Mobility of fluorescent derivatives of cytochrome c in mitochondria.

Motion of cytochrome c bound to giant (2-10-micron diam) mitochondria isolated from the waterbug Lethocerus indicus was examined using the technique of fluorescence recovery after photobleaching. Fluorescent cytochrome c was exchanged for native cytochrome c through partly damaged outer membrane. Recovery profiles were not statistically different when the fluorescence from iron-free cytochrome c or fluorescein-labeled cytochrome c was used and were essentially the same in the presence or absence of an uncoupler. In the presence of excess porphyrin cytochrome c, the apparent diffusion coefficient was 6 X 10(-11) cm2/s in 0.3 M sucrose-mannitol-EDTA and 3 X 10(-10) cm2/s in 0.10 M KCl/0.10 M sucrose. At concentrations of porphyrin cytochrome c that are stoichiometric with cytochrome c oxidase and for mitochondria in which excess cytochrome c was washed away, two components were observed in the recovery profile. The diffusion coefficient of the fast component was 1 X 10(-10) cm2/s. The second component showed no recovery during the time scale of measurement (D less than 10(-12) cm2/s). We speculate on the origin of the immobile fraction.

Mitochondrial respiratory chain of Tetrahymena pyriformis: the properties of submitochondrial particles and the soluble b and c type pigments.

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 degree C there are two b-type pigments with half-reduction potentials of --0.04 and --0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a2 with Em7.2 of 0.245 and 0.345 V. EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spine ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g =6, and a g=2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed. Cytochrome c553 is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c553 is a negatively charged protein at neutral pH with an Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with KD of 0.8.10(-6) M. Reduced cytochrome c553 serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c553 shows the presence of a low spin ferric heme with the dominant resonance signal at g=3.28. A pigment with an alpha absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and Em7.2 of--0.17 V and exhibits a high spin ferric heme signal at g=6.

Mitochondrial respiratory chain of Tetrahymena pyriformis. The thermodynamic and spectral properties.

The mitochondria isolated from the ciliate protozoon Tetrahymena pyriformis carry an oxidative phosphorylation with P/O ratio of 2 for succinate oxidation and P/O ratio of 3 for the oxidation of the NAD-linked substrates. The respiration is more than 90% inhibited with 1 mM cyanide while antimycin A and rotenone inhibit at concentrations of 1000-fold higher than those effective in mammalian mitochondria. Using a combination of spectral studies and potentiometric titrations, the components of the respiratory chain were identified and characterized with respect to the values of their half-reduction potentials. In the cytochrome bc1 region of the chain a cytochrome c was present with an Em7.2 of 0.225 V and two components with absorption maxima at 560 nm and the half-reduction potential values of -0.065 and -0.15 V at pH 7.2. The cytochrome with the more positive half-reduction potential was identified as the analogue of the cytochrome(s) b present in mitochondria of higher organisms, while the cytochrome with the more negative half-reduction potential was tentatively identified as cytochrome o. In addition ubiquinone was present at a concentration of approx. 4 nmol per mg mitochondrial protein. In the spectral region where cytochromes a absorb at least three cytochromes were found. A cytochrome with an absorption maximum at 593 nm and a midpoint potential of -0.085 V at pH 7.2 was identified as cytochrome a1. The absorption change at 615-640 nm, attributed usually to cytochrome a2, was resolved into two components with Em7,2 values of 0,245 and 0.345 V. It is concluded that the terminal oxidase in Tetrahymena pyriformis mitochondria is cytochrome a2 which in its two component structure resembles cytochrome aa3.

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes.

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, iysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 plus or minus 10 mV (b561), and 0 plus or minus mV (566) and cytochrome c1 (Em7.2 equals +280 plus or minus 5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40-60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b566 is pH-dependent above pH 7.0 ( minus 60 mV/pH unit) while that of b561 is essentially pH-independent from pH 6.7-8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b566(bT), b561 (bK) and c1 respectively is made on the basis of redox midpoint potentials. In addition, the preparation contains four distinct types of iron-sulfur centers: S1 and S2 (Em7.4 equals minus 260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c1 complex: Rieske's iron-sulfur protein (Em7.4 equals +280 mV) and Ohniski's Center 5 (Em7.4 equals +35 mV).

Studies of the orientation of the mitochondrial redox carriers. III. Orientation of the gx and gy axes of the hemes of cytochrome oxidase with respect to the plane of the membrane in oriented membrane multilayers.

The EPR absorption properties of the hemes of cytochrome oxidase and their liganded derivatives were examined in oriented multilayers from isolated oxidase, mitochondrial membranes and membrane fragments of a bacterium, Paracoccus denitrificans. The hemes of the oxidase in all the systems investigated were oriented normal to the plane of the multilayers. The directions of the g signals corresponding to the gx and gy axes of the g tensor were found to be different in low-spin ferric heme in fully oxidized oxidase and in half-reduced liganded oxidase. It is suggested that this different orientation of gx and gy in fully oxidized oxidase and half-reduced liganded oxidase arises because the respective EPR signals belong to two different hemes, those of cytochrome a and a3.

Regulation of cellular energy metabolism: the Crabtree effect.

The Crabtree effect (inhibition of respiration by glycolysis) is observed in cells with approximately equal glycolytic and respiratory capacities for ATP synthesis. Addition of glucose to aerobic suspensions of glucose-starved cells (Sarcoma 180 ascites tumor cells) causes a burst of respiration and lactate production due to ATP utilization for glucose phosphorylation by hexokinase and phosphofructokinase. This burst of activity is followed by inhibition of both respiration and glycolysis, the former to below the value before glucose addition (Crabtree effect). Both the respiratory rate and the glycolytic flux appear to be regulated by the cytosolic [ATP]/[ADP][Pi] albeit by completely different mechanisms. Respiration is regulated by the free energy of hydrolysis of ATP, such that the rate increases as the [ATP]/[ADP][Pi] decreases and decreases as the [ATP]/[adp][Pi] increases. The regulatory enzymes of glycolysis are activated by ADP (AMP) and Pi and inhibited by ATP. Thus both respiration and glycolysis increase or decrease as the [ATP]/[ADP][Pi] decreases or increases. The parallel regulation of both ATP-producing pathways by this common metabolite ratio is consistent with the cytoplasmic [ATP]/[ADP][Pi] being an important determinant of homeostatic regulation of cellular energy metabolism.

Studies on the orientations of the mitochondrial redox carriers. I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane.

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45 degrees between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2-1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a3. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90 degrees.

Studies on the orientations of the mitochondrial redox carriers. II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated, oriented mitochondrial multilayers.

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c1, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the "tightly bound" mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90 degrees.

Nitric oxide diffusion coefficients in solutions, proteins and membranes determined by phosphorescence.

The reactivity of nitric oxide under a given condition is a complex function of its diffusivity and the concentration of reacting partners. Quenching by NO of luminescence from Ru and Pd chelates of mesoporphyrin IX, two molecules which exhibit phosphorescence at room temperature, was utilized to evaluate the gas concentration and apparent diffusion coefficients. The properties of Ru-mesoporphyrin, a dye not previously employed as a probe for O2 or NO, were determined and the assay was verified and used to quantify NO produced by decomposition of nitrosocysteine. The pseudo-second order quenching constants were obtained from Stern-Volmer plots measured under various conditions and used to calculate diffusion coefficients for nitric oxide in solutions, proteins and membranes. The diffusion coefficients were greater at 37 than at 25 degrees C and, at a given temperature, smaller in proteins and membranes than in water. The conclusion is that NO and O2 closely resemble each other in diffusivity but that NO is slightly less lipophilic, resulting in somewhat faster apparent diffusion in protein and slower diffusivity in lipid, relative to O2. Taking a mean diffusion coefficient for NO of 10(-7) cm2s-1, then within 10 s the mean path is 10(-3) cm, or less than the diameter of a single cell. However, at low NO and O2 concentrations, the halflife of NO will be considerably longer than 10 s, and consequently the path of NO diffusion much greater.

The structure of a cytochrome oxidase-lipid model membrane.

The structure of "membranous cytochrome oxidase" has been investigated by X-ray diffraction, optical polarization spectroscopy and EPR spectroscopy. These studies indicate that the cytochrome oxidase molecules are oriented symmetrically in the membrane profile with a significant portion of their mass occurring within the extravesicular surface of the membrane; the oxidase molecultes span the membrane profile; the distribution of the oxidase molecules over the plane of these membranes is non-crystalline; the oxidase molecules contain bundles of alpha-helical polypeptide chain segments where the average orientation of the helices is normal to the membrane plane; and the average heme orientation within the oxidase molecules is such that the normal to the heme plane lies in the plane of the membrane.

Cellular effects of endotoxin in vitro: mobility of endotoxin in the plasma membrane of hepatocytes and neuroblastoma cells.

Lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) was used to examine interactions between endotoxin and plasma membrane in isolated rat hepatocytes and mouse neuroblastoma NB41A3 cells. At the same endotoxin to cell ratio, hepatocytes bound more toxin than did neuroblastoma cells. At a dose of 12 micrograms/mg dry wt, a bound mobile fraction of between 60 and 75% of FITC-LPS was found on hepatocytes at 25 degrees C with a lateral diffusion coefficient (D) of 4.0 X 10(-9) cm2/s. In neuroblastoma cells, the mobile fraction was larger (85-90%), with D 1.0 X 10(-8) cm2/s. D was temperature-dependent between 10 and 37 degrees C and increased from 1.8 X 10(-9) to 1.0 X 10(-8) cm2/s in hepatocytes and from 9.4 X 10(-9) to 1.9 X 10(-8) cm2/s in neuroblastoma cells. In both types of cell, nonviable (cells which did not exclude Trypan blue) as compared to viable cells showed different recovery patterns and 100% of the probe molecules were mobile. These results suggest that: (1) endotoxin binding to mammalian cells consists of two subpopulations with different mobilities; (2) binding of the immobile fraction is dependent on cellular integrity; and (3) the differences in binding, lateral mobility, and size of the immobile fraction in hepatocytes and neuroblastoma cells may be due to variations in membrane composition and/or number of binding sites.

The location of redox centers in biological membranes determined by resonance x-ray diffraction. I. Observation of the resonance effect.

We have developed resonance X-ray diffraction methods to locate for the first time intrinsic metal atoms associated with redox centers within biological membrane systems. The study of membranes containing dilute concentrations of resonant scatterers has been made possible by the development of synchrotron radiation sources of X-rays. The technique permits altering the scattering power of a particular atom relative to others by varying the incident X-ray energy. Thus, this method may be used to locate a metal atom within a complex integral protein without chemical modification of the membrane. We present resonance diffraction data taken with synchroton radiation for two different membrane systems: cytochrome oxidase incorporated into lipid vesicles and a photosynthetic reaction center-cytochrome c complex also reincorporated into lipid vesicles.

The location of redox centers in biological membranes determined by resonance x-ray diffraction. II. Analysis of the resonance diffraction data.

In the preceding paper (Stamatoff, J., Eisenberger, P., Blasie, J.K., Pachence, J.M., Tavormina, A., Erecinska, M., Dutton P.L. and Brown, G. (1982) Biochim. Biophys. Acta 679, 177-187), we described the observation of resonance X-ray scattering effects from intrinsic metal atoms associated with redox centers in membrane proteins on the lamellar X-ray diffraction from oriented multilayers of reconstituted membranes. In this paper, we discuss the possible methods of analysis of such data and present the results of our model refinement analysis concerning (a) the location of the cytochrome c heme iron atom in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex and (b) the location of the heme a and a3 iron atoms in the profile structure of a reconstituted membrane containing cytochrome oxidase. The former results are of special importance because they provide a test of the validity of the resonance diffraction data and the methods of analysis, since the location of cytochrome c in the reaction center-cytochrome c membrane profile is known independently of the resonance diffraction experiments.

GABA production in rat islets of Langerhans.

Homogenates of pancreatic islets catalyzed breakdown of L-glutamate to GABA with a rate of 0.24 +/- 0.04 nmol.min-1 x mg-1 protein at 37 degrees C. The formation of GABA was stimulated by addition of pyridoxal phosphate in the range 0.05-1 microM (0.97 +/- 0.02 nmol.min-1 x mg protein-1 at a saturating cofactor concentration), which indicates that the process was catalyzed by glutamic acid decarboxylase. The half-maximal effect was obtained with 0.1 microM PLP. Kinetic analyses of the results showed that the Vmax and Km for the reaction were 1.12 nmol.min-1 x mg protein-1 and 0.66 mM, respectively. The pH optimum was 7.0. Subcellular fractionation revealed that 51% of GAD activity was present in the cytosol, 17% in microsomes, 9% in secretory granules, 5% in mitochondria, and 11% in cell debris. Comparison of the kinetic properties of the cytosolic and microsomal forms of the enzyme showed that their Km for glutamate was the same, but that the cytosolic GAD had a lower Km for PLP. GABA synthesis in the nominal absence of PLP was enhanced by malate (twofold increase at 5 mM) and citrate (threefold increase at 5 mM), but was unaffected by ATP and chloride. However, if the islet homogenate was prepared and incubated in the presence of PLP, neither malate nor citrate influenced enzyme activity. Aspartate and AOA were powerful inhibitors of glutamate breakdown. Freshly isolated islets contained approximately 4 mM GABA, whereas the concentration was < 0.1 mM in whole pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes.

The relationships between Na/K pump activity and adenosine triphosphate (ATP) production were determined in isolated rat brain synaptosomes. The activity of the enzyme was modulated by altering [K+]e, [Na+]i, and [ATP]i while synaptosomal oxygen uptake and lactate production were measured simultaneously. KCl increased respiration and glycolysis with an apparent Km of about 1 mM which suggests that, at the [K+]e normally present in brain, 3.3-4 mM, the pump is near saturation with this cation. Depolarization with 6-40 mM KCl had negligible effect on ouabain-sensitive O2 uptake indicating that at the voltages involved the activity of the Na/K ATPase is largely independent of membrane potential. Increases in [Na+]i by addition of veratridine markedly enhanced glycoside-inhibitable respiration and lactate production. Calculations of the rates of ATP synthesis necessary to support the operation of the pump showed that greater than 90% of the energy was derived from oxidative phosphorylation. Consistent with this: (a) the ouabain-sensitive Rb/O2 ratio was close to 12 (i.e., Rb/ATP ratio of 2); (b) inhibition of mitochondrial ATP synthesis by Amytal resulted in a decrease in the glycoside-dependent rate of 86Rb uptake. Analyses of the mechanisms responsible for activation of the energy-producing pathways during enhanced Na and K movements indicate that glycolysis is predominantly stimulated by increase in activity of phosphofructokinase mediated via a rise in the concentrations of adenosine monophosphate [AMP] and inorganic phosphate [Pi] and a fall in the concentration of phosphocreatine [PCr]; the main moving force for the elevation in mitochondrial ATP generation is the decline in [ATP]/[ADP] [Pi] (or equivalent) and consequent readjustments in the ratio of the intramitochondrial pyridine nucleotides [( NAD]m/[NADH]m). Direct stimulation of pyruvate dehydrogenase by calcium appears to be of secondary importance. It is concluded that synaptosomal Na/K pump is fueled primarily by oxidative phosphorylation and that a fall in [ATP]/[ADP][Pi] is the chief factor responsible for increased energy production.

Intracellular and extracellular changes of [Ca2+] in hypoxia and ischemia in rat brain in vivo.

Changes in intra- and extracellular free calcium concentration were evaluated with ion-selective microelectrodes during periods of anoxia and ischemia in three different regions of intact rat brain. Recordings stable for at least 2 min and in most cases for 4-6 min were chosen for analysis. Under normoxic conditions neuronal [Ca2+]i varied between less than 10(-8) and 10(-7) M from cell to cell but no systematic regional differences were observed. Elimination of O2 or interruption in blood flow caused, within 30-60 s, slight intracellular alkalinization followed by a small rise in [Ca2+]i, a mild degree of hyperpolarization, and disappearance of electrical activity in the cortex, in that order. It is postulated that a decline in cellular energy levels, as manifested by H+ uptake associated with creatine phosphate hydrolysis, leads to an increase in [Ca2+]i, which activates Ca2(+)-dependent K+ channels and consequently enhances gK. 2-4 min later there was a sudden, large rise in [K+]e, a fall in [Ca2+]e and a rapid elevation of [Ca2+]i. The magnitude of the latter was greatest in a high proportion of hippocampal neurons in area CA1 and some cortical cells, while it was smallest and relatively delayed in thalamic neurons. In the hippocampus area CA1 increases in [Ca2+]i to as much as 6-8 x 10(-4) were observed; some of these could be reversed when O2 or blood flow were restored to normal. Pretreatment of animals with ketamine and MK-801, antagonists of excitatory amino acid transmitters, markedly slowed and decreased the rises in [Ca2+]i. The effects of the two agents were most pronounced in the hippocampus. It is concluded that the receptor-operated channels are largely responsible for Ca2+ entry into certain cells during hypoxia/ischemia. This pathway may be of primary importance in parts of the hippocampus and cortex, regions of the brain that are particularly vulnerable to O2 deprivation and which receive high glutamatergic input and have an abundance of excitatory amino acid receptors.

Ion homeostasis in rat brain in vivo: intra- and extracellular [Ca2+] and [H+] in the hippocampus during recovery from short-term, transient ischemia.

Changes in intra- and extracellular [Ca2+] and [H+], together with alterations in tissue PO2 and local blood flow, were measured in areas CA1 and CA3 of the hippocampus during recovery (up to 8 h) after an 8-min period of low-flow ischemia. Restoration of blood supply was followed by an immediate rise in flow and tissue PO2 above normal, with large fluctuations in both persisting for up to 4 h. In area CA1, [Ca2+]i decreased rapidly from an ischemic mean value of 30 microM to a control mean level of 73.1 nM in 20-30 min, whereas normalization of [Ca2+]e took approximately 1 h. Recovery of [Ca2+]i was accelerated by preischemic administration of a calcium antagonist, nifedipine, and a free radical scavenger, N-tert-butyl-alpha-phenylnitrone (PBN), but not by MK-801, a blocker of N-methyl-D-aspartate receptors. There was a secondary rise in [Ca2+]i in many cells beginning approximately 2 h after reperfusion. This was attenuated somewhat by PBN but not clearly influenced by either nifedipine or MK-801. Changes of [Ca2+]i in area CA3 were much smaller and slightly slower than in area CA1 and were not affected by the drugs mentioned above. In both areas CA1 and CA3, pHe and pHi fell during ischemia to an average value of 6.2, from which there was a rapid initial recovery in the first 5-10 min when blood flow was restored. Thereafter tissue pH rose slowly and did not reach control levels for approximately 1 h, and in some microareas not at all. It is concluded that (a) effective mechanisms for restoring normal [Ca2+]i remain intact after 8 min of low-flow ischemia; (b) in neurons of area CA1, some insidious change in the homeostasis of calcium triggers a secondary rise in its free cytosolic concentration, which may be causally related to activation of irreversible cell damage; and (c) the changes in [Ca2+]i and [Ca2+]e during and following 8 min of ischemia can be adequately accounted for by movements of a fixed pool of Ca between intra- and extracellular compartments, and possible mechanisms are discussed.

Amino acid neurotransmitters in the CNS. Relationships between net uptake and exchange in rat brain synaptosomes.

Carefully isolated, metabolically competent rat brain synaptosomes accumulate acidic amino acid neurotransmitters down to very low external levels. This supports the suggestion that nerve endings are involved in terminating transmission at the synapses and in maintaining low levels of these molecules in the external environment in the brain. At saturating levels of acidic amino acids, the rate of inward and outward movements of the Na+-amino acid complex (exchange) is much faster than the net uptake. The transmembrane gradients of aspartate and glutamate approach each other under all conditions explored which indicates that these two amino acids share the same transport system.

Osmotic effects on bacterial transport and energetics.

Paracoccus denitrificans suspended in media containing 20-300 mM NaCl swelled progressively as the salt concentration was decreased. The increase in intracellular water volume was accompanied by an enhancement of respiration and a stimulation of the rates of net potassium and alpha-aminoisobutyric acid accumulation. It is postulated that influx of water and consequent lowering of intracellular solute concentration trigger transport mechanisms which are destined to restore the original ion and metabolite balance. Since a number of transport reactions operate against the electrochemical gradient of their substrates, energy utilization increases. The increased ATP usage and lowering of [ATP] stimulates the activity of the respiratory chain and increases oxygen uptake and energy production.

Amino acid neurotransmitters in the CNS. Characteristics of the acidic amino acid exchange.

D-Aspartate exchange, defined as amino acid-stimulated D-[3H]aspartate efflux, was investigated in a preparation of rat brain synaptosomes. The efflux of radiolabelled D-aspartate was found to be enhanced by micromolar concentrations of externally added D- and L-aspartate, L-glutamate, L-cysteate and L-cysteinesulphinate. The stimulation of release by external amino acids followed Michaelis-Menten kinetics; the apparent Km values (in microM) were: 14.65 +/- 0.98 for D-aspartate; 8.00 +/- 1.5 for L-aspartate; 22.31 +/- 1.62 for L-glutamate; 6.76 +/- 0.3 for L-cysteate and 7.89 +/- 1.23 for L-cysteinesulphinate. The Vmax values for efflux were 2.16-4.06 nmol/min per mg protein. The exchange process was found to require external NaCl but was very little affected by increase in the external [K+]. The demonstration of exchange as a part of the transport process provides support for the suggestion that in synaptosomal preparations a substantial portion of influx and efflux of amino acid neurotransmitters occurs via a reversible membrane carrier.