The intracellular location of pyruvate carboxylase, citrate synthase and 3-hydroxyacyl-CoA dehydrogenase in lactating rat mammary gland.

The intracellular location of pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat mammary gland was investigated by using a fractional-extraction technique. The results indicate a mitochondrial location for all three enzymes.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

A novel syndrome affecting multiple mitochondrial functions, located by microcell-mediated transfer to chromosome 2p14-2p13

We have studied cultured skin fibroblasts from three siblings and one unrelated individual, all of whom had fatal mitochondrial disease manifesting soon after birth. After incubation with 1 mM glucose, these four cell strains exhibited lactate/pyruvate ratios that were six times greater than those of controls. On further analysis, enzymatic activities of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, NADH cytochrome c reductase, succinate dehydrogenase, and succinate cytochrome c reductase were severely deficient. In two of the siblings the enzymatic activity of cytochrome oxidase was mildly decreased (by approximately 50%). Metabolite analysis performed on urine samples taken from these patients revealed high levels of glycine, leucine, valine, and isoleucine, indicating abnormalities of both the glycine-cleavage system and branched-chain alpha-ketoacid dehydrogenase. In contrast, the activities of fibroblast pyruvate carboxylase, mitochondrial aconitase, and citrate synthase were normal. Immunoblot analysis of selected complex III subunits (core 1, cyt c(1), and iron-sulfur protein) and of the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins, decreasing the possibility that an import and/or assembly factor is involved. To elucidate the underlying molecular defect, analysis of microcell-mediated chromosome-fusion was performed between the present study's fibroblasts (recipients) and a panel of A9 mouse:human hybrids (donors) developed by Cuthbert et al. (1995). Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. These results indicate that the underlying defect in our patients is under the control of a nuclear gene, the locus of which is on chromosome 2. A 5-cM interval has been identified as potentially containing the critical region for the unknown gene. This interval maps to region 2p14-2p13.     

A study on the physical interaction between the pyruvate dehydrogenase complex and citrate synthase

In this paper, physicochemical evidence is given for the association between the pyruvate dehydrogenase complex (EC 1.2.4.1) and citrate synthase (EC 4.1.3.7) with two gel chromatographic techniques with poly(ethylene glycol) co-precipitation and with ultracentrifugation. Experiments with active enzyme gel chromatography indicate that citrate synthase also associates with pyruvate dehydrogenase complex in its functioning state. Citrate synthase binds to the isolated transacetylase core of pyruvate dehydrogenase complex, but in the binding to the whole pyruvate dehydrogenase complex the two other components of the complex are also involved. One pyruvate dehydrogenase complex can bind 10-11 citrate synthase dimers, and the dissociation constant is about 5.7-6.0 microM as determined by two independent methods. The association between the pyruvate dehydrogenase complex and citrate synthase raises the possibility of the dynamic compartmentation of acetyl-CoA in the mitochondria which results in the direction of acetyl-CoA from pyruvate towards citrate.     

Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD(+)-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.     

The role of enzymes of pyruvate and citrate metabolism in control of gluconeogenesis in oocytes and embryos of the loach,Misgurnus fossilis L.

Summary Cessation of gluconeogenesis during oocyte maturation inMisgurnus fossilis L. is accompanied by an increase of pyruvate dehydrogenase activity (EC 1.2.4.1). The activity of other enzymes of citrate and pyruvate metabolism (citrate synthetase, EC 4.1.3.7, pyruvate carboxylase, EC 6.4.1.1., malate dehydrogenase, EC 1.1.1.37) remains constant during oocyte maturation and early embryogenesis.In the course of oocyte maturation the levels of acetyl-CoA, pyruvate and citrate remained unchanged, but the level of malate and oxaloacetate underwent drastic increase. The level of phosphoenolpyruvate increased about two-fold. The mitochondrial (NAD⁺)/(NADH) ratio was calculated by measurement of intermediates of the glutamate dehydrogenase reaction and it was found to increase six-fold during oocyte maturation. The lower mitochondrial (NAD⁺)/(NADH) ratio in oocytes compared to that in the embryos is likely to be responsible for the transfer of reducing equivalents from mitochondria to cytoplasm, while in embryos transfer in the opposite direction takes place.     

Pyruvate carboxylase in lactating rat and rabbit mammary gland

1. Pyruvate carboxylase [pyruvate-carbon dioxide ligase (ADP), EC 6.4.1.1] was found in cell-free preparations of lactating rat and rabbit mammary glands, and optimum assay conditions for this enzyme were determined. 2. Subcellular-fractionation studies with marker enzymes showed pyruvate carboxylase to be distributed between the mitochondrial and soluble fractions of lactating rat mammary gland. Evidence is presented that the soluble enzyme is not an artifact due to mitochondrial damage. 3. In contrast, pyruvate carboxylase in lactating rabbit mammary gland is confined to the mitochondrial fraction. 4. The final product of pyruvate carboxylase action in the mitochondrial and particle-free supernatant fractions of lactating rat mammary gland was shown to be citrate. 5. The effects of freeze-drying, ultrasonic treatment and freezing-and-thawing on the specific activity of mitochondrial pyruvate carboxylase were investigated.     

Changes induced by Fe deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris) leaves

The effects of iron deficiency and iron resupply on the metabolism of leaf organic acids have been investigated in hydroponically grown sugar beet. Organic acid concentrations and activities in leaf extracts of several enzymes related to organic acid metabolism were measured. Enzymes assayed included phosphoenol pyruvate carboxylase (PEPC; EC 4.1.1.31), different Krebs cycle enzymes: malate dehydrogenase (MDH; EC 1.1.1.37), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2), citrate synthase (CS; EC 4.1.3.7) and isocitrate dehydrogenase (ICDH; EC 1.1.1.42), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and two enzymes related to anaerobic metabolism (lactate dehydrogenase [LDH]; EC 1.1.1.27, and pyruvate decarboxylase [PDC]; EC 4.1.1.1). Iron concentration in leaves was severely decreased by iron deficiency. Iron resupply caused an increase in iron concentrations, reaching levels similar to the controls in 96 h. Iron deficiency induced a 2.3-fold (from 16 to 37 mmol m⁻²) increase in leaf total organic acid concentration. Organic anion concentrations were still 4-fold higher than the controls 24 h after resupply and decreased to values similar to those found in the controls after 96 h. All measured enzymes had increased activities in extracts of iron-deficient leaves when compared to the controls and generally decreased to control values 24 h after iron addition. These data provide evidence that organic acid accumulation in iron-deficient leaves is likely not due to an enhancement in leaf carbon fixation. Instead, this accumulation could be associated with organic acid export from the roots to the leaves via xylem.     

Enzymic changes in rabbit and rat mammary gland during the lactation cycle

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.     

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.     

The Activities of Some Energy-Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions

Abstract: The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (nonsynaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD-linked malate dehydrogenase (EC 1.1.1.37), D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.     

Differential effects of 2-oxo acids on pyruvate utilization and fatty acid synthesis in rat brain

1. The effects of 2-oxo-4-methylpentanoate, 2-oxo-3-methylbutanoate and 2-oxo-3-methylpentanoate on the activity of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase, (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old rats were investigated. 2. The pyruvate dehydrogenase enzyme activity was competitively inhibited by 2-oxo-3-methylbutanoate with respect to pyruvate with a K(i) of 2.04mm but was unaffected by 2-oxo-4-methylpentanoate or 2-oxo-3-methylpentanoate. 3. The citrate synthase activity was inhibited competitively (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~7.2mm) and 2-oxo-3-methylbutanoate (K(i)~14.9mm) but not by 2-oxo-3-methylpentanoate. 4. The acetyl-CoA carboxylase activity was not inhibited significantly by any of the 2-oxo acids investigated. 5. The fatty acid synthetase activity was competitively inhibited (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~930mum) and 2-oxo-3-methylpentanoate (K(i)~3.45mm) but not by 2-oxo-3-methylbutanoate. 6. Preliminary experiments indicate that 2-oxo-4-methylpentanoate and 2-oxo-3-phenylpropionate (phenylpyruvate) significantly inhibit the ability of intact brain mitochondria from 14-day-old rats to oxidize pyruvate. 7. The results are discussed with reference to phenylketonuria and maple-syrup-urine disease. A biochemical mechanism is proposed to explain the characteristics of these diseases.     

Influence of 1,2,3-benzene-tricarboxylate on pyruvate metabolism in rat-liver mitochondria.

1,2,3-Benzene-tricarboxylate, a known inhibitor of the mitochondrial tricarboxylate carrier, was found to inhibit pyruvate carboxylation as well as the transport of citrate out of the matrix in rat liver mitochondria incubated with pyruvate. The inhibition of pyruvate carboxylation was observed with both intact mitochondria and with the solubilized pyruvate carboxylase. The inhibition of the pyruvate carboxylase by 1,2,3-benzene-tricarboxylase was not mediated via one of the parameters known to regulate the activity of the enzyme and therefore a direct inhibition of the enzyme by the tricarboxylate was assumed. Since the pyruvate carboxylase is exclusively localized in the mitochondrial matrix space it was concluded that 1,2,3-benzene-tricarboxylate penetrates into this compartment.     

The localization of some coenzyme A-dependant enzymes in rat liver mitochondria

1. CoA, acetyl-CoA, l-carnitine and acetyl-l-carnitine when added to rat liver mitochondria equilibrate with approximately two-thirds of the total intramitochondrial water. The mitochondrial space calculated to be freely permeable to these solutes was identical with that obtained for sucrose. 2. Acetyl-CoA is rapidly deacylated by rat liver mitochondria at 0 degrees C, and special precautions are required to measure its mitochondrial permeation. 3. Rat liver mitochondria were separated into fractions that correspond to the inner membrane, the outer membrane, and the soluble proteins of the matrix and intermembrane compartment. Soluble enzymes considered to be located in the matrix were citrate synthase (EC 4.1.3.7), palmitoyl-CoA dehydrogenase (EC 1.3.2.2), electron-transferring flavoprotein, medium-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.2), l-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35) and 3-keto-acyl-CoA thiolase (EC 2.3.1.16). Carnitine palmitoyltransferase (EC 2.3.1.-) is largely associated with the inner-membrane fraction. A long-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.3) is associated with the outer-membrane fraction.     

Regulation of enzyme activity in plants by reversible phosphorylation

This paper reviews the seven specific plant enzymes which have been shown or suggested, to date, to undergo reversible covalent modification by regulatory phosphorylation, including mitochondrial pyruvate dehydrogenase (EC 1.2.4.1), chloroplastic pyruvate, orthophosphate dikinase (EC 2.7.9.1) and ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39), cytoplasmic phosphoenolpyruvate carboxylase (EC 4.1.1.31) and 6-phosphofructo-2-kinase (EC 2.7.1.105), microsomal hydroxymethylglutaryl - CoA reductase (EC 1.1.1.34), and quinate: NAD+ oxidoreductase (EC 1.1.1.24).     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Carbon metabolism in Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41

The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41, grown either at an atmospheric content of CO2 in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO2 content increased to 5-10%. Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate cycle enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cellular extracts of strain 41. During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5-10% resulted in the activation of growth and iron oxidation, a 20-30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.     

Density gradient and differential centrifugation methods for chloroplast purification and enzyme localization in leaf tissue

Intact chloroplasts, isolated by differential-centrifugation and sucrose density-gradient methods, have been used to study the degree of apparent artifactual adsorption of citrate synthase (EC 4.1.3.7) to the organelles. Unfractionated homogenates layered directly on to sucrose density gradients gave elution profiles showing definite citrate synthase activity in the intact and broken plastid regions, along with the major mitochondrial peak. Nonreversible triose-phosphate dehydrogenase (EC 1.2.1.9), a cytosolic marker, showed no activity in any particulate region of the gradient. Crude chloroplast pellets and twice washed (resedimented and resuspended) chloroplasts layered on to the gradient gave progressively reduced citrate synthase activity in the plastid regions. In addition, the peak in the mitochondrial region of the gradient was virtually eliminated when washed chloroplasts were fractionated on the gradient. Differences in protein binding behavior on the chloroplasts may necessitate the inclusion of a washing step in chloroplast purification procedures. Moreover, repeated sedimentation and resuspension can also be a useful procedure to reduce mitochondrial contamination of chloroplast preparations.Work supported by the Rubber Research Institute of Malaysia     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.