Leucine transport in plasma membrane vesicles of Saccharomyces cerevisiae

Yeast plasma membrane vesicles were obtained by the fusion of liposomes with purified yeast membranes by means of the freeze thaw-sonication technique. Beef heart mitochondria cytochrome-c oxidase was incorporated into the vesicles. Addition of substrate (ascorbate/TMPD/cytochrome c) generated a membrane potential negative inside, and an alkaline pH gradient inside the vesicle, that served as the driving force for leucine transport. Both delta pH and delta psi could drive leucine transport. When delta pH was increased in the presence of valinomycin and potassium, at the expense of delta psi, leucine uptake increased by 10%.     

Calcium and proton transport in membrane vesicles from barley roots

Ca²⁺ uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of ⁴⁵Ca²⁺ was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca²⁺ uptake coincided with the peak of proton transport by the tonoplast H⁺-ATPase. Depending on the concentration of Ca²⁺ in the assay, Ca²⁺ uptake was inhibited 50 to 75% by those combinations of ionophores and solutes that eliminated the pH gradient and membrane potential. However, 25 to 50% of the Ca²⁺ uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that ⁴⁵Ca uptake was driven by the low affinity, high capacity tonoplast Ca²⁺/nH⁺ antiporter and also by a high affinity, lower capacity Ca²⁺-ATPase. The Ca²⁺-ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca²⁺ transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-man-nosyl synthase activities. A small shoulder of Ca²⁺ uptake in the plasma membrane region of the gradient was inhibited by vanadate and erythrosin B and may represent the activity of a separate plasma membrane Ca²⁺-ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca²⁺ concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca²⁺ created 800- to 2000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca²⁺ on ATP-generated pH gradients are discussed and the suggestion is made that Ca²⁺ dissipates pH gradients by a different mechanism than is responsible for Ca²⁺ uptake into tonoplast vesicles.     

Amino acid transport in plasma membrane vesicles from isolated rat liver parenchymal cells

Plasma membrane vesicles were prepared from isolated rat liver parenchymal cells. The transport of several amino acids was studied and found to be identical to that in membrane vesicles from whole liver tissue.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

The association of proton movement with galactose transport into subcellular membrane vesicles of Escherichia coli.

1. Subcellular membrane vesicles were prepared from a strain of Escherichia coli constitutive for the GalP galactose-transport system. 2. The addition of substrates of the GalP transport system to vesicle suspensions promoted alkaline pH changes, which provided direct evidence for the coupling of sugar and proton transport. 3. Respiration-energized galactose transport was progressively inhibited at pH values above 6.0, and was abolished by agents that render the membrane permeable to protons. 4. The combined effects of valinomycin, the nigericin-like compound A217 and pH on galactose transport suggested that both delta pH and delta psi components of the protonmotive force contributed to energization of galactose transport. 5. These results substantiate the conclusion that the GalP transport system operates by a chemiosmotic mechanism.     

Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver

Summary Thel-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by thel-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. Thel-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li⁺ ions, but not by K⁺ or choline ions. Sodium transport is sensitive to-SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhancel-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.     

Spheroplast-derived membrane vesicles from Rhodobacter capsulatus cells catalyzing nucleotide transport

Rodobacter capsulatus cells, which were cultured anaerobically in high light intensity, had fewer foldings in the cytoplasmic membrane than those which were grown in lower light intensities. Spheroplast-derived membrane fractions obtained from cells cultured under high light intensity contained a high yield of large right-side-out membrane vesicles. The right-side-out vesicles catalyzed reversible light-induced proton efflux as did intact cells. Nucleotide transport activity was also catalyzed by these membrane vesicles. This activity was indirectly monitored by measurement of photophosphorylation or hydrolysis of externally added diphospho- and triphosphonucleosides. These enzymatic activities occur inside the cytoplasmic membrane of spheroplasts and membrane vesicles and therefore require the transport of the externally added reagents. The indirect measurements of transport were complemented by the demonstration of direct uptake of radiolabeled nucleotides into the membrane vesicles. These data support the suggestion that a nucleotide transporter located in the cytoplasmic membrane of R. capsulatus bacteria mediates these activities.     

Electron transport and electrochemical proton gradient in membrane vesicles of Clostridium thermoautotrophicum.

Membrane vesicles of Clostridium thermoautotrophicum containing carbon monoxide dehydrogenase generated a proton motive force when exposed to CO. This proton motive force, with a value of -140 mV, consisted of only an electrical potential at pH 7.5 and above and of an electrical potential and pH gradient at a lower pH. The proton motive force drove the uptake of L-alanine by the vesicles to a concentration of 300 times that of the medium.     

Trans-stimulation and driving forces for GSH transport in sinusoidal membrane vesicles from rat liver

Sinusoidal membrane vesicles from rat liver were employed to study the characteristics of GSH transport. Saturable concentration dependent uptake was best described by the sum of a high and low Km transport. Preloading with GSH markedly stimulated the initial uptake of GSH. GSH transport was electrogenic; uptake was enhanced by an inwardly directed K+ gradient which could be blocked by the K+-channel blocker, Ba2+. The other cations such as Na+, Li+ were poor substitutes for K+. These results therefore show that net GSH transport involves movement of K+.     

Nucleoside transport in cells and membrane vesicles from Escherichia coli K12.

Osmotic shock treatment of cells of Escherichia coli K12 caused a reduction in the transport of nucleosides into the cells. The strains used carried mutations in the nucleoside catabolizing enzymes. This indicated that the decrease in transport capacity was not due to loss of these enzymes during the shock treatment. Membrane vesicles, prepared from the same strains, showed a limited transport of cytidine, deoxycytidine, and uridine. Transport of purine nucleosides and of thymidine was very low in vesicles lacking the appropriate nucleoside phosphorylases and no significant stimulation was observed if the nucleoside phosphorylases were present in the membrane vesicles. These results all indicate that components outside the cytoplasmic membrane are important for nucleoside transport. Selection for resistance to fluorodeoxycytidine yielded mutants which were unable to transport any nucleoside, even when the nucleoside phosphorylases were present in high amounts. This finding is consistent with a requirement for a specific transport process prior to the initial enzymatic attack on the incoming nucleoside.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

Sodium-dependentl-serine transport in plasma membrane vesicles isolated from Ehrlich cells by two-phase compartmentation

Summary Plasma membrane vesicles were prepared from Ehrlich cells using two-phase system compartmentation. The highly pure plasma membrane vesicles obtained presented a negligible mitochondrial contamination and were suitable for studies of amino acid transport.l-Serine transport showed a clear ionic specificity, maximum incorporation being observed when an inwardly directed NaSCN gradient was used. Na⁺-dependentl-serine transport was dependent on assay temperature and membrane potential, and it seemed to be carried out by two different transport systems. An essential sulfhydryl group seemed to be involved in the transport process.     

Vitamin B12 transport by rat liver lysosomal membrane vesicles

Vitamin B12 (hydroxycobalamin) is endocytosed by mammalian cells as a complex with transcobalamin II and then processed to free B12 in lysosomes. The mechanism by which free B12 becomes available for subsequent cellular metabolism has been uncertain. Lysosomal transport of cyanocobalamin (B12) was examined using membrane vesicles prepared from Percoll gradient purified lysosomes. B12 uptake by vesicles was dependent upon pH and was inhibited by the protonophore CCCP. Transport exhibited saturation kinetics with a Km of 3.5 microM and temperature dependence with a Q10 of 1.8. Uptake of B12 was dependent upon divalent cations and was inhibited by EDTA. Preparation of vesicles in the presence of 100 microM B12 resulted in stimulation of uptake consistent with a mechanism of countertransport. Excess cyanocobalamin, adenosylcobalamin, methylcobalamin, or cobinamide dicyanide inhibited uptake of B12. Trans-stimulation studies showed that only the first three compounds are actually transported species with cyanocobalamin as the preferred substrate. We conclude that lysosomes have a specific transport system for vitamin B12 that results in release of this enzyme cofactor to the cytoplasm.     

Ionic requirements for taurocholate transport in rat liver plasma membrane vesicles

As part of the enterohepatic circulation, taurocholate is taken up by hepatocytes by a Na⁺-gradient-dependent, carrier-mediated process. The dependence of taurocholate uptake on the presence of a Na⁺ gradient, outside greater than inside, has been studied in isolated rat liver plasma membranes. The uptake is specific for sodium, and a cotransport stoichiometry of 2 Na⁺ per taurocholate taken up was found. The presence of K⁺ ions inside the vesicles was also found to be essential for maximum Na⁺-stimulated uptake of taurocholate, although a K⁺ gradient is not required. Mg²⁺ was almost as effective as K⁺ in this regard. The symport of Na⁺ and taurocholate during uptake was shown to be electrogenic, so that K⁺ may act as an exchange counterion preventing the accumulation of positive charge within the vesicles.Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

Sodium transport in basolateral membrane vesicles from rat enterocytes

Basolateral membranes purified from rat jejunal enterocytes and enriched 14 times in (Na, K)-ATPase, are present as unsealed and right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1, as determined by detergent activation of ATPase activity. Entrance of 1 mM Na into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. Carrier-mediated Na transport across the basolateral membrane can be trans-stimulated and cis-inhibited by K and further stimulated by ATP (activation of the Na pump). The ATP effect can be suppressed by vanadate and strophanthidin and enhanced by bleomycin (19% increase), which positively also acts on (Na, K)-ATPase activity (16% increase). In addition to the Na pump this study demonstrates the existence of a carrier-mediated Na transport trans-stimulated by K. There appears to be no cotransport of Na-K.