Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Mammalian Amino Acid Transport System y+ Revisited: Specificity and Cation Dependence of the Interaction with Neutral Amino Acids

A reevaluation of the specificity of system y⁺, the classical transporter for cationic amino acids is presented. System y⁺ has been defined as a transporter for cationic amino acids that binds neutral amino acids with lower affinity in the presence of Na⁺. The discovery of other transporters for cationic amino has suggested that some properties, originally attributed to system y⁺, may relate to other transport systems. Uncertainty concerns mainly, the affinity for neutral amino acids and the cation dependence of this interaction. Neutral amino acids (13 analogues tested) were found to bind to system y⁺ in human erythrocytes with very low affinity. Inhibition constants (K_iy, mm) ranged between 14.2 mm and >400 mm, and the strength of interaction was similar in the presence of Na⁺, K⁺ or Li⁺ (145 mm). In choline medium, no interaction was detected up to 20 mm of the neutral amino acid. Guanidinium ion (5 mm, osmolarity maintained with choline) potentiated neutral amino acid binding; the effect was most important in the case of l-norvaline which aligned with guanidinium ion is equivalent to arginine. This suggests cooperative interaction at the substrate site. The specificity of system y⁺ was shown to be clearly distinct from that of system y⁺L, a cationic amino acid transporter that accepts neutral amino acids with high affinity in the presence of Na⁺ and which influenced the classical definition of system y⁺. Received: 28 September 1998/Revised: 21 December 1998     

Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper

Chick brain microsomal ATPase was strongly inhibited by Cu²⁺. (Na⁺ + K⁺)-ATPase was more susceptible to low levels of Cu²⁺ than Mg²⁺-ATPase. The inhibition of (Na⁺ + K⁺)-ATPase could be partially protected from Cu²⁺ in the presence of ATP in the preincubation period. When Cu²⁺ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu²⁺ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu²⁺ (1.0 μM) on (Na⁺ + K⁺)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K⁺ (5 mM), V with Na⁺ was markedly decreased in the presence of Cu²⁺ and K_m was about twice that of the control. However, at high K⁺ concentration (20 mM), the K_m for Na⁺ was not affected. At both low (25 mM) and high (100 mM) Na⁺, Cu²⁺ displayed non-competitive inhibition of the enzyme with respect to K⁺.On the basis of these data, we suggest that Cu²⁺ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu²⁺ interacts reversibly with the enzyme.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

The effect of metal ions on the amidolytic activity of human factor Xa (Activated Stuart-Prower Factor)

The effect of metal ions on human activated Factor X (Factor X_a) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca²⁺, Mn²⁺, and Mg²⁺ enhanced Factor X_a amidolytic activity with K_m values of 30 μm, 20 μm, and 1.4 mm, respectively. Na⁺ activation of Factor X_a amidolytic activity was also found. The K_m for Na⁺ activation was 0.31 m. Both the divalent metal ions and Na⁺ increased the affinity of Factor X_a for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor X_a. However, K⁺ was a competitive inhibitor of the Na⁺ activation (K_i = 0.14 m). Lanthanide ions inhibited Factor X_a amidolytic activity. Gd³⁺ inhibition of Factor X_a hydrolysis of S2222 was noncompetitive and had a K_i of 3 μm. The lanthanide ion inhibition could not be reversed by Ca²⁺ even when Ca²⁺ was present in a 1000-fold excess over its K_m indicating nonidentity of the Factor X_a lanthanide and Ca²⁺ binding sites. It is concluded that the Factor X_a Ca²⁺ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg²⁺, Na⁺, and K⁺ may be physiological regulators of Factor X_a activity.     

Characterization of microsomal ATPases from developing human placenta

Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na⁺ + K⁺ + Mg²⁺, Mg²⁺, and Ca²⁺ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg²⁺-dependent and Na⁺ + K⁺ + Mg²⁺-dependent ATPases possess nearly the same K_m (apparent) for ATP, while the Ca²⁺-dependent enzyme shows a different one. Mg²⁺-dependent ATPase shows higher substrate affinity than Ca²⁺-dependent ATPase, although the V_max of the Mg²⁺-dependent enzyme is lower than that of the latter. However, for each enzyme, the K_m remains almost constant and V_max varies during ontogenic development. V_max of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, l-phenylalanine, l-leucine, and l-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.     

Glutamate,aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain

To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K_m of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K⁺ and Rb⁺ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na⁺; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na⁺ and K⁺ gradients. Monensin inhibits uptake by selectively dissipating the Na⁺ gradient. For both glutamate and GABA transport, the Na⁺ and K⁺ gradients are synergistic and not additive.     

The Interaction of Na+ and K+ in Voltage-gated Potassium Channels : Evidence for Cation Binding Sites of Different Affinity

Voltage-gated potassium (K⁺) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K⁺ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K⁺ channel that is highly selective for K⁺ over Na⁺ in physiological solutions, but conducts Na⁺ in the absence of K⁺. Na⁺ and K⁺ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na⁺ currents inactivated more rapidly and deactivated more slowly than K⁺ currents. Currents carried by 160 mM Na⁺ were inhibited by external K⁺ with an apparent IC₅₀ <30 μM. K⁺ also altered both inactivation and deactivation kinetics of Na⁺ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K⁺ were inhibited by external Na⁺, with an apparent IC₅₀ of ∼100 mM. In contrast to the effects of low [K⁺] on Na⁺ current kinetics, Na⁺ did not affect K⁺ current kinetics, even at concentrations that inhibited K⁺ currents by 40–50%. These data suggest that Na⁺ block of K⁺ currents did not involve displacement of K⁺ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K⁺ channels, differential K⁺ and Na⁺ conductance, and the concentration dependence of K⁺ block of Na⁺ currents and Na⁺ block of K⁺ currents. Based on our results, we hypothesize that the permeation pathway contains a single high affinity binding site, where selectivity and ionic modulation of gating occur.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

Transport of l-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of l-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris·HCl of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in part buffered saline. l-Methionine is actively transported in WI38 by saturable, chemically specific mechanisms which are temperature, pH and, in part, Na⁺ dependent, and are reactive with both l- and d-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent K_M of 21.7 μM and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent K_M of 547 μM and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na⁺- free media or after treatment with ouabain suggested that system 1 is Na⁺ independent and that system 2 is Na⁺ dependent. Preloading of cells with unlabeled l-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled l- or d-methionine or l-phenylalanine, but not in the presence of l-arginine. l-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na⁺-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na⁺-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K_m=18.7 μM; V_max=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K_m=11.5 μM and V_max=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na⁺-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na⁺-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

High-affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes

2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na⁺-independent glucose transport system with a K_m, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with K_i's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity K_m, of 75 mM, where the K_l, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.     

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

The binding of [³H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10⁺² nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na⁺ was required for the binding of phloridzin at the high affinity sites. Na⁺ decreased the apparent K_i for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na⁺. Ca²⁺ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K₁ was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent K_i of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na⁺, whereas the interaction of d-glucose with the membranes had a prominent Na⁺-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na⁺ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.     

Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na⁺,K⁺-ATPase α₂ and α₃ isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na⁺,K⁺-ATPase mutations, extending the C terminus by either 28 residues (“+28” mutation) or an extra tyrosine (“+Y”), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na⁺ and K⁺ concentrations ([Na⁺]_i and [K⁺]_i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na⁺ affinity without disturbance of K⁺ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α₂ despite a high expression level. A significant rise of [Na⁺]_i and reduction of [K⁺]_i was detected in cells expressing mutants with reduced Na⁺ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na⁺ affinity were found to reduce [Na⁺]_i. It is concluded that the Na⁺ affinity of the Na⁺,K⁺-ATPase is an important determinant of [Na⁺]_i.     

Characterization of the cardiac Na/K pump by development of a comprehensive and mechanistic model

A large amount of experimental data on the characteristics of the cardiac Na⁺/K⁺ pump have been accumulated, but it remains difficult to predict the quantitative contribution of the pump in an intact cell because most measurements have been made under non-physiological conditions. To extrapolate the experimental findings to intact cells, we have developed a comprehensive Na⁺/K⁺ pump model based on the thermodynamic framework (Smith and Crampin, 2004) of the Post-Albers reaction cycle combined with access channel mechanisms. The new model explains a variety of experimental results for the Na⁺/K⁺ pump current (I_NaK), including the dependency on the concentrations of Na⁺ and K⁺, the membrane potential and the free energy of ATP hydrolysis. The model demonstrates that both the apparent affinity and the slope of the substrate-I_NaK relationship measured experimentally are affected by the composition of ions in the extra- and intracellular solutions, indirectly through alteration in the probability distribution of individual enzyme intermediates. By considering the voltage dependence in the Na⁺- and K⁺-binding steps, the experimental voltage-I_NaK relationship could be reconstructed with application of experimental ionic compositions in the model, and the view of voltage-dependent K⁺ binding was supported. Re-evaluation of charge movements accompanying Na⁺ and K⁺ translocations gave a reasonable number for the site density of the Na⁺/K⁺ pump on the membrane. The new model is relevant for simulation of cellular functions under various interventions, such as depression of energy metabolism.     

The Extracellular K+ Concentration Dependence of Outward Currents through Kir2.1 Channels Is Regulated by Extracellular Na+ and Ca2+

It has been known for more than three decades that outward Kir currents (I_K1) increase with increasing extracellular K⁺ concentration ([K⁺]_o). Although this increase in I_K1 can have significant impacts under pathophysiological cardiac conditions, where [K⁺]_o can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K⁺]_o dependence of Kir2.1-mediated outward I_K1 was due to [K⁺]_o-dependent inhibition of outward I_K1 by extracellular Na⁺ and Ca²⁺. This could be accounted for by Na⁺/Ca²⁺ inhibition of I_K1 through screening of local negative surface charges. Consistent with this, extracellular Na⁺ and Ca²⁺ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I_K1 in a similar [K⁺]_o-dependent manner as Na⁺/Ca²⁺. Site-directed mutagenesis studies identified Asp¹¹⁴ and Glu¹⁵³ as the source of surface charges. Reducing K⁺ activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na⁺ and Ca²⁺, suggesting that in addition to exerting a surface electrostatic effect, Na⁺ and Ca²⁺ might inhibit outward I_K1 by inhibiting K⁺ activation. This study identified interactions of K⁺ with Na⁺ and Ca²⁺ that are important for the [K⁺]_o dependence of Kir2.1-mediated outward I_K1.     

Discrimination of single transport systems. The Na plus-sensitive transport of neutral amino acids in the Ehrlich cell

Uptake of methionine, α-aminoisobutyric acid, and α-(methyl-amino)-isobutyric acid has been shown to occur by at least two transport systems, one sensitive and the other insensitive to the Na⁺ concentration. For α-aminoisobutyric acid and its N-methyl derivative, the Na⁺-insensitive uptake is not concentrative and its rate increases almost linearly with concentration within the range examined. In contrast, the Na⁺-insensitive uptake of methionine is concentrative and subject to inhibition by such amino acids as phenylalanine, leucine, and valine, although not in a manner to indicate that the uptake is mediated by a single agency. This component is not produced by a residual operation of the Na⁺-requiring transport system, handicapped by the absence of Na⁺ or by its having combined with α-aminoisobutyric acid. The increase in the rate of methionine uptake is linear with concentration only above about 16 mM methionine. The Na⁺-sensitive uptakes of methionine, α-aminoisobutyric, and α-(methylamino)-isobutyric acid appear to occur by the same population of transport-mediating sites. Both K_m and V _max of the Na⁺-sensitive uptake of these three amino acids change with changes in the concentration of Na⁺, an effect which is shown to have a theoretical basis. A similarity in the values of Vmax for ten amino acids entering principally by the Na⁺-sensitive agency indicates that differences in their K_m values probably measure differences in their affinities for that transport-mediating system.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.