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.     

Isocitrate Dehydrogenase and Glutamate Synthesis in Acetobacter suboxydans

Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered alpha-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 x g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to alpha-ketoglutarate which can then undergo a transamination to glutamate.     

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.     

Evidence for autotrophic CO2 fixation via the reductive tricarboxylic acid cycle by members of the epsilon subdivision of proteobacteria

Based on 16S rRNA gene surveys, bacteria of the epsilon subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the epsilon subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO(2) fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO(2) fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the alpha- and beta-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO(2) fixation in epsilon-proteobacteria. Since epsilon-proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO(2) fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.     

Aspects of carbon metabolism in Chloroflexus

When fluoroacetate was added to aerobic, washed cells of Chloroflexus, O₂ uptake was strongly inhibited and citrate accumulated. Under anaerobic conditions in the light, fluoroacetate inhibited CO₂ uptake and caused citrate accumulation. The results are taken as evidence for the operation of a tricarboxylic acid cycle in Chloroflexus both under aerobic conditions in the dark and anaerobically in the light. 2. Organic compounds are assimilated into the storage materials polyglucose and poly--hydroxybutyric acid by washed cells of Chloroflexus. The type of storage product formed from acetate depends upon the availability of reducing power. 3. Low activities of the key enzymes of the reductive pentose phosphate cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase were detected in cell free extracts of photoheterotrophically grown Chloroflexus.Abbreviations RuBP Ribulose-1,5-bisphosphate - TCA tricarboxylic acid - PHB poly--hydroxybutyric acid     

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.     

The CO2 assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic,aerobic hydrogen-oxidizing bacterium,Hydrogenobacter thermophilus

The incorporation of ¹⁴CO₂ by the cell suspensions of an extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus was studied. After short time incubation of the cell suspensions with ¹⁴CO₂, the radiactivity was initially present in aspartate, glutamate, succinate, phosphorylated compounds, citrate, malate and fumarate. All of these compounds except phosphorylated compounds were related to the members of the tricarboxylic acid cycle. The proportion of labelled aspartate onglutamate in total radioactivity on each chromatogram decreased with incubation time, while the percentage of the radioactivity incorporated in phosphorylated compounds increased with time up to 10 s. These indicated that aspartate and glutamate is derived from primary products of CO₂ fixation.In cell-free extracts of Hydrogenobacter thermophilus, the two key enzymes in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase could not be detected. The key enzymes of the reductive tricarboxylic acid cycle, fumarate reductase and ATP citrate lyase were present. Activities of phosphoenolpyruvate synthetase and pyruvate carboxylase were also detected. The referse reactions (dehydrogenase reactions) of -ketoglutarate synthase and pyruvate synthase could be detected by using methyl viologen as an electron acceptor.These findings strongly suggested that a new type of the reductive tricarboxylic acid cycle operated as the CO₂ fixation pathway in Hydrogenobacter thermophilus.     

Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cyclase.

Carbon-14 was incorporated into oxalate and CO2 from either citrate-1,5-14C, succinate-1,4-14C, or fumarate-1,4-14C by cultures of Aspergillus niger pregrown on a medium which contained glucose as the sole carbon source and which did not allow citrate accumulation. In cell-free extracts of mycelium forming oxalate and CO2 from added citrate the following enzymes of the tricarboxylic acid (TCA) cycle were identified: citrate synthase CE 4.1.3.7), aconitate hydratase (EC4.2.1.3), NAD and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41, 1.1.1.42), (alpha-oxoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.99.1), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37). The in vitro activity of aconitate hydratase and of NADP-dependent isocitrate dehydrogenase was shown to be almost identical to the rate of in vivo degradation of citrate or to exceed this rate. The degradation of citrate to oxalate was inhibited completely by 9 mM fluoroacetate. It is concluded that the TCA cycle is involved in the formation of oxalate from citrate.     

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.     

Physiological basis for meso-tartrate sensitivity in some strains of Salmonella typhimurium.

meso-Tartrate inhibited the growth of non-meso-tartrate-utilizing strains of Salmonella typhimurium in peptone water media and mineral salts media with some, but not all, carbon sources. C-R intermediates of the tricarboxylic acid cycle or compounds readily converted to them and substrates metabolized independently of the C-6 part of the cycle spared bacteria from the inhibitory effects of meso-tartrate when added to cultures along with meso-tartrate. Experiments with cell-free extracts of non-meso-tartrate-utilizing strains from batch and continuous cultures showed that meso-tartrate was a competitive inhibitor of isocitrate dehydrogenase and isocitrate lyase activities and also inhibited citrate synthase and malate synthase activities. The synthesis of these enzymes was not inhibited by meso-tartrate. The isocitrate enzymes of meso-tartrate-utilizing strains of S. typhimurium were similarly inhibited by meso-tartrate, but inhibition of the growth of meso-tartrate-utilizing strains was demonstrable only in uninduced cultures in which the intracellular concentrations of meso-tartrate were high.     

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.     

Enzymes for purine synthesis and scavenging in pathogenic mycobacteria and their distribution in Mycobacterium leprae

Three enzymes catalysing the synthesis of four intermediates (phosphoribosylglycinamide, phosphoribosylaminoimidazole-succinocarboxamide, phosphoribosylaminoimidazole-carboxamide and AMP) in the purine biosynthetic pathway were detected in extracts of Mycobacterium microti and M. avium, even when the organisms had been grown in mice. However only one of the three enzymes, adenylosuccinate AMP-lyase (catalysing the synthesis of the last two of the four intermediates listed above) was detected in M. leprae. Phosphoribosyltransferases, which convert adenine, guanine and hypoxanthine to the corresponding nucleoside monophosphates, and adenosine kinase were the major enzymes for purine scavenging in all mycobacteria studied. In contrast to enzymes in the synthetic pathway, evidence for metabolic regulation of the purine-scavenging enzymes was obtained. In particular, 20-80-fold differences in the activities of guanine phosphoribosyltransferase and adenosine kinase were observed when M. microti was grown in media with or without purines, or in mice. In M. leprae, activities of all phosphoribosyltransferases were low in comparison with activities in M. microti and M. avium (specific activity less than 2% when comparisons were made between extracts of host-grown mycobacteria). However, activity of adenosine kinase was higher in host-grown M. leprae than in host-grown M. microti or M. avium.     

Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo. Evidence for restricted mobility of a multienzyme complex

It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle in the mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching to measure the diffusional mobilities of chimeras consisting of green fluorescent protein (GFP) fused to the C terminus of four tricarboxylic acid cycle enzymes: malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinyl-CoA synthetase. The GFP-enzyme chimeras were localized selectively in the mitochondrial matrix in transfected Chinese hamster ovary (CHO) and COS7 cells. Laser photobleaching using a 0.7-microm diameter spot demonstrated restricted diffusion of the GFP-enzyme chimeras. Interestingly, all four chimeras had similar diffusional characteristics, approximately 45% of each chimera was mobile and had a diffusion coefficient of 4 x 10(-8) cm(2)/s. In contrast, unconjugated GFP in the mitochondrial matrix (targeted using COX8 leader sequence) diffused freely (nearly 100% mobility) with a greater diffusion coefficient of 20 x 10(-8) cm(2)/s. The mobility of the GFP-enzyme chimeras was insensitive to substrate source, ATP depletion, or inhibition of the adenine nucleotide translocase. These results indicate similar mobility characteristics of unrelated tricarboxylic acid cycle enzymes having different sizes and physical properties, providing biophysical evidence for a diffusible multienzyme complex in the mitochondrial matrix.     

Amino Acid Catabolism and Malic Enzyme in Differentiating Dictyostelium discoideum

Amino acids produced from protein degradation are the major energy source for differentiation and aging in Dictyostelium discoideum. Considering the reactions involved in the conversion of amino acids from an average protein into tricarboxylic acid cycle intermediates, a route from a cycle intermediate (probably malate) to acetyl coenzyme A is required for the complete utilization of amino acids. Citrate was isolated from cells pulse-labeled with (14)C-labeled amino acids and was cleaved with citrate lyase. When cells were pulse-labeled with [U-(14)C]-glutamate the specific radioactivity of the acetate and oxaloacetate portions of citrate were consistent with the conclusion that one-third of the carbon flowing through the tricarboxylic acid cycle is removed for the synthesis of acetyl coenzyme A. The data were also consistent with the patterns of carbon flux required to maintain steady-state levels of cycle intermediates in cells catabolizing amino acids. It is suggested that the malic enzyme (EC 1.1.1.40) catalyzes the synthesis of acetyl coenzyme A from malate and is responsible for the observed citrate labeling pattern. In cell extracts the activity of this enzyme increased markedly with the onset of differentiation. The properties of partially purified (40-fold) malic enzyme isolated at culmination indicated that the enzyme was allosteric and was positively affected by aspartate and glutamate. Thus, amino acid production from protein degradation would stimulate a reaction essential for the efficient utilization of these amino acids for energy.     

Phosphorylation of citrate lyase ligase in Clostridium sphenoides and regulation of anaerobic citrate metabolism in other bacteria

Since anaerobic bacteria cannot take advantage of citrate oxidation through the reactions of the tricarboxylic acid cycle special enzymes are needed for its fermentation. The activity of citrate lyase (the key enzyme of the citrate fermentation pathway) is in most cases strictly controlled by acetylation/deacetylation and configurational changes. In order to efficiently regulate citrate metabolism the activity of various regulatory enzymes, that modulate citrate lyase activity, are in turn under stringent control. Covalent modification by phosphorylation/dephosphorylation and electron transport dependent processes are some of the regulatory mechanisms that are here involved. L-Glutamate, which signals the availability of citrate, plays a central role in the regulation of citrate metabolism by influencing the enzymes that are acting in a complex cascade system.     

Metabolism of Yarrowia lipolytica grown on ethanol under conditions promoting the production of alpha-ketoglutaric and citric acids: a comparative study of the central metabolism enzymes

A comparative study of the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing alpha-ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.     

Enzymes of the tricarboxylic acid cycle in Obeliscoides cuniculi (Nematoda; Trichostrongylidae) during parasitic development

The specific activities of the enzymes of the tricarboxylic acid cycle; citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were determined in early fifth-stage, young and mature adult Obeliscoides cuniculi, the rabbit stomach worm. ∝-Ketoglutarate dehydrogenase activity could not be determined in any fraction. Fumarate reductase activity was found only in the mitochondrial fraction while all other enzymes, including an NADP-dependent malic enzyme were localized in the cytoplasm. Glutamate dehydrogenase, acid and alkaline phosphatase activities were also recorded. High levels of those enzymes acting in the “reversed” direction, i.e. MDH and fumarase relative to the enzymes of the “forward” direction, i.e. citrate synthase, aconitase and isocitrate dehydrogenase suggests that under anaerobic conditions a modified tricarboxylic acid cycle can operate. Some variations in specific activities were apparent as the worms matured but no qualitative differences were observed.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

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.     

Activity and androgenic control of enzymes associated with the tricarboxylic acid cycle, lipid oxidation and mitochondrial shuttles in the epididymis and epididymal spermatozoa of the rat.

1. Enzyme activities (units/g wet wt.) were determined in the caput and cauda epididymidis and in epididymal spermatozoa of the rat. 2. The activity of most enzymes in the cauda was between 50 and 100% of that in the caput, except that ATP citrate lyase was barely detectable in the cauda. 3. Spermatozoa, unlike epididymal tissue, contained sorbitol dehydrogenase but lacked ATP citrate lyase. NADP+-malate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, succinate dehydrogenase, carnitine acetyltransferase and citrate synthase were 5 to 400 times as active in spermatozoa as in epididymal tissue. 4. 2-Oxoglutarate dehydrogenase was the least active member of the tricarboxylic acid cycle in all tissues and most closely matched the measured flux through the cycle. 5. The concentrations of hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase were equivalent to the more active enzymes of the tricarboxylic acid cycle, indicating the capacity for extensive lipid oxidation, and the presence of 3-hydroxybutyrate dehydrogenase suggests that these tissues can also oxidize ketone bodies. 6. Transfer of reducing equivalents from cytoplasm to mitochondrion is unlikely to occur by means of the glycerol phosphate cycle because mitochondrial glycerol 3-phosphate dehydrogenase is relatively inactive in epididymal tissue, whereas the cytoplasmic enzyme has little activity in spermatozoa, but transfer may be accomplished by the malate-aspartate shuttle. 7. Transfer of acetyl units from mitochondrion to cytoplasm could be effected by the pyruvate-malate cycle in the caput of androgen-maintained rats, but not in the other tissues because of the low activity of ATP citrate lyase. Acetyl unit transfer could take place via acetylcarnitine, mediated by carnitine acetyltransferase. 8. Castration resulted in a decrease in the concentration of nearly all enzymes, although subsequent administration of testosterone restored concentrations to values similar to those in animals maintained by endogenous androgen. The extent to which enzyme concentration was changed by an alteration in androgen status was highly variable, but was most marked in the case of pyruvate carboxylase.