CONTROL OF THE REDOX STATE OF THE PYRIDINE NUCLEOTIDES IN THE RAT CEREBRAL CORTEX. EFFECT OF ELECTROSHOCK-INDUCED SEIZURES

—The concentrations of most of the intermediates of glycolysis and of the tricarboxylic acid cycle were determined in the cerebral cortex of rats, frozen 10 s after the induction of a generalized seizure by electroshock. The apparent equilibrium constant for the combined glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactic dehydrogenase reactions, i.e. K_app= [Lactate] [3-Phosphoglycerate] [ATP]/[Pyruvate] [Glyceraldehyde-3-phosphate] [ADP] [HPO²₄], was evaluated and found to be similar to the value reported for the in vitro system at pH 7. During an estimated 4–5-fold increase in glycolytic flux imposed by the seizure, this system remained close to equilibrium. In control cortex the components of the aldolase reaction were deviated 80-fold from equilibrium but shifted slightly toward equilibrium during the seizure. The components of the aspartate aminotransferase reaction were maintained in equilibrium in both the control and the seizure states. Of 4 reactions used to assess the cytoplasmic and mitochondrial redox states, only the lactic dehydrogenase reaction was considered reliable in the acutely changing situation of the seizure, and yielded a calculated decrease in the cytoplasmic [NAD⁺]/[NADH] ratio. This change, coupled with an observed decrease in the [ATP]/[ADP] [HPO²₄] ratio during the seizure, supports the concept that in brain, as in liver (Krebs & Veech , 1969), the phosphate potential determines the redox state of the tissue.     

The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6-phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α-oxoglutarate and malate, were essentially the same in all regions studied. α-Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased. These findings are interpreted as indicating regional variations in nueronal activity during audiogenic seizures. During the period when clinical seizure activity is apparent neuronal activity increases in the subcortical regions. This is reflected by an increase in energy utilization and an increase in glycolytic flux in these areas. However, a concomitant increase in citric acid cycle flux does not seem to occur during this period. Citric acid cycle flux does appear to increase after the seizure is over.     

ENZYMES OF THE TRICARBOXYLIC ACID CYCLE IN SEA URCHIN EGGS AND EMBRYOS

Changes in the activity of some enzymes of the tricarboxylic acid cycle during development of sea urchins were investigated. Unfertilized eggs showed substantial activity of citrate synthase, aconitase, NAD- and NADP-specific isocitrate dehydrogenases, fumarase and malate dehydrogenase. During development, the activity of citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase and malate dehydrogenase increases gradually, whereas the activity of fumarase remains rather constant. There is no close correlation between changes in the enzyme activity and the increase in oxygen consumption during development. Citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase are mainly localized in the mitochondrial fraction, whereas fumarase and malate dehydrogenase are present in both mitochondrial and cytosol fractions. The intracellular localization of these enzymes does not change during development. A possible mechanism for the regulation of some enzymes of the tricarboxylic acid cycle in sea urchin eggs is discussed.     

Elimination and replenishment of tricarboxylic acid-cycle intermediates in myocardium.

1. The contribution of Co2 fixation to the anaplerotic mechanisms in the myocardium was investigated in isolated perfused rat hearts. 2. K+-induced arrest of the heart was used to elicit a transition in the concentrations of the intermediates of the tricarboxylic acid cycle. 3. Incorporation of 14C from [14]bicarbonate into tricarboxylic acid-cycle intermediates was measured and the rates of the reactions of the cycle were estimated by means of a linear optimization program which solves the differential equations describing a simulation model of the tricarboxylic acid cycle and related reactions. 4. The results showed that the rate of CO2 fixation is dependent on the metabolic state of the myocardium. Upon a sudden diminution of cellular ATP consumption, the pool size of the tricarboxylic acid-cycle metabolites increased and the rate of label incorporation from [14C]bicarbonate into the cycle metabolites increased simultaneously. The computer model was necessary to separate the rapid equilibration between bicarbonate and some metabolites from the potentially anaplerotic reactions. The main route of anaplerosis during metabolite accumulation was through malate + oxaloacetate. Under steady-state conditions there was a constant net outward flow from the tricarboxylic acid cycle via the malate + oxaloacetate pool, with a concomitant anaplerotic flow from metabolites forming succinyl-CoA (3-carboxypropionyl-CoA).     

On the metabolism of ornithine in synaptosomal preparations

The present results show that ornithine is metabolized to glutamate by isolated synaptosomes from mouse cerebral cortex. Under the experimental conditions used the glutamate was channelled further to the tricarboxylic acid cycle, and to a lesser degree to GABA. The possible significance of these metabolic pathways are discussed. Results of an earlier study suggest an excessive metabolism of ornithine via putrescine to GABA in synaptosomes. Those results could not be verified in the present study and a possible reason for the disagreement is demonstrated. However, the present results suggest that putrescine, which is known to be produced from ornithine elsewhere in the nervous tissue, may be metabolized to GABA in synaptosomes.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.     

Metabolism of [U-13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling

Abstract: Metabolism of [U-¹³C₅]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify ¹³C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-¹³C₅]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-¹³C₅]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-¹³C₂]glutamate was observed in astrocytes incubated with [U-¹³C₅]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using ¹³CO₂ produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.     

Brain metabolism of exogenous pyruvate

Pyruvate given in large doses may be neuroprotective in stroke, but it is not known to what degree the brain metabolizes pyruvate. Intravenous injection of [3-13C]pyruvate led to dose-dependent labelling of cerebral metabolites so that at 5 min after injection of 18 mmoles [3-13C]pyruvate/kg (2 g sodium pyruvate/kg), approximately 20% of brain glutamate and GABA were labelled, as could be detected by 13C nuclear magnetic resonance spectrometry ex vivo. Pyruvate, 9 mmoles/kg, was equivalent to glucose, 9 mmoles/kg, as a substrate for cerebral tricarboxylic acid (TCA) cycle activity. Inhibition of the glial TCA cycle with fluoroacetate did not affect formation of [4-13C]glutamate or [2-13C]GABA from [3-13C]pyruvate, but reduced formation of [4-13C]glutamine by 50%, indicating predominantly neuronal metabolism of exogenous pyruvate. Extensive formation of [3-13C]lactate from [2-13C]pyruvate demonstrated reversible carboxylation of pyruvate to malate and equilibration with fumarate, presumably in neurones, but anaplerotic formation of TCA cycle intermediates from exogenous pyruvate could not be detected. Too rapid injection of large amounts of pyruvate led to seizure activity, respiratory arrest and death. We conclude that exogenous pyruvate is an excellent energy substrate for neurones in vivo, but that care must be taken to avoid the seizure-inducing effect of pyruvate given in large doses.     

Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.Subject terms: Virology, Molecular evolution     

Nonfunctional Tricarboxylic Acid Cycle and the Mechanism of Glutamate Biosynthesis in Acetobacter suboxydans

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.     

Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart

The role of the metabolite disposal mechanisms in the regulation of the tricarboxylic acid cycle pool size was studied in isolated perfused rat hearts oxidizing 2 mM propionate. Malate and succinate accumulated during the propionate metabolism. A further 118% increase in the malate concentration and 600% increase in the succinate concentration and a slight inhibition of the propionate uptake were observed during a subsequent KCl-induced arrest of the heart metabolizing propionate. When the mechanical activity of the heart was restored, the malate and succinate concentrations returned to the same levels as before the arrest of the heart, but the propionate uptake did not rise significantly. The mean disposal rates of the tricarboxylic acid cycle metabolites during the cardiac arrest and subsequent restoration of the activity were 1.4 and 2.4 μmol/min per g dry weight, respectively. During cardiac arrest the malate carbon disposed was almost totally recovered as C₃ compounds, whereas after the increase in the ATP-consumption most of it was oxidized. The results show that propionate is oxidized by heart muscle at an appreciable rate but the disposal rate of the tricarboxylic acid cycle intermediates is not tightly regulated by the cellular energy state. Although the metabolite pool size of the tricarboxylic acid cycle responds to change in the ATP consumption, the energy state appears to have a greater effect on the fate of the C₃ compounds formed than on the actual rate of C₄ compound disposition.     

Qualitative and quantitative changes in phospholipid spectra of the brain of rats with corazol-induced epilepsy

The enhancement of the sum of phospholipids in seizure period was observed during initially generalized corazol seizure in white rats' cerebral cortex. This sum fell in an hour after epileptic fit. It is noted that the maintenance of lysophosphatidylcholines and phosphatidylserines enhanced during the fit, and the maintenance of phosphatidylcholines and phosphatidylethanolamines fell either in cerebral cortex, or in cerebellum. At the same time the enhancement of the mixed fraction of sphingomyelin and phosphatidylinositide level and cardiolipins-1 in the cerebral cortex was observed. The maintenance of the latter in cerebellum fell during an hour after the attack. The quantity of cardiolipins-2 in cerebellum enhanced during the attack. It is suggested that the effect of corazol seizure leads to the enhancement of lisoforms at the expense of the intensification of disintegration of other fractions of lisoforms.     

Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin-degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin

A proteomic differential display technique was utilized to study cellular responses of Phanerochaete chrysosporium exposed to vanillin, one of the key intermediates found during lignin biodegradation. Intracellular proteins were resolved by 2-DE and target protein spots were identified using MALDI-MS after in-gel tryptic digestions. Upon addition of vanillin to P. chrysosporium, up-regulation of homogentisate 1,2-dioxygenase, 1,4-benzoquinone reductases, aldehyde dehydrogenase, and aryl-alcohol dehydrogenase, which seem to play roles in vanillin metabolism, was observed. Furthermore, enzymes involved in glycolysis, the tricarboxylic acid cycle, the pentose-phosphate cycle, and heme biosynthesis were also activated. Up-regulation of extracellular peroxidase was also observed. One of the most unique phenomena against exogenous vanillin was a switch from the glyoxylate cycle to the tricarboxylic acid cycle, where a drastic increase in isocitrate dehydrogenase activity was observed. The exogenous addition of other aromatic compounds also caused an increase in its activity, which in turn triggered NAD(P)H production via the action of dehydrogenases in the tricarboxylic acid cycle, heme biosynthesis via the action of aminolevulinic acid synthase on succinyl-CoA, and energy production via activation of the mitochondrial electron transfer system. These metabolic shifts seem to be required for activating a metabolic system for aromatic compounds.     

Physiology and metabolism of pathogenic neisseria: tricarboxylic acid cycle activity in Neisseria gonorrhoeae.

Tricarboyxlic acid cycle activity was examined in Neisseria gonorrhoeae CS-7. The catabolism of glucose in N. gonorrheae by a combination of the Entner-Doudoroff and pentose phosphate pathways resulted in the accumulation of acetate, which was not further catabolized until the glucose was depleted or growth became limiting. Radiorespirometric studies revealed that the label in the 1 position of acetate was converted to CO2 at twice the rate of the label in the 2 position, indicating the presence of a tricarboxylic acid cycle. Growth on glucose markedly reduced the levels of all tricarboxylic acid cycle enzymes except citrate synthase (EC 4.1.3.7). Extracts of glucose-grown cells contained detectable levels of all tricarboxylic acid cycle enzymes except aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), and a pyridine nucleotide-dependent malate dehydrogenase (EC 1.1.1.37). Extracts of cells capable of oxidizing acetate lacked only the pyridine nucleotide-dependent malate dehydrogenase. In lieu of this enzyem, a particulate pyridine nucleotide-independent malate oxidase (EC 1.1.3.3) was present. This enzyme required flavin adenine dinucleotide for activity and appeared to be associated with the electron transport chain. Radiorespirometric studies utilizing labeled glutamate demonstrated that a portion of the tricarboxylic acid cycle functioned during glucose catabolism. In spite of the presence of all tricarboxylic acid cycle enzymes, N. gonorrhoeae CS-7 was unable to grow in medium supplemented with cycle intermediates.     

Importance of Glutamine for γ-Aminobutyric Acid Synthesis in Rat Neostriatum In Vivo

This work was carried out to evaluate the importance of glial cells in providing precursors for the in vivo synthesis of gamma-aminobutyric acid (GABA). Fluorocitrate, which selectively inhibits the tricarboxylic acid cycle in glial cells, was administered locally in rat neostriatum. Inhibition of the glial cell tricarboxylic acid cycle led to a decrease both in glutamine level and in gamma-vinyl GABA (GVG)-induced GABA accumulation, an observation indicating reduced GABA synthesis. The role of glutamine, which is synthesized in glial cells as a precursor for GABA, was further investigated by inhibition of glutamine synthetase with intrastriatally administered methionine sulfoximine. In this case, the glutamine level was reduced to near zero values, and the GVG-induced GABA accumulation was only half that of normal. The results show that glutamine is an important precursor for GABA synthesis, but it cannot be the sole precursor because it was not possible to depress the GVG-induced GABA accumulation completely.     

Pathways of glucose catabolism during germination of Streptomyces spores

Abstract The participation of the different glucose-catabolic pathways during germination of Streptomyces antibioticus spores was studied. In dormant spores, glucose is catabolized through the pentose phosphate (PP) and the Embden-Meyerhof-Parnas (EMP) pathways, with an active tricarboxylic acid cycle. The relative participation of each catabolic pathway is regulated by germinative or non-germinative conditions. During spore germination, the pentose phosphate pathway continuously increased in its participation in the glucose catabolism and it was the major glucose-catabolic pathway in the exponential phase of growth. In addition, it showed the existence of an active tricarboxylic acid cycle in dormant spores, which was being drained for biosynthetic purposes.     

Role of glutamate in the sporogenesis of Bacillus cereus.

Bacillus cereus T, sporulating in a chemically defined medium under optimum conditions, requires substrate quantities of glutamate during the first 4 h of sporogenesis. Seventy percent of the glutamate utilized was catabolized to CO2 during this period, with the remaining glutamate carbon assimilated into various spore constituents, principally protein and nucleic acid. The importance of glutamate as the primary source of reducing potential and energy for early stages of spore formation was investigated. Although the relative efficiency at which tricarboxylic acid cycle intermediates substituted for glutamate was suggestive of oxidation via the tricarboxylic acid cycle, only partial inhibition of glutamate oxidation by fluoroacetate was observed.     

Effect of acetate and octanoate on tricarboxylic acid cycle metabolite disposal during propionate oxidation in the perfused rat heart

Tricarboxylic acid cycle pool size is determined by anaplerosis and metabolite disposal. The regulation of the latter during propionate metabolism was studied in isolated perfused rat hearts in the light of the characteristics of NADP-linked malic enzyme, which is inhibited by acetyl-CoA. The acetyl-CoA concentration was varied by infusions of acetate and octanoate, and the rate of metabolite disposal was calculated from a metabolic balance sheet compiled from the relevant metabolic fluxes. Propionate addition increased the tricarboxylic acid cycle pool size 4-fold and co-infusion of acetate or octanoate did not change it further. Propionate caused a decrease in the CoA-SH concentration and a 10-fold increase in the propionyl-CoA concentration. A paradoxical increase in the CoA-SH concentration was observed upon co-infusion of acetate in the presence of propionate, an effect probably caused by competitive inhibition of propionate activation. A more pronounced decline in the propionyl-CoA concentration was observed upon the co-infusion of octanoate. In a metabolic steady state, acetate and octanoate reduced propionate disposal only slightly, but did not increase the tricarboxylic acid cycle pool size. The results are in accord with the notion that the tricarboxylic acid pool size is mainly regulated by the anaplerotic mechanisms.     

Anaerobic incorporation of radioactivity from 2,3-14C-succinic acid into citric acid cycle intermediates and related compounds in the sea musselMytilus edulis L.

Summary Sea mussels were exposed to nitrogen for various periods (0, 1, 3 and 6 days) and subsequently injected with 2,3-¹⁴C-succinic acid. After 2.5 h anaerobic incubation concentrations of succinate, some amino acids and volatile fatty acids were determined as well as the distribution of radioactivity.Conversion of the precursor decreased from 80 to 40%, due to increased dilution with endogenous succinate, accumulated during the anaerobic preincubation period.More than 80% of the activity of the converted 2,3-¹⁴C-succinic acid was incorporated into malate, aspartate, glutamate, alanine and propionate. This indicates that succinate is not only an end product of anaerobic glycogen breakdown, but remains an active intermediate of the tricarboxylic acid cycle, which can still operate under anaerobic conditions.Concentration and radioactivity of propionate were markedly increased after prolonged anoxia, which gives evidence that succinate is actively converted to propionate during anaerobiosis.Observed accumulation of glutamate during anoxia is explained by incomplete oxidation of pyruvate, which leaves the tricarboxylic acid cycle at the stage of 2-ketoglutarate.     

Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor.