Organization of Krebs tricarboxylic acid cycle enzymes in mitochondria

Sonic oscillation of mitochondria usually leads to the release of a number of Krebs tricarboxylic acid cycle enzymes. These enzymes have, therefore, been referred to as soluble matrix enzymes. In the present report, we show that gentle sonic or osmotic disruption can be used to obtain a mitochondrial preparation where these enzymes appear to be organized in a large complex of proteins. Using citrate synthase as a marker for these enzymes, we show that the proposed complex is easily sedimented at 32,000 X g in 30 min. The exposed citrate synthase in these complexes can be inhibited by its antibody, indicating that the enzymes are not merely entrapped in substrate-permeable vesicles. The effects of pH, temperature, ionic strength, and several metabolites on the ability to obtain the sedimentable citrate synthase have been tested. These studies indicate that the complex is stable at conditions presumed to exist in situ. Electron microscopic studies show that gentle sonic oscillation gives rise to an efflux of mitochondrial matrix contents which tend to remain attached to the original membranes. The sedimentable fraction also contained four other presumably soluble Krebs tricarboxylic acid cycle enzymes: aconitase, NAD+-isocitrate dehydrogenase, fumarase, and malate dehydrogenase.     

Reversible transdominant inhibition of a metabolic pathway. In vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes in yeast

The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an "assembly mutation," i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15-amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context.     

Cross-linking of mitochondrial matrix proteins in situ

Different cross-linkers (10 mM) of varying specificity and arm length were found to cross-link mitochondrial matrix proteins in situ in 2 min at pH 7.4. As seen by SDS-polyacrylamide electrophoresis, the disappearance of individual protein bands was accompanied by concomitant appearance of polymeric aggregates that failed to enter the 4% spacer gel. The disorganization of the mitochondrial matrix infrastructure either by swelling or sonication of the mitochondria resulted in a decrease in the rate of cross-linking. Leakage of citrate synthase, malate dehydrogenase and fumarase was found to be reduced when cross-linked mitochondria were made permeable with toluene. On lysing the cross-linked mitochondria, a major part of the matrix protein (75%) was found to sediment with the membrane fraction. The activities of citrate synthase, malate dehydrogenase and fumarase in rat liver mitochondria were also found to increase in the precipitates with a concomitant decrease in their activities in the soluble matrix fraction. These results indicate that the cross-linker enters the mitochondria and cross-links matrix proteins including Krebs cycle enzymes either to the mitochondrial membranes, or to themselves resulting in very large molecular weight complexes. These results are interpreted to mean that in liver mitochondria, the Krebs cycle enzymes are preferentially located near the membrane.     

Induction of glyconeogenic enzymes by gibberellin a(3) in endosperm of castor bean seedlings

Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to gibberellin A(3) (GA(3)) (100 micromolar) for periods up to 20 hours. Endosperm homogenates were fractionated on linear sucrose gradients and enzymes in mitochondria, glyoxysome, and cytosol fractions were assayed. Gibberellin treatment resulted in increases in the activities of enzymes in all three compartments. There were also enzymes in all three compartments which were not affected by exogenous applications of GA(3). The isozymes of l-asparate-alpha-ketoglutarate aminotransferase in both mitochondria and glyoxysomes were induced coordinately, whereas the isozymes of citrate synthase and malate dehydrogenase were not. All gluconeogenic enzymes in glyoxysomes are induced by GA(3). With the exception of the mitochondrial malate dehydrogenase isozyme, all enzymes of the tricarboxylic acid cycle believed to participate in glyconeogenesis were increased. The cytosolic enzymes malate dehydrogenase, phosphoenolpyruvate carboxykinase, and fructose bisphosphatase were induced, but the levels of pyruvate kinase and enolase were not affected by GA(3) treatment.     

Correlation between the malate dependent progesterone and citrate biosynthesis in the mitochondrial fraction of human term placenta. The stimulatory effect of ADP and ATP

The relationship between malate dependent conversion of cholesterol to progesterone and citrate biosynthesis in human term placental mitochondria has been investigated. It has been shown that ADP and ATP (but not AMP) stimulate, significantly, both progesterone and citrate formation. The stimulatory effect of these adenine nucleotides was dependent on the presence of Mn2+ in the incubation medium. When Mn2+ was omitted or replaced by Mg2+ only negligible stimulatory effect of ADP and ATP was observed. Atractyloside and oligomycin were without effect on ADP and ATP stimulated progesterone and citrate production. Other dinucleotides tested as: GDP, UDP and CDP stimulated both progesterone and citrate formation only slightly. In all the experiments presented the rate of progesterone biosynthesis was found to be significantly correlated with the rate of citrate production. The experimental results presented in this paper suggest that the stimulatory effect of ADP and ATP on malate dependent progesterone biosynthesis is a consequence of an increased conversion of malate to tricarboxylic Krebs cycle intermediates. The possible mechanism by which ATP and ADP stimulate the citrate formation in human placental mitochondria is discussed.     

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.     

Organization of citric acid cycle enzymes into a multienzyme cluster

The possibility that some of the enzymes of the citric acid cycle may be loosely associated into a multienzyme cluster has been investigated using extracts prepared by gentle disruption of cells. Gel filtration and sucrose density gradient centrifugation have shown that five sequential enzymes of the cycle specifically associate into a cluster: fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase. Ultrasonication destroys the abilities of the enzymes to associate. The cluster could catalyse the sequence of reactions leading from fumarate to oxoglutarate and has been found in extracts of several bacterial species as well as rat liver mitochondria.     

Enzymes of the tricarboxylic acid cycle in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate pathway were investigated in adults and infective larvae of Ancylostoma ceylanicum and Nippostrongylus brasiliensis, and their activities were compared with those obtained in rat liver. A complete sequence of enzymes of the TCA cycle, with most of them showing activities quite similar to those in the rat liver homogenate, was detected in adults of both species. All the enzymes except fumarase and malate dehydrogenase were located predominantly in mitochondria where they showed a variable distribution of activities between the soluble and the membranes fractions. Malate dehydrogenase and fumarase were found in both the mitochondria and the 9,000-g supernatant fraction. Succinyl CoA synthetase, which was present in minimum activity, appeared rate limiting. Enzymes of the glyoxylate pathway, particularly isocitrate lyase, seemed to aid the functioning of the Krebs cycle by allowing the formation of succinate from isocitrate. The infective larvae of both species also were found equipped with all the enzymes of the Krebs cycle. Nonetheless, only isocitrate lyase of the glyoxylate pathway could be detected in these parasites.     

Organization of Krebs tricarboxylic acid cycle enzymes

Binding of enzymes of the Krebs TCA cycle to biological membranes was characterized with respect to intracellular location, susceptibility to various chemical and physical treatments, and extractability as a macromolecular component of the mitochondrial inner membrane. It was shown that citrate synthase and malate dehydrogenase bind to the inner membrane in an ionic strength-sensitive, saturable, and specific manner to a relatively thermostabile component manifested on the inner (matrix) surface of the inner membrane of the mitochondrion. From these data several arguments in support of the physiological applicability of these processes were deduced, and the question of whether these two enzymes bind to the same or different membrane components was considered. Also, experiments preliminary to purification of the citrate synthase binding component were presented.     

Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes.

The level of aspartate aminotransferase in liver mitochondria was found to be approximately 140 microM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (M(r) = 1.6 x 10(5) to 2.7 x 10(6)) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase-binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes. The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase. The level of malate dehydrogenase was found to be so high (140 microM) in liver mitochondria, compared with that of citrate synthase (25 microM) and the pyruvate dehydrogenase complex (0.3 microM), that there would also be a sufficient supply of oxalacetate to citrate synthase-pyruvate dehydrogenase.     

Metabolic effects of mislocalized mitochondrial and peroxisomal citrate synthases in yeast Saccharomyces cerevisiae

Genes CIT1 and CIT2 from Saccharomyces cerevisiae encode mitochondrial and peroxisomal citrate synthases involved in the Krebs tricarboxylic acid (TCA) cycle and glyoxylate pathway, respectively. A Deltacit1 mutant does not grow on acetate, despite the presence of Cit2p that could, in principle, bypass the resulting block in the TCA cycle. To elucidate this absence of cross-complementation, we have examined the ability of Cit1p to function in the cytosol, and that of Cit2p to function in mitochondria. A cytosolically localized form of Cit1p was also incompetent for restoration of growth of a Deltacit1 strain on acetate, suggesting that mitochondrial localization of Cit1p is essential for its function in the TCA cycle. Cit2p was able, when mislocalized in mitochondria, to restore a wild-type phenotype in a strain lacking Cit1p. We have purified these two isoenzymes as well as mitochondrial malate dehydrogenase, Mdh1p, and have shown that Cit2p was also able to mimic Cit1p in its in vitro interaction with Mdh1p. Models of Cit1p and Cit2p structures generated on the basis of that of pig citrate synthase indicate very high structural and electrostatic surface potential similarities between the two yeast isozymes. Altogether, these data indicate that metabolic functions may require structural as well as catalytic roles for the enzymes.     

Purification and Characterization of Glyoxysomal Enzymes from Germinating Pumpkin Cotyledons

Four glyoxysomal enzymes, malate synthase, malate dehydrogenase,3-hydroxyacyl-CoA dehydrogenase and citrate synthase, were purifiedfrom glyoxysomes of germinating pumpkin cotyledons. Molecularweights of their subunits were as follows: malate synthase,60,000; malate dehydrogenase, 33,000; 3-hydroxyacyl-CoA dehydrogenase,72,000 and citrate synthase, 45,000. Malate synthase and 3-hydroxyacyl-CoAdehydrogenase activities were exclusively localized in glyoxysomes,whereas malate dehydrogenase and citrate synthase activitieswere found in both glyoxysomes and mitochondria. Monospecificantibodies against malate dehydrogenase and citrate synthaseinhibited their activities present in glyoxysomes but in mitochondria.Immunocytochemical analysis using the protein A-gold techniquecombined with Lowicryl K4M embedding showed that the antigenicsites for these enzymes were found exclusively in glyoxysomes.These data indicates that malate dehydrogenase and citrate synthasepresent in glyoxysomes are immunologically different from thosein mitochondria, respectively. ¹ This is paper No. 9 in the series "Analytical Studies on MicrobodyTransition". ³ Present address: Meiji Institute of Health Science, Naruta,Odawara, Kanagawa 250, Japan. ⁵ Present address: Department of Biology, Faculty of Science,Kobe University, Rokkoudai, Nada, Kobe 657, Japan. (Received December 23, 1987; Accepted January 27, 1988)     

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.     

Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. A triple mutant of CS (Deltacit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas Deltaaco1- and Deltaidh12-deficient mutants showed higher accumulation of citrate and increased levels of Al tolerance. Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a 2- to 3-fold increase in citrate levels, and the transformants showed enhanced Al tolerance. A gene for Arabidopsis mitochondrial CS was overexpressed in canola using an Agrobacterium tumefaciens-mediated system. Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared with the wild type. Root growth experiments revealed that transgenic lines have enhanced levels of Al tolerance. The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 micro M Al. Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

The interaction of yeast citrate synthase with yeast mitochondrial inner membranes

The specific interaction of yeast citrate synthase with yeast mitochondrial inner membranes was characterized with respect to saturability of binding, pH optimum, effect of ionic strength, temperature response, and inhibition by oxalacetate. The binding ability of the inner membranes is inhibited by proteolysis and heat treatment, which implies that the membrane component(s) responsible for binding is a protein. A protein fraction from inner membranes when added to liposomes will bind citrate synthase. In addition, the binding of yeast fumarase, mitochondrial malate dehydrogenase, and cytosolic malate dehydrogenase to yeast inner membranes was examined. For these studies the yeast mitochondrial matrix enzymes, citrate synthase (from two types of yeast), malate dehydrogenase, and fumarase, as well as cytosolic malate dehydrogenase, were purified using rapid new techniques.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

Physiology and metabolism of two alkane oxidizing and citric acid producing strains of Candida parapsilosis

Summary The activity of enzymes of the tricarboxylic acid (TAC) and glyoxylate (GC) cycles in Candida parapsilosis (wild type KSh 21 and mutant 337) were studied under different physiological and metabolic conditions. C. parapsilosis differed in most of its enzyme activities from other non-citric acid producing yeasts. Furthermore, pH-value, temperature and age of culture proved to act differently on both strains of the tested organism.The addition of trans-aconitate increased not only the growth but also the activities of citrate synthase and some other enzymes while that of aconitase decreased enormously.The high citrate synthase activity might be connected with the role of citrate in the transport of acetyl groups.Abbreviations CS citrate synthase - AC aconitase - ICDH isocitrate dehydrogenase - GDH glutamate dehydrogenase - Fum fumarase - MDH malate dehydrogenase - ICL isocitrate lyase - MS malate synthase     

Kinetic advantage of the interaction between the fatty acid beta-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C4, C8 and C14)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD+ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 microM in gently sonicated and 15 microM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD(+)-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD+ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of beta-oxidation enzymes in situ. Respiration-linked oxidation of butyryl-, octanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

Poly-β-hydroxybutyrate (PHB) biosynthesis,tricarboxylic acid activity and PHB content in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) root nodule

An experiment was conducted to assess the relationship between poly-β-hydroxybutyrate (PHB) biosynthesis and tricarboxylic acid (TCA) activity in desi and kabuli chickpea (Cicer arietinum L.) genotypes. The specific activities of enzymes of PHB metabolism viz., β-ketothiolase (PHB-A), acetoacetyl coenzyme A reductase (PHB-B) and PHB synthase (PHB-C), and those of tricarboxylic acid cycle (citrate synthase (CS) and malate dehydrogenase (MDH) under symbiosis were measured in bacteroids and compared with the PHB accumulation in the nodule and the root. The significant positive correlation was observed between shoot and nodule mass and PHB-A, PHB-B, and PHB-C activities. However, nodule and shoot weights were not significantly correlated with PHB content either in the roots or nodules. The same was true for PHB levels and citrate synthase activity. MDH activity showed a significant negative correlation with nodule PHB. A marked variation and an age dependant increase in malate dehydrogenase activity were measured. A higher capacity for malate oxidation by an increased MDH is likely alter the balance between malate decarboxylation and oxidation, resulting in a higher steady-state concentration of oxaloacetate and that may favor the utilization of acetyl-CoA in the TCA cycle rather than for the synthesis of PHB.