Pyruvate and ketone-body transport across the mitochondrial membrane. Exchange properties, pH-dependence and mechanism of the carrier.

The effects of exchangeable ions and pH on the efflux of pyruvate from preloaded mitochondria are reported. Efflux obeys first-order kinetics, and the stimulation of efflux by exchangeable ions such as acetoacetate and lactate obeys Michaelis--Menten kinetics. The apparent Km value +/- S.E. for acetoacetate was 0.56 +/- 0.14 mM (n = 5) and that for lactate 12.3 +/- 2.3 mM (n = 6). The Vmax. values +/- S.E. at 0 degrees C were 16.2 +/- 2.0 and 21.9 +/- 2.7 nmol/min per mg of protein. The exchange of a variety of other substituted monocarboxylates was also studied. Efflux was also stimulated by increasing the external pH. The data gave a pK for the transport process of 8.35 and a Vmax. of 3.31 +/- 0.14 nmol/min per mg. The similarity of the Vmax. values for various exchangeable ions but the difference of this from the Vmax. in the absence of exchangeable ions may indicate that transport of pyruvate occurs with H+ and not in exchange for an OH- ion. The inhibition of transport by alpha-cyano-4-hydroxycinnamate took several seconds to reach completion at 0 degrees C. It is proposed that inhibition occurs by binding to the substrate site and subsequent reaction with an -SH group on the inside of the membrane. The inhibitor can be displaced by substrates that can also enter the mitochondria independently of the carrier and so compete with the inhibitor for the substrate-binding site on the inside of the membrane. A mechanism for transport is proposed that invokes a transition state of pyruvate involving addition of an -SH group to the 2-carbon of pyruvate. Evidence is presented that suggests that ketone bodies may cross the mitochondrial membrane either on the carrier or by free diffusion. The physiological involvement of the carrier in ketone-body metabolism is discussed. The role of ketone bodies and pH in the physiological regulation of pyruvate transport is considered.     

Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine.

1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.     

Transport of pyruvate nad lactate into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride-independent carrier.

The kinetics and activation energy of entry of pyruvate and lactate into the erythrocyte were studied at concentrations below 4 and 15mM respectively. The Km and Vmax. values for both substrates are reported, and it is shown that pyruvate inhibits competitively with respect to lactate and vice versa. In both cases the Km for the carboxylate as a substrate was the same as its Ki as an inhibitor. Alpha-Cyano-4-hydroxycinnamate and its analogues inhibited the uptake of both lactate and pyruvate competitively. Inhibition was also produced by treatment of cells with fluorodinitrobenzene but not with the thiol reagents or Pronase. At high concentrations of pyruvate or lactate (20mM), uptake of the carboxylate was accompanied by an efflux of Cl-ions. This efflux of Cl- was inhibited by alpha-cyano-4-hydroxycinnamate and picrate and could be totally abolished by very low (less than 10 muM) concentrations of the inhibitor of Cl- transport, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid. This inhibitor titrated out the chlordie efflux induced by pyruvate, bicarbonate, formate and fluoride, in each case total inhibition becoming apparent when approximately 1.2x10(6) molecules of inhibitor were present per erythrocyte, that is, about one inhibitor molecule per molecule of the Cl- carrier. Evan when Cl- efflux was totally blocked pyruvate and lactate uptake occurred. Kinetic evidence is presented which suggests that the Cl- carrier can transport pyruvate and lactate with a high Km and high Vmax., but that an additional carrier with a low Km and a low Vmax. also exists. This carrier catalyses the exchange of small carboxylate anions with intracellular lactate, is competitively inhibited by alpha-cyano-4-hydroxycinnamate and non-competitively inhibited by picrate. The Cl- carrier shows a reverse pattern of inhibition. It is concluded that net efflux of lactic acid from the cell must occur on the Cl- carrier and involve exchange with HCO3 - followed by loss of CO2. The low Km carrier might be used in pyruvate/lactate or acetoacetate/beta-hydroxybutyrate exchanges involved in transferring reducing power across the cell membrane. The possibility that the Cl- carrier exists in cells other than the erythrocyte is discussed. It is concluded that its presence in other cell membranes together with a low intracellular Cl- concentration would explain why the pH in the cytoplasm is lower than that of the blood, and why permeable carboxylate anions do not accumulate within the cell when added from outside.     

A mitochondrial monocarboxylate transporter in rat liver and heart and its possible function in cell control.

Several hydroxy- and keto-substituted monocarboxylates were found to undergo co- as well as counter-exchange across the mitochondrial membrane. The results argue against a simple Donnan system and may be explained by the existence of a transporter for monocarboxylates. In support of this explanation it was apparently possible to 'pump' pyruvate to the sucrose-inaccessible space by using the dicarboxylate transporter. Further, several aromatic and aliphatic analogues of pyruvate, but not of di- or tri-carboxylate transport inhibitors, have been shown to prevent pyruvate-exchange reactions. Palmitoylcarnitine was found to have a much stronger affinity for the carrier than either carnitine or pyruvate and the possible consequences of this for carnitine-palmitoylcarnitine exchange and on the control of the pyruvate dehydrogenase complex are explored. In view of the range of transport inhibitors and substrates it is suggested that the carrier has a fairly broad specificity. 'Inhibitor-stop' kinetic studies show that the speed of translocation of pyruvate at 1 degrees C is of the same order as malate. The possible correlation between the role of a hydroxy-keto acid transporter in substrate exchange and some whole animal experiments is briefly discussed. It is proposed that for reasons of control the cell will require membrane monocarboxylate transporters no less than di- or tri-carboxylate carriers.     

Blood-brain barrier transport of the alpha-keto acid analogs of amino acids

A number of alpha-keto acid analogs of amino acids have been found to penetrate the blood-brain barrier (BBB). Pyruvate, alpha-ketobutyrate, alpha-ketoisocaproate, and alpha-keto-gamma-methiolbutyrate all cross the BBB by a carrier-mediated process and by simple diffusion. Under normal physiological conditions, diffusion accounts for roughly 15% or less of total transport. Aromatic alpha-keto acids, phenylpyruvate, and p-hydroxyphenylpyruvate do not penetrate the BBB, nor do they inhibit the transport of other alpha-keto acids. Evidence based primarily on inhibition studies indicates that the carrier-mediated transport of alpha-keto acids occurs via the same carrier demonstrated previously for propionate, acetoacetate, and beta-hydroxybutyrate transport, commonly referred to as the monocarboxylate carrier. As a group, the alpha-keto acid analogs of the amino acids have the highest affinity for the carrier, followed by propionate and beta-hydroxybutyrate. Starvation for 4 days induces transport of alpha-keto acids, but transport is suppressed in rats fed commercial laboratory rations and subjected to portacaval shunts. The mitochondrial pyruvate translocator inhibitor alpha-cyanocinnamate has no effect on the BBB transport of alpha-keto acids.     

The monocarboxylate carrier from bovine heart mitochondria: partial purification and its substrate-transporting properties in a reconstituted system

The monocarboxylate (pyruvate) carrier from bovine heart mitochondria was extracted from submitochondrial particles with Triton X-114 in the presence of cardiolipin. By a single hydroxylapatite chromatography step a 125-fold purification of the carrier protein could be achieved. High pyruvate/pyruvate-exchange activity was recovered, when the protein was reconstituted into phospholipid vesicles. No transport activity was observed, when the isolation occurred in the absence of phospholipids. The 2-cyano-4-hydroxycinnamate sensitive pyruvate exchange reaction was strongly temperature sensitive and dependent on the amount of protein reconstituted. Other 2-ketoacids caused competitive inhibition of the pyruvate uptake. Inhibitors of other mitochondrial carries, however, had very low or no effect on the monocarboxylate exchange. The influence of different -SH group reagents on the measured pyruvate/pyruvate-exchange in the reconstituted system was similar to the one observed with intact mitochondria. It is concluded that the described procedures for extraction, purification and reconstitution of the mitochondrial monocarboxylate carrier conserved the functional properties of the protein.     

Effects of pH on beta-hydroxybutyrate transport in rat erythrocytes and thymocytes.

Entry of beta-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or alpha-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [beta-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry Km from 29 to 16 to 10 mM, entry V being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external beta-hydroxybutyrate enhanced efflux slightly. At high external pH, external beta-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal beta-hydroxybutyrate (betaHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled betaHB-/OH- exchange, betaHB- and OH- competing for influx, C:OH- moving faster than C: betaHB-, empty C being immobile. They are also compatible with coupled betaHB-/H+ copermeation, empty C moving inward faster than H+:C:betaHB-, H+:C being immobile, and C:betaHB- (without H+) being so unstable as not to be formed in significant amounts (relative to C, H+:C, and H+:C:betaHB-).     

The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes.

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart are discussed.     

The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.

1. Studies on the kinetics of pyruvate transport into mitochondria by an 'inhibitor-stop' technique were hampered by the decarboxylation of pyruvate by mitochondria even in the presence of rotenone. Decarboxylation was minimal at 6 degrees C. At this temperature the Km for pyruvate was 0.15 mM and Vmax. was 0.54nmol/min per mg of protein; alpha-cyano-4-hydroxycinnamate was found to be a non-competitive inhibitor, Ki 6.3 muM, and phenyl-pyruvate a competitive inhibitor, Ki 1.8 mM. 2. At 100 muM concentration, alpha-cyano-4-hydroxycinnamate rapidly and almost totally inhibited O2 uptake by rat heart mitochondria oxidizing pyruvate. Inhibition could be detected at concentrations of inhibitor as low as 1 muM although inhibition took time to develop at this concentration. Inhibition could be reversed by diluting out the inhibitor. 3. Various analogues of alpha-cyano-4-hydroxycinnamate were tested on rat liver and heart mitochondria. The important structural features appeared to be the alpha-cyanopropenoate group and the hydrophobic aromatic side chain. Alpha-Cyanocinnamate, alpha-cyano-5-phenyl-2,4-pentadienoate and compound UK 5099 [alpha-cyano-beta-(2-phenylindol-3-yl)acrylate] were all more powerful inhibitors than alpha-cyano-4-hydroxycinnamate showing 50% inhibition of pyruvate-dependent O2 consumption by rat heart mitochondria at concentrations of 200, 200 and 50 nM respectively. 4. The specificity of the carrier for its substrate was studied by both influx and efflux experiments. Oxamate, 2-oxobutyrate, phenylpyruvate, 2-oxo-4-methyl-pentanoate, chloroacetate, dichloroacetate, difluoroacetate, 2-chloropropionate, 3-chloropropionate and 2,2-dichloropropionate all exchanged with pyruvate, whereas acetate, lactate and trichloroacetate did not. 5. Pyruvate entry into the mitochondria was shown to be accompanied by the transport of a proton (or by exchange with an OH-ion). This proton flux was inhibited by alpha-cyano-4-hydroxycinnamate and allowed measurements of pyruvate transport at higher temperatures to be made. The activation energy of mitochondrial pyruvate transport was found to be 113 kJ (27 kcal)/mol and by extrapolation the rate of transport of pyruvate at 37 degrees C to be 42 nmol/min per mg of protein. The possibility that pyruvate transport into mitochondria may be rate limiting and involved in the regulation of gluconegenesis is discussed. 6. The transport of various monocarboxylic acids into mitochondria was studied by monitoring proton influx. The transport of dichloroacetate, difluoroacetate and oxamate appeared to be largely dependent on the pyruvate carrier and could be inhibited by pyruvate-transport inhibitors. However, many other halogenated and 2-oxo acids which could exchange with pyruvate on the carrier entered freely even in the presence of inhibitor.     

alpha-Ketobutyrate metabolism in perfused rat liver: regulation of alpha-ketobutyrate decarboxylation and effects of alpha-ketobutyrate on pyruvate dehydrogenase

The oxidative decarboxylation and subsequent production of glucose from alpha-ketobutyrate were studied using perfused livers from fasted rats. The production of 14CO2 from alpha-keto-[1-14C]butyrate increased monotonically while the production of glucose from alpha-ketobutyrate was biphasic as the perfusate concentration of alpha-ketobutyrate was increased. The biphasic gluconeogenic response using alpha-ketobutyrate as the gluconeogenic precursor was similar to that observed with propionate. The decarboxylation of alpha-ketobutyrate was found to be exquisitely sensitive to the effects of the monocarboxylate transport inhibitor, alpha-cyanocinnamate. Infusion of beta-hydroxybutyrate caused a substantial inhibition of alpha-ketobutyrate decarboxylation while dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, did not stimulate the metabolism of alpha-ketobutyrate but was inhibitory. The effects of alpha-ketobutyrate infusion on pyruvate decarboxylation were tested and it was found that at low perfusate pyruvate concentrations (ca. 0.25 mM) increasing alpha-ketobutyrate led to increasing inhibition of pyruvate decarboxylation, while at high perfusate pyruvate concentrations (ca. 2.5 mM) an initial inhibition was apparent which did not increase substantially with increasing alpha-ketobutyrate concentrations. The results obtained indicate that the regulation of alpha-ketobutyrate metabolism by oxidative decarboxylation differs significantly from that of pyruvate. In addition, while the rate of gluconeogenesis using alpha-ketobutyrate as a precursor was remarkably similar to that using propionate as a gluconeogenic precursor, the effects of alpha-ketobutyrate on the oxidative decarboxylation of pyruvate were qualitatively different from the effects of propionate on pyruvate metabolism.     

Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier.

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.     

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.     

Mannitol and glucose movement across the Golgi membrane of lactating-rat mammary gland.

1. Purified Golgi-membrane vesicles of lactating-rat mammary gland were penetrated by glucose. 3-O-methylglucose, mannose, fructose, sorbitol and mannitol, but not by lactose or sucrose. 2. The kinetics of mannitol uptake and release were followed at 2-6 degrees C with the aid of fine filters (0.45 micrometers pore size) to separate the vesicles from the medium. 3. Mannitol efflux exhibited apparent first-order kinetics with k approximately 1 min-1. Neither saturability, nor inhibition by excess sorbitol or glucose, could be observed. 4. Mannitol efflux at 18 degrees C was about seven times faster than at 1 degrees C, and rates at higher temperatures were too fast to be measured. The rate of glucose efflux at 2-6 degrees C exceeded that of mannitol severalfold. 5. These findings imply a channel or carrier of definite, but limited, specificity straddling the Golgi membrane and able to supply glucose for lactose synthesis.     

Kinetics of lactate and pyruvate transport in cultured rat myotubes

Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The initial rate of decrease in intracellular pH (pHi) upon lactate or pyruvate incubation was used to determine total transport (carrier mediated and diffusion). Both lactate and pyruvate transport could be inhibited by a combination of 0.5 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid, 5 mM mersalyl and 10 mM alpha-cyano-4-hydroxycinnamate. The kinetic parameters, Km and Vmax, for carrier-mediated transport of lactate were 9.9+/-1.1 mM and 0. 69+/-0.02 mmol l-1 s-1, respectively. For pyruvate, Km and Vmax were 4.4+/-1.3 mM and 0.30+/-0.05 mmol l-1 s-1, respectively. The diffusion component of the total transport was 0.0040+/-0.0005[S] (n=4) and 0.0048+/-0.0003[S] (n=4) for lactate and pyruvate, respectively. Furthermore, it was observed that the two monocarboxylate transporter isoforms present in mature skeletal muscles, MCT1 and MCT4 (formerly called MCT3 (M.C. Wilson, V.N. Jackson, C. Heddle, N.T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O.F. Hutter, A.P. Halestrap, Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes.     

L-lactate transport in Ehrlich ascites-tumour cells.

Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids.     

Effect of an unstirred layer on the membrane permeability of anandamide

To study the effect of an unstirred layer (UL), we have investigated the exchange efflux kinetics of anandamide at 0 degrees C, pH 7.3, from albumin-free as well as from albumin-filled human red blood cell ghosts to media of various BSA concentrations ([BSA](o)). The rate constant (k(m)) of unidirectional flux from the outer membrane leaflet to BSA in the medium increased with the square root of [BSA](o) in accordance with the existence of a UL, which is a water layer adjacent to the membrane that is not subject to the same gross mixing that takes place in the rest of the medium. From k(m), it is possible to calculate the rate constant of anandamide dissociation from BSA (k(1)) if we know the membrane binding of anandamide, the equilibrium dissociation constant of BSA-anandamide complexes, and the diffusion constant of anandamide. We estimated k(1) to be 3.33 +/- 0.27 s(-1). The net flux of [(3)H]anandamide is balanced by an equal and opposite movement of nonradioactive anandamide in exchange efflux experiments. This means that our results are also valid for uptake. We show that for anandamide with rapid membrane translocation, UL causes a significant resistance to cellular uptake. Depicting the rate of anandamide uptake as a function of equilibrium water phase concentrations results in a parabolic uptake dependence. Such apparent "saturation kinetics" is often interpreted as indicating the involvement of transport proteins. The validity of such an interpretation is discussed.     

Pyruvate/malate antiporter in rat liver mitochondria.

To gain some insight into the process by which both acetylCoA and NADPH, needed for fatty acid synthesis, are obtained, in the cytosol, from the effluxed intramitochondrial citrate, via citrate lyase and malate dehydrogenase plus malic enzyme respectively, the capability of externally added pyruvate to cause efflux of malate from rat liver mitochondria was tested. The occurrence of a pyruvate/malate translocator is here shown: pyruvate/malate exchange shows saturation features (Km and Vmax values, measured at 20 degrees C and at pH 7.20, were found to be about 0.25 mM and 2.7 nmoles/min x mg mitochondrial protein, respectively) and is inhibited by certain impermeable compounds. This carrier, together with the previously reported tricarboxylate and oxodicarboxylate translocators proved to allow for citrate and oxaloacetate efflux due to externally added pyruvate.