Cystine-glutamate transport interactions in rat renal cortical tubules,brushborder vesicles,and cultured renal tubule cells

Glutamate had no significant effect on the uptake of 0.025 mM cystine by isolated rat renal cortical tubules and brushborder membrane vesicles in contrast to lysine which significantly inhibits cystine transport. Glutamate, however, markedly inhibited cystine uptake by rat renal tubule cells grown in a serum-free, hormonally defined media for 5 days. Lysine also inhibited cystine transport in these cultured renal tubule cells.     

Effect of cystine loading and cystine dimethylester on renal brushborder membrane transport

The effect of loading renal tubule cells with cystine was studied by incubating them with cystine dimethylester. Proline uptake into brushborder membrane vesicles isolated from the cystine loaded cells was not different from that observed into brushborder vesicles isolated from tubules incubated in buffer alone. Incubating brushborder membranes with 2 mM cystine dimethylester for 10 minutes reduced the uptake of proline by 27% after 15 seconds of incubation and by 21% after 60 seconds of incubation. There was no effect after 20 minutes of incubation. Pre-incubating brushborder membrane vesicles with cystine dimethylester had no statistically significant effect on the affinity of priline for the carrier, but did reduce the maximal rate of proline uptake by 49%.     

Presence of a sodium-dependentd-glucose transport system in the kidney of the atlantic hagfish (Myxine glutinosa)

Summary A membrane fraction, rich in brushborder membranes, was prepared from the archinephric duct of the atlantic hagfish (Myxine glutinosa) and the uptake ofd-glucose and other sugars into the membrane vesicles was investigated by a rapid filtration technique. Uptake ofd-glucose was found to be sodium-dependent, phloridzin-inhibitable and osmotically sensitive. A sodium gradient dependent overshoot was demonstrated at 25° C as well as at the more physiological temperature of 4°C. The sodium dependentd-glucose transport was inhibited by -methyl-d-glucoside, but not by 2-deoxy-d-glucose. Furthermore at the same concentration of sugars the initial uptake ofd-glucose was 7.2-fold higher thanl-glucose uptake.d-glucose transport across the membrane in the presence of a sodium gradient was stimulated when SCN^– replaced Cl^– and inhibited when gluconate replaced Cl^–.d-glucose uptake in the presence of a sodium- and potassium gradient was decreased by the addition of valinomycin. In addition, the presence of ad-glucose gradient enhanced sodium uptake into the vesicles as compared to a mannitolgradient. Phloridzin inhibited thed-glucose dependent sodium flux. Thus an electrogenic stereospecific sodium glucose co-transport system, with properties similar to that found in the kidney of higher vertebrates is present in this primitive vertebrate and might participate in secondary-active sugar reabsorption in the archinephric duct.     

Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles

Summary Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes withK _m values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8–7.1 Glutamic acid uptake also occurred by a two-component system withK _m values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The lowK _m system for glutamic acid had a pH optimum of 7.2–7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20–30% by 3 mM phenylalanine, valine, -aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.     

Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney

Summary A Na⁺-dependent hexose transport system with similar characteristics to that observed in the kidney is retained in a cultured epithelial cell line from pig kidney (LLC-PK₁). The active transport of methyl-d-glucoside ( MGP), a nonmetabolizable sugar, which shares the glucose-galactose transport system in kidney cells is mediated through a Na⁺-dependent, substrate-saturable process. The kinetic analysis of the effect of Na⁺ on the uptake of MGP indicated that the Na⁺-sugar cotransport system is an affinity type system in which the binding of either sugar or Na⁺ to carrier increases the affinity for the other ligand without affecting theV _max. The sequence of selectivity for different sugars studied by the inhibition produced in the uptake of MGP is very similar to that reported in rat kidney, rabbit kidney cortex slices, and rabbit renal brush border membrane vesicles. Phlorizin, even at very low concentration, almost completely inhibits MGP uptake. Conversely, phloretin at the same low concentration stimulated the sugar accumulation by inhibition of efflux, probably at the level of the basolateral membrane. Sulfhydryl group inhibitors also blocked the MGP uptake, suggesting that these groups were required for normal functioning of the sugar carrier system. This sugar transport system is an important functional marker to study the molecular events associated with the development of polarization in epithelial cells.     

Cystine uptake by rat jejunal brushborder membrane vesicles

The presence of a sodium-stimulated, saturable uptake process for L-cystine is described in brushborder membrane vesicles isolated from rat jejunal mucosa. Concentration-dependence studies indicate the presence of a single transport system for cystine withK _m=0.053 mM andV _max=0.633 nmol/mg/15 s. Lysine completely inhibits the uptake of cystine.     

The effect of azaserine on glutamine uptake by rat renal brush-border membranes.

Azaserine added directly to isolated rat renal brush-border membrane vesicles inhibits uptake of L-glutamine. Azaserine acts as a non-competitive inhibitor of the low-Km system for glutamine transport, but has no effect on the high-Km system. Preincubation of the vesicles with azaserine at 37 degrees C min is not required for transport inhibition to occur, although it is a requirement for gamma-glutamyl transpeptidase inhibition. Removal of azaserine from the vesicle preparation by repeated resuspensions in buffer results in a reversal of the transport inhibition but not in reversal of enzyme inhibition. Azaserine also inhibits vesicle uptake of L-proline and alpha-methyl D-glucoside, indicating a generalized effect on membrane transport systems. The data cast doubt on the postulate that gamma-glutamyl transpeptidase might act as the carrier mechanism for glutamine reabsorption by renal cortical cells.     

Glucose uptake into plasma membrane vesicles from the maternal surface of human placenta

Summary Glucose uptake into plasma membrane vesicles from the maternal surface of the human placenta was measured with the Millipore filtration technique. Uptake ofd-glucose was dependent on the osmolarity of the incubation medium surrounding the vesicles. Uptake ofd-glucose exceeded that ofl-glucose. The uptake ofd-glucose was not enhanced by placing 100mm NaCl or NaSCN in the medium outside the vesicles (none inside) at the onset of uptake determinations.d-glucose transport was inhibited by cytochalasin B; phloretin, phlorizin, and 1-fluoro-2,4-dinitrobenzene.d-glucose uptake was inhibited by 2-deoxy-d-glucose, 3-O-methyl-d-glucose and to a lesser extent byd-galactose. It was not inhibited by -methyl-d-glucoside. Cytochalasin B binding to the vesicles was 30% inhibited in the presence of 80mm d-glucose. The results indicate that the system for facilitated transport ofd-glucose at the maternal face of the placenta is distinctly different from that on the brush-border membrane of intestine or renal tubule and more closely resembles that of human erythrocyte.     

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK _m approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK _d 0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK _i values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK _d 1.05±0.13nM and apparentK _i values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [³H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM _r on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.     

Histochemical detection of α-d-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-α-d-glucoside

Summary 5-Br-4-Cl-3-Indoxyl--d-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of -d-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fichsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid -d-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane -d-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane -d-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid -d-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal -d-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.Supported by the German Research Foundation (Sfb 174) and the BMFT (Project CMT 35)     

On the uptake of biotin by the rat renal tubule

Little is known of biotin handling by transporting epithelium. Accordingly, we have examined the characteristics of biotin uptake by rat renal tubular epithelium. Renal cortical slices showed concentrative, temperature-sensitive uptake of biotin. Renal brushborder membrane vesicles exhibited an "overshoot" phenomenon with uptake of 1.9 nM biotin in the presence of a 100 mM NaCl gradient. This overshoot was reduced in magnitude with reduction of the sodium gradient to 50 mM. Biocytin significantly reduced uptake by the vesicles. Concentration-dependent studies yielded an apparent transport Km of 200 nM. We conclude that biotin is actively transported by the rat renal proximal tubule by a system which is at least partially Na+ dependent, and shared by biocytin.     

Generation of succinyl-coenzyme A in photosynthetic bacteria

Pathways of succinyl-Coenzyme A (succinyl-CoA) formation in various photosynthetic bacteria were investigated through several approaches, including determination of activity levels of relevant enzymes. Extracts of photosynthetically grown cells of representative Rhodospirillaceae and Chromatium vinosum showed -ketoglutarate dehydrogenase (KGD) activities sufficient to account for generation of the succinyl-CoA required for biosynthetic metabolism. Except as noted below, the observed ratios of fumarate reductase/succinate dehydrogenase activities were low, consistent with the conclusion that these organisms produce succinyl-CoA oxidatively from -ketoglutarate (KG), rather than by reductive metabolism of fumarate. On the other hand, the green bacterium Chlorobium limicola appears to produce succinyl-CoA by the reductive pathway; in this organism, KGD activity could not be detected, and a high fumarate reductase/succinate dehydrogenase ratio was observed. Results obtained with Rhodopseudomonas gelatinosa suggest that this otherwise typical member of the Rhodospirillaceae may be able to generate succinyl-CoA via both arms of the citric acid cycle, that is, oxidatively from KG, and reductively from fumarate. To further explore the several physiological roles of the conversion: KGsuccinyl-CoA in Rhodopseudomonas capsulata, a mutant (strain KGD 11) almost completely blocked in KGD activity was isolated and studied in detail. Under anaerobic photosynthetic conditions, KGD 11 grows readily on succinate as the sole carbon source; in contrast to the wild type parent, however, it cannot grow with l-glutamate as the source of carbon. The R. capsulata parental strain can grow in darkness as an aerobic heterotroph on various carbon/energy sources including pyruvate, D,L-malate, or succinate. Mutant KGD 11, however, is unable to grow aerobically on the substrates noted. These results indicate that the energy for aerobic dark growth of R. capsulata is provided by respiratory phosphorylation fueled by citric acid cycle function, and that this requires a substantial level of KGD activity. The present findings also indicate that citric acid cycle sequences in most of the Rhodospirillaceae prominently used in current research are geared to operate in the oxidative direction, as in nonphotosynthetic aerobic heterotrophs.Abbreviations CoA coenzyme A - FR fumarate reductase - KG -ketoglutarate - KGD -ketoglutarate dehydrogenase - SD succinate dehydrogenase     

Lectin binding by microvillous membranes and coated-pit regions of human syncytial trophoblast

Summary The distribution of saccharides on the microvillous membrane of the human syncytial trophoblast was investigated using ferritin conjugates of four lectins: concanavalin A (specific for -d-manno- and -d-glucopyranosyl residues), wheatgerm agglutinin (specific forN-acetylglucosamine),Limulus polyphemus lectin (specific forN-acetylneuraminic acid), andLotus tetragonolobus lectin (specific for -l-fucose). Concanavalin A and wheatgerm agglutinin (WGA) reacted strongly with the surface membrane and ferritin deposits were also observed in coated pit regions of the membrane. Lectins fromL. polyphemus andL. tetragonolobus, however, reacted only weakly with the microvillous border and neither reacted with coated pits.Enhanced agglutinability of trophoblast cells in comparison with other foetal cells from the same conceptus was seen with WGA. This agglutination was inhibited by addition of acetylglucosamine or by a solubilized membrane fraction which was bound by a column of WGA-Sepharose. The membrane fraction which did not bind to the column did not inhibit agglutination. Electrophoresis of the WGA-bound membrane proteins revealed six subunits, the major band having an apparent mol. wt. of 55 000. A protein of this mol. wt was also seen in coated vesicles isolated from equivalent human placentae.     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Glucose-dependent respiration in suspensions of rabbit cortical tubules

Summary The effects of glucose on cellular respiration were examined in suspensions of rabbit cortical tubules. When glucose was removed from the bathing fluid, oxygen consumption (QO₂) decreased from 18.6±0.8 to 15.7±0.5 nmol O₂/mg protein·min (P<0.01). The transported but nonmetabolized analogue of glucose, -methyl-d-glucoside (MG), was found to support QO₂ to the same extent as glucose. These observations were also evident in the presence of butyrate, a readily oxidized substrate of the renal cortex. Additional studies with nystatin and ouabain indicated that glucose-related changes in QO₂ were the result of changes in Na, K-ATPase associated respiration. The effect of glucose was localized to the luminal membrane since phlorizin (10^–5 m), a specific inhibitor of liminalk glucose-sodium cotransport, also significantly reduced QO₂ by 10±1%. Phlorizin inhibition of QO₂ was also evident in the presence of MG but was abolished when glucose was removed from the bathing medium. Finally, measurement of NADH fluorescence showed that addition of glucose (5mm) to a tubule suspension causes an oxidation of NAD. These data are all consistent with glucose acting to increase respiration by stimulating sodium entry at the luminal membrane (via glucose-sodium cotransport) followed by increased sodium pump activity and its associated increase in mitochondrial respiration.     

Identification and comparsion of bile acid-binding polypeptides in ileal basolateral membrane

Summary Bile acid-binding polypeptides were examined using basolateral membrane vesicles and enterocytes isolated from rat ileum. The uptake of a photolabile taurocholate derivative, (7,7,-azo-3, 12-dihydroxy-5[3-³H]cholan-24-oyl)-2-aminoethanesulfonate, 7,7-azo-TC, in ileal vesicles preloaded with paraaminohippurate (PAH) was stimulated with respect to uptake in unpreloaded vesicles. The PAH-transstimulated uptake of 7,7-azo-TC was inhibited by taurocholate and vice versa. Irradiation of membrane vesicles in the presence of 7,7-azo-TC irreversibly inhibited PAH-transtimulated taurocholate uptake. Photoaffinity labeling of basolateral membrane vesicles directly with [³H] 7,7-azo-TC and separation of proteins by SDS-PAGE revealed incorporation of radioactivity into several polypeptides. Photoaffinity labeling of vesicles in the presence of taurocholate inhibited the labeling of 54,000 and 59,000 mol. wt. polypeptides. The efflux of taurocholate from ileal enterocytes wascis-inhibited by 7,7-azo-TC andtransstimulated by PAH. Irradiation of enterocytes in the presence of 7,7-azo-TC inhibited taurocholate efflux greater than the presence of 7.7-azo-TC in the dark. When enterocytes that were irradiated in the presence of [³H] 7,7-azo-TC were fractionated and the resultant basolateral membrane fraction was subjected to SDS-PAGE, incorporation of radioactivity into the 54,000 and 59,000 mol. wt. polypeptides was seen. In contrast, when the brush-border membrane fraction was subjected to SDS-PAGE, greatest incorporation of radioactivity was seen in the previously described 99,000 mol. wt. polypeptide. These studies suggest that 7,7-azo-TC shared transporters with natural bile acid and identified polypeptides that may be involved in bile acid and identified polypeptides that may be involved in bile acid transport across the basolateral membrane and differ from that seen in the brush-border membrane of the ileal epithelial cell.     

Biosynthesis of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane by methanogenic bacteria

In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX l-methionine-d,l-sulfoximine - MTA-5 mixed alkyltrimethylammonium bromide - Mops morpholinopropane sulfonic acid     

Transport of amino acids in gamma-glutamyl transpeptidase-implanted human erythrocytes

gamma-Glutamyl transpeptidase purified from hog kidney cortex was implanted in the human erythrocyte membrane by incubation of erythrocytes at 37 degrees c with gamma-glutamyl transpeptidase-incorporated dipalmitoyl phosphatidylcholine vesicles. Membranes prepared from these implanted cells exhibited 4- to 5-fold increase in gamma-glutamyl transpeptidase activity. The association/insertion of gamma-glutamyl transpeptidase into erythrocyte membrane was further demonstrated by antibody to gamma-glutamyl transpeptidase. Implantation of gamma-glutamyl transpeptidase into erythrocyte membrane led to stimulation of uptake of glutamate and alanine, which are normally transported at a slow rate in human erythrocytes. The uptake of these amino acids in the implanted system was inhibited by inhibitors (serine-borate and azaserine) of transpeptidase activity as well as by antibody to gamma-glutamyl transpeptidase. These results in the implanted human erythrocytes demonstrate that gamma-glutamyl transpeptidase enzyme can mediate the translocation of amino acids and provide further evidence in support of its postulated role in the transport of amino acids in natural membranes.     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

The influence of pH on cystine and dibasic amino acid transport by rat renal brushborder membrane vesicles

The uptake of cystine and lysine by rat renal brushborder membrane vesicles was examined at various intravesicular and extravesicular hydrogen ion concentrations to discern whether ionic species are determinants of specificity for the shared transport system and whether hydrogen ion gradients play a role in determining uptake values. When intravesicular and extravesicular pH are identical, the highest uptake of cystine occurred at pH 7.4, with lesser uptake at pH 6.0 and 8.3. Since cystine is electroneutral at pH 6.0 and 90% anionic at pH 8.3, it appears that neither form of the amino acid is a preferred species for transport. A similar relationship between pH and uptake occurs for lysine, which is cationic at pH below 8.5. This suggests that pH affects the functioning of the membrane carrier system independent of ionic species of the substrate. There is no apparent relationship of cystine uptake to hydrogen ion gradients across the membrane. Over the range of extravesicular pH studied, optimal cystine uptake occurred whenever the intravesicular pH was 7.4. Competitive interactions between unlabeled amino acids and labeled cystine were not affected by the extravesicular pH and, therefore, did not seem determined by the ionic species of cystine.