Krebs Cycle Enzymes Research Articles

Apocytochromeband other mitochondrial DNA sequences are differentially expressed during the life cycle ofTrypanosoma brucei

Cytochromes and Krebs cycle enzymes are not detected in bloodstream forms of Trypanosoma brucei but are present in procyclic forms. We have analyzed transcription of mitochondrial sequences which contain the apocytochrome b gene and several other open reading frames (ORFs). Multiple transcripts map to individual DNA sequences located on both DNA strands. Larger low abundance transcripts map to multiple ORFs and may be precursor RNAs. Small abundant transcripts map to G + C rich sequences that do not have obvious protein coding functions. The larger of two presumptive apocytochrome b transcripts is strikingly more abundant in procyclic than bloodstream forms and other mitochondrial transcripts are also differentially abundant between these two forms. In addition, many mitochondrial transcripts appear to be differentially polyadenylated between bloodstream and procyclic forms. We suggest that the mechanisms which regulate the production of the mitochondrial respiratory system in T. brucei involve differential expression of mitochondrial genes.

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.

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids.

The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5-3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), alpha-ketoglutaric acid (alpha-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with alpha-KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with alpha-KGA than through reductive amination of alpha-KGA. The experimental data proved that intact bacteroids possess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon. The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N2-fixation, and sucrose available from leaves.

Multiple enzymic lesions in obligate methanotrophic bacteria

Most methane-utilizing bacteria are obligate methylotrophs [1-4]. Possible reasons for their obligate dependence on Cl-SUbstrates, as in case of strict autotrophs, are the metabolic blocks in energetic and biosynthetic pathways [1-8]. For instance, Type I obligate methanotrophs, with the ribulosemonophosphate pathway of formaldehyde assimilation, possess an incomplete Krebs cycle, lacking a-ketoglutarate dehydrogenase. As a result they are unable to oxidize and grow upon polycarbon compounds. In contrast, Type II obligate methanotrophs, with the serine:pathway, have a complete set of the Krebs cycle enzymes but lack hexokinase and 6-phosphogluconate dehydrogenase. However, the absence of a-ketoglutarate dehydrogenase in Type I methanotrophs appears to be an insufficient reason for explaining their failure to grow on multicarbon compounds, since this enzyme may be completely repressed in typical heterotrophic bacteria, e.g., Escherichia coli growing anaerobically on glucose [6,7]. The cause for obligateness in Type II methanotrophs is also not clear, since the lack of hexokinase and 6phosphogluconate dehydrogenase only impairs growth of these bacteria on sugars. In order to explore this problem of obligateness further, we set out to analyze possible metabolic routes of pyruvate and acetate in both types of obligate methanotrophs. Preliminary results of this research have been reported [3,9]. 2.1. Organisms and growth conditions

The ratio of 3‐hydroxyacyl‐CoA dehydrogenase to lipoamide dehydrogenase activity in individual muscle fibers: Mitochondrial specialization for source of energy

3-Hydroxyacyl-CoA dehydrogenase (HAD) has been widely used to assess the capacity for fatty acid oxidation by different muscle fiber types, with various Krebs cycle enzymes as a base for comparison. We have measured this enzyme in individual lyophilized fibers of the guinea pig soleus and the white and red portions of the vastus lateralis, and compared its activity in each fiber with that of lipoamide dehydrogenase (LAD), which as a part of the pyruvate dehydrogenase complex fulfills a function similar to HAD in forming acetyl-CoA, but from pyruvate and, thus, mainly from a carbohydrate source. The mean HAD/LAD ratio was 17.2 +/- 3 in the red vastus, 24.9 +/- 3 in the white vastus, and 43.7 +/- 10 in the soleus, all differences being highly significant. The two types of fast fibers were not distinguished from one another by the enzyme ratio within either the white or the red portion of the vastus lateralis. Data from all of the fast fibers taken together indicate a close correlation (0.93) between the two enzymes, whereas values from the soleus indicate a specialization of the mitochondria of the slow muscle fibers for the oxidation of fatty acids.

Glutamate as the common precursor for the aglycon of the naturally occurring C-nucleoside antibiotics.

Pyrazofurin is one of four naturally occurring C-nucleoside antibiotics; it is elaborated by Streptomyces candidus. The biosynthesis of the pyrazole ring of pyrazofurin has been studied by using 13C- and 14C-labeled acetate. Carbon-13 incorporation into pyrazofurin was observed by proton-decoupled 13C Fourier transform NMR spectroscopy. The incorporation of 14C from [1-14C]acetate was 0.7%. The enrichment of carbons 3, 4, and 5 of pyrazofurin from [2-13C]acetate by S. candidus confirms earlier findings that acetate is converted to glutamate by the combined action of the Krebs cycle and malic enzyme [Elstner, E. F., Suhadolnik, R. J., & Allerhand, A. (1973) J. Biol. Chem. 248, 5385]. Malic enzyme will give rise to [1,2-13C]acetate from [2-13C]acetate. The [1,2-13C]acetate is then converted to glutamate labeled with 13C in carbons 2--5. The 13C incorporation data indicate that carbons 1, 2, 3, and 4, but not 5, of glutamate serve as the four-carbon donor for the carboxamide carbon, C-5, C-4, and C-3, respectively, of the pyrazole ring of pyrazofurin.

Effect of Xiphidiocercarial Infection on the Activities of Glycolytic and Oxidative Enzymes in Lymnaea luteola

The activities of aldolase, malate dehydrogenase, cytochrome oxidase, and NADH oxidase were studied in the foot, mantle, and digestive gland of the freshwater gastropod, Lymnaea luteola, infected with xiphidiocercaria of Prosthogonimus sp. The aldolase:MDH ratio was reduced in all three tissues of infected snails. The overall decrease of NADH oxidase in all the infected tissues and the significant reduction of cytochrome oxidase activity in the infected digestive gland might lead to the accumulation of NADH. It was concluded that the TCA cycle probably was activated in order to supply organic acids that can be utilized either by the host or by the parasite. Marshall et al. (1974) demonstrated in- creased levels of several glycolytic enzymes in the parasitized digestive gland of the proso- branch Littorina saxatilis, and Mengebier and Wood (1967, 1969) reported reduced ac- tivities of glycolytic and oxidative enzymes in infected Crassostrea virginica. Though Mc- Manus and James (1975a, b) studied the in- termediary metabolism of the gastropod host, Littorina saxatilis, separate from parasites, their main concern was to compare the en- zyme systems and metabolic pathways be- tween the digestive gland of the host and the daughter sporocysts of Microphallus similis. They showed that the distribution of the Krebs cycle enzymes was similar both in the host digestive gland and the daughter sporo- cysts. Studies on Lymnaea luteola revealed that the adenylate pool size and the oxidation of substrates were altered in infected Lymnaea luteola (Narayanan and Venkateswara Rao, 1978a, b). Therefore, study of changes in the activity profiles of the enzymes catalyzing im- portant metabolic steps seems warranted. Such changes in Lymnaea luteola infected

Cultivation of Dyskinetoplastic Trypanosoma brucei

Dyskinetoplastic (Dk) mutant African trypanosomes are able to grow in the mammalian host, but not in the insect host or in culture media in which normal African trypanosomes grow as procyclic trypomastigotes (midgut forms). This inability of Dk mutants to grow as procyclic trypomastigotes is the result of alterations of the kinetoplast (mitochondrial) genetic system and the consequent respiratory deficiencies. Normal African trypanosomes lack Krebs cycle and cytochrome enzymes when in the mammalian host. Hence, Dk mutant African trypanosomes can grow in the mammalian host because the energy-generating respiratory systems affected by the Dk alterations are not used by bloodstream trypomastigotes. Most other kinetoplastid flagellates are dependent on these mitochondrial respiratory systems at all stages of their life cycles and, thus, Dk mutants of these other kinetoplastids are nonviable. (See Hajduk, 1978, Prog. Mol. Subcell. Biol. 6: 158-200.) When transferred into appropriate culture media, African trypanosomes transform into procyclic trypomastigotes and concomitantly lose infectivity to the mammalian host. Hirumi et al. (1977, Science 196: 992-994) devised a culture system which supports the growth of infective bloodstream trypomastigotes. This system employs cultivation of the trypanosomes along with cultured mammalian cells. In this report, I describe the cultivation of Dk mutant Trypanosoma brucei in a system similar to that devised by Hirumi et al. The Dk stock used in these experiments was isolated following acriflavin treatment of T. brucei 14/2/164 in mice as described by Stuart (1971, J. Cell Biol. 49: 189-195). No trypanosomes with a Giemsa-staining kinetoplast have been observed in this stock, despite repeated examination over the past 12 yr. Additionally, molecular hybridization, using physically purified and recombinant kDNA sequences, has shown that this stock lacks most if not all kDNA sequences (Stuart, 1979, Ibid. 83: 362a). Blood containing about 5 x 107 trypanosomes per milliliter was harvested by heart puncture from rats 3 days after inoculation. The trypanosomes were separated from the rat blood cells using aseptic techniques. Infected blood was centrifuged (3,000 rpm, 3 min) in a clinical centrifuge. A small portion of the trypanosome layer was carefully removed and suspended in 0.5 ml 0.06 M sodium phosphate buffer, pH 8.0 containing 0.25% NaCl and 0.5% glucose, then passed through 0.1 ml DEAE, equilibrated in the same buffer, in a 1-ml disposable syringe. The cells were eluted directly into RPMI-1640 medium containing 20% fetal bovine serum (heat inactivated), penicillin (50 U/ml), streptomycin (50 ,ug/ml), and fungizone (2.5 ,ug/ml). Medium was maintained at 37 C. The isolation of the trypanosomes in this way took about 5 min, and resulted in substantial numbers of trypanosomes with which to initiate the cultures.

Antitumor Agents XLI: Effects of Eupaformosanin on Nucleic Acid, Protein, and Anaerobic and Aerobic Glycolytic Metabolism of Ehrlich Ascites Cells

The major effect of eupaformosanin as an antineoplastic agent on Ehrlich ascites cell metabolism was to inhibit deoxyribonucleic acid synthesis, specifically at deoxyribonucleic acid polymerase and thymidylate synthetase enzymatic sites. Both pyrimidine and purine systems of Ehrlich ascites were marginally inhibited. Ribonucleic acid synthesis and messenger and ribosomal polymerase activities also were suppressed. Cyclic adenosine monophosphate levels were increased significantly, which correlated with the drastic reduction of histone phosphorylation. Eupaformosanin also suppressed a number of glycolytic and Krebs cycle enzymes as well as oxidative phosphorylation in vitro. All of the inhibited enzymes are known thiol-bearing enzymes that can undergo a Michael-type addition with the α-methylene-γ-lactone moiety of eupaformosanin, as shown with other sesquiterpene lactones.

Influence of Krebs Cycle Acids on Clavine Alkaloid Production and Changes in Activity of Krebs and Glyoxylate Cycle Enzymes During Fermentation

The shake-flask cultures of a Claviceps purpurea strain with high clavine-alkaloid production were investigated. At the early fermentation the Krebs cycle played a dominant role as an amphibolic pathway. The intensive alkaloid synthesis was preceded by a decrease of the Krebs cycle activity and significant rise of the glyoxylate bypass. Pyruvate entered the cell predominatly via glyoxylate bypass. 2-ketoglutarate, isocitrate, citrate, fumarate, malate, and succinate markedly stimulated the alkaloid synthesis. Catabolite repression is assumed to take inhibitory part in regulation of the alkaloid synthesis. Some Krebs cycle acids might participate in overcoming of influence of the catabolite repression. In Schüttelkultur wurde die Clavin-Synthese eines Claviceps-purpurea -Stammes mit intensiver Alkaloidsynthese untersucht. Zu Beginn der Fermentation hat der Krebszyklus erstrangige Bedeutung. Intensiver Alkaloidsynthese ging ein Rückgang der Aktivität des Krebszyklusses und eine Erhöhung der des Glyoxalatzyklusses voraus. Pyruvat tritt in die Zelle hauptsächlich über den Glyoxylatzyklus ein. 2-Ketoglutarat, Isozitrat, Zitrat, Fumarat, Malat und Sukzinat stimulieren die Alkaloidbildung. Die katabolische Repression spielt eine hemmende Rolle bei der Steuerung der Alkaloidsynthese. Einige Säuren des Krebszyklusses beteiligen sich an der Hemmung der katabolischen Repression bei der Alkaloidbildung.

Enzymatic properties of mitochondria isolated from normal and vitamin B 12-deficient rats

The mitochondria from livers of normal and vitamin B 12-deprived animals were compared. The rate of oxygen uptake (states 3 and 4) of mitochondria from vitamin B 12-deprived livers was higher than that of normal mitochondria while the respiratory control index of B 12-deprived mitochondria was lower than normal. Most Krebs cycle enzyme activities were increased in the hepatic mitochondria from B 12-deprived animals. Two-dimensional gel electrophoresis of mitochondrial matrix proteins also indicated that an increase in Krebs cycle enzyme quantities occurred but the increase was not general for all mitochondrial proteins.

Glycolytic, Krebs cycle and pentose phosphate cycle enzymes in spermatozoa of the buffalo (Bubalus bubalis).

Various enzymes of glycolysis (hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase), the Krebs cycle (isocitrate, succinic and malate dehydrogenases), and the pentose phosphate cycle (glucose-6-phosphate and 6-phosphogluconate dehydrogenases) were studied in buffalo spermatozoa by biochemical and cytochemical methods. The enzymes of glycolysis were found to be loosely bound whereas those of the Krebs and pentose phosphate cycles were strongly bound to mitochondrial membranes. All the enzymes studied were localized histochemically in the mid-piece.

49] Measurement of matrix enzyme activity in Situ in isolated mitochondria made permeable with toluene

This chapter discusses that studies on isolated enzymes cannot accurately reflect their precise in vivo behavior. As the knowledge concerning the concentrations of metabolic intermediates has increased, it has become possible to show that regulatory data obtained from studies on enzymes in vitro do not agree with the known metabolic behavior of enzymes in tissues. In an attempt to explain the apparent discrepancies, the existence of microenvironments within cells has been postulated. The major difficulty with testing such hypotheses in animal cells stems from the impermeability of cells to most cofactors and substrates so that behavior of enzymes in vivo cannot be tested. Toluene has been employed to make isolated microbial cells permeable to normally non-penetrating substrates and cofactors. Because these cells have rigid cell walls, no problem was encountered in relation to cell breakage. In a similar manner, yeast cells, which also possess a rigid wall, have been made permeable to metabolic intermediates by toluene treatment, and the control properties of several enzymes were studied. It presents a method of making rat mitochondria permeable to substrates and stabilizing them so that the individual properties of Krebs cycle enzymes can be studied.

Pathophysiology of Aspirin Overdosage Toxicity, With Implications for Management

The principal pathophysiologic effect of toxic doses of salicylates are characterized by (1) stimulation of the respiratory center of the brain, leading to hyperpnea and respiratory alkalosis; (2) uncoupling of oxidative phosphorylation, leading to increased oxygen utilization and glucose demand, increased oxygen utilization and glucose demand, increased glyconeogenesis, and increased heat production; (3) inhibition of Krebs cycle enzymes, leading to decreased glucose availability and increased organic acids; (4) alterations in lipid metabolism and amino acid metabolism, enhancing metabolic acidosis; and (5) increased fluid and electrolyte losses, leading to dehydration, sodium depletion, potassium depletion, and loss of buffer capacity. The principal toxic manifestations of respiratory alkalosis and metabolic acidosis, altered glucose availability and depletion, fluid and electrolyte losses, and hypermetabolism result in serious morbidity and are potentially fatal. Therapy of salicylate intoxication should be aimed principally at replacement of fluid electrolytes, correction of acidemia, administration of glucose, and prevention of further salicylate absorption and enhancement of salicylate elimination.

Coexistence of isocitrate lyase and NADP-isocitrate dehydrogenase in [formula omitted] mitochondria

Glyoxylate and Krebs cycle marker enzymes cobanded on sucrose gradients after isopycnic centrifugation of mitochondrial pellets. Mitochondrial heterogeneity was investigated by treating particles with a Nitro Blue Tetrazolium reaction mixture designed to specifically increase the buoyant density of mitochondria containing the Krebs cycle enzyme isocitrate dehydrogenase. The banding of Krebs cycle enzymes at a heavier buoyant density was accompained by the glyoxylate cycle marker isocitrate lyase. Both isocitrate-metabolizing enzymes appear to be compartmented within the same mitochondria. This strongly suggests that regulation of one or both of these enzymes is necessary for directing carbon flow through the two different cycles.

Biochemical correlates of flying behaviour in bats

The activities of glycolytic and Krebs cycle enzymes, and the distribution and functional properties of lactate dehydrogenase isoenzymes, were determined in pectoral muscles from a range of bats that differ in flying behaviour. Correlations were found between flying patterns, and the degree to which the pectoral muscle depended upon anaerobic glycolysis during fight.

Metabolic intermediates, enzymes and lysosomal activity in aortas of spontaneously hypertensive rats.

Metabolic intermediate levels, glycolytic and Krebs cycle enzyme activities and lysosomal acid hydrolase activities were measured in aortas of spontaneously hypertensive (SHR) versus normotensive (WKY) rats. In the hypertensive aortas the level of lactate, the ratio of lactate to glucose and of lactate to malate was higher in the SHR than WKY aortas. In the hypertensive aortas the obvious shift of metabolism toward higher rate of glycolysis was associated with decreased activity of malate dehydrogenase and espically of lipoamide dehydrogenase. The latter is an essential compoenent of the alpha-ketoglutarate and pyruvate dehydrogenase enzyme complexes and it appears that these complexes are among the sites of arterialmetavolism which are primarily altered by the elevated blood pressure, resulting in increased production of lactate. The activity of the marker lysosomal enzyme N-acetyl-beta-glucosaminidase was unequivocally elevated in the hypertensive aortas. The activity of beta-glucuronidase exhibited incogruous differences between the SHR and WKY aortas and the activity of aortic acid phosphatase did not differ in the two rat strains. The results are discussed in relation to arterial injury, permeability, and atherogenesis.

Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: The glycosome

FEBS LettersVolume 80, Issue 2 p. 360-364 Full-length articleFree Access Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: The glycosome Fred R. Opperdoes, Fred R. Opperdoes Section for Medical Enzymology and Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Eerste Constantijn Huygensstraat 20 Amsterdam The NetherlandsSearch for more papers by this authorPiet Borst, Piet Borst Section for Medical Enzymology and Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Eerste Constantijn Huygensstraat 20 Amsterdam The NetherlandsSearch for more papers by this author Fred R. Opperdoes, Fred R. Opperdoes Section for Medical Enzymology and Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Eerste Constantijn Huygensstraat 20 Amsterdam The NetherlandsSearch for more papers by this authorPiet Borst, Piet Borst Section for Medical Enzymology and Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Eerste Constantijn Huygensstraat 20 Amsterdam The NetherlandsSearch for more papers by this author First published: August 15, 1977 https://doi.org/10.1016/0014-5793(77)80476-6Citations: 456AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume80, Issue2August 15, 1977Pages 360-364 This article also appears in:50th Anniversary ReferencesRelatedInformation

Purification and properties of squid mantle adenylate kinase. Role of NADH in control of the enzyme

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from the mantle muscle of the squid, Loligo pealeii, was purified over 170-fold to homogeneity as judged by polyacrylamide and starch gel electrophoresis. The tissue contains a single isozyme of adenylate kinase, the enzyme from cytoplasmic and mitochondrial compartments (90 and 10% of total activity, respectively) being identical in physical and kinetic properties. Molecular weight was found to be 27,000 +/- 400. The enzyme shows a pH optimum of 8.2 in the forward (APD utilizing) and 7.4 in the reverse direction. Michaelis constants for ADP, ATP, and AMP are 0.70, 0.13, and 0.15 mM, respectively, with optimal Mg2+:adenylate ratios being 1:2 for ADP and 1:1 for ATP. A comparison of mass action ratios with the equilibrium constant indicated that squid adenylate kinase is held out of equilibrium in resting, but not active, muscle. A search for metabolic modulators of adenylate kinase revealed that NADH (Ki of 0.1 mM) was the only modulator which exerted a significant effect within its in vivo concentration range. The data presented indicate that NADH inhibition is the factor maintaining adenylate kinase in a nonequilibrium state in resting muscle and that release of this inhibition can serve to integrate adenylate kinase into the known scheme of intermediary metabolism in this tissue. A sharp drop in NADH levels at the onset on muscular work co-ordinates that activation of aerobic metabolism in this tissue and allows adenylate kinase to return to equilibrium function. At equilibrium, the enzyme can function to ampligy the concentration of AMP, a potent activator and deinhibitor of key glycolytic and Krebs cycle enzymes. The effect of modulators of adenylate kinase in preventing denaturation by heat or proteolysis revealed that NADH and substrates induced conformational changes in the enzyme which rendered it less susceptible to denaturation. The conformation state induced by NADH differed from that induced by substrate.

An Attempt at the Cytochemical Localization of Citrate Synthase in Rat Heart Muscle

Most of the Krebs cycle enzymes are believed to exist in ‘soluble’ form in the mitochondrial matrix except succinate dehydrogenase which is a part of the inner membrane (1). Citrate synthase, one of the Krebs cycle enzymes, is present in rat liver, kidney and heart mitochondria proportional to the amount of the inner membrane area rather than the matrix volume (2). Recent studies on model systems support the idea that the ‘soluble’ Krebs cycle enzymes are probably localized on the inner surface (matrix side) of the inner membrane (3-5). Localization of these enzymes in mitochondria by cytochemical or immunocytochemical methods would be crucial to determine the validity of the above hypothesis. The present study describes cytochemical localization of citrate synthase in rat heart muscle.