM OF THE ASPARTYL MOIETY OF N-ACETYL-l-ASPARTIC ACID IN THE RAT BRAIN

The metabolism of N-acetyl-l -aspartic acid (NAA) was studied in rat brain. [Aspartyl-U-¹⁴C]NAA was metabolized predominantly by deacylation. Studies of NAA biosynthesis from l -[U-¹⁴C]aspartic acid have confirmed previous reports that NAA turns over slowly in rat brain. However, intracerebrally-injected N-acetyl-l -[U-¹⁴C]asparticacid was rapidly metabolized. Exogenous NAA appears to be taken up rapidly into a small, metabolically-active pool. This pool serves as substrate for a tricarboxylic acid cycle associated with the production of glutamate for the biosynthesis of glutamine. The bulk of the NAA content in brain appears to be relatively inactive metabolically.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Sequence analyses of Type I and Type II chains in human hair and epithelial keratin intermediate filaments: promiscuous obligate heterodimers, Type II template for molecule formation and a rationale for heterodimer formation

Sequence comparisons have been undertaken for all hair and epithelial keratin IF chains from a single species--human. The results lead to several new proposals. First, it is clear that not only is the chain structure of the molecule an obligate heterodimer but promiscuous association of Type I and Type II chains must occur in vivo. Second, the higher predicted content of alpha-helix in Type II chains in solution relative to that expected for Type I chains suggests that it is the Type II chains that precede their Type I counterparts and that they may serve as templates for molecule formation. Third, heterodimer formation leads naturally to greater structural and functional specificity, and this may be required not only because keratin IF have more interacting partners in its cell type than other types of IF have in theirs but also because hair and skin IF have two distinct structures that relate to the "reducing" or "oxidizing" environment in which they can find themselves. The transition between the two forms may require specific head/tail interactions and this, it is proposed, would be more easily accomplished by a heterodimer structure with its greater in-built specificity.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Complex I of winter wheat mitochondria respiratory chain is the most sensitive to uncoupling action of plant stress-related uncoupling protein CSP 310

The influence of stress uncoupling protein CSP 310 on functional stability of different mitochondrial respiratory chain complexes was analysed using various substrates of the tricarboxylic acid cycle. Complex I was the most sensitive to CSP 310 uncoupling action whilst other complexes were more stabile. It is proposed that the key point of CSP 310 uncoupling action is complex I of plant mitochondrial respiratory chain.