Maintenance of glucagon-stimulated system A amino acid transport activity in rat liver plasma membrane vesicles

Plasma membrane vesicles prepared from intact rat liver or isolated hepatocytes retain transport activity by systems A, ASC, N, and Gly. Selective substrates for these systems showed a Na+-dependent overshoot indicative of energy-dependent transport, in this instance, driven by an artificially-imposed Na+ gradient. Greater than 85% of Na+-dependent 2-aminoisobutyric acid (AIB) uptake was blocked by an excess of 2-(methylamino)isobutyric acid (MeAIB) with an apparent Ki of 0.6 mM. Intact hepatocytes obtained from glucagon-treated rats exhibited a stimulation of system A activity and plasma membrane vesicles isolated from those same cells partially retained the elevated activity. Transport activity induced by substrate starvation of cultured hepatocytes was also evident in membrane vesicles prepared from those cells. The membrane-bound glucagon-stimulated system A activity decays rapidly during incubation of vesicles at 4 degrees C (t1/2 = 13 h), but not at -75 degrees C. Several different inhibitors of proteolysis were ineffective in blocking the decay of transport activity. Hepatic system N transport activity was also elevated in plasma membrane vesicles from glucagon-treated rats, whereas system ASC was essentially unchanged. The results indicate that both glucagon and adaptive regulation cause an induction of amino acid transport through a plasma membrane-associated protein.     

Energy coupling to net K+ transport in Escherichia coli K-12.

Energy coupling for three K+ transport systems of Escherichia coli K-12 was studied by examining effects of selected energy sources and inhibitors in strains with either a wild type or a defective (Ca2+, Mg2+)-stimulated ATPase. This approach allows discrimination between transport systems coupled to the proton motive force from those coupled to the hydrolysis of a high energy phosphate compound (ATP-driven). The three K+ transport systems here studied are: (a) the Kdp system, a repressible high affinity (Km=2 muM) system probably coded for by four linked Kdp genes; (b) the Trka system, a constitutive system with high rate and modest affinity (Km=1.5 mM) defined by mutations in the single trkA gene; and (c) the TrkF system, a nonsaturable system with a low rate of uptake (Rhoads, D.B., Waters, F.B., and Epstein, W. (1976) J. Gen. Physiol. 67, 325-341). Each of these systems has a different mode of energy coupling: (a) the Kdp system is ATP-driven and has a periplasmic protein component; (b) the TrkF system is proton motive force-driven; and (c) the TrkA system is unique among bacterial transport systems described to date in requiring both the proton motive force and ATP for activity. We suggest that this dual requirement represents energy fueling by ATP and regulation by the proton motive force. Absence of ATP-driven systems in membrane vesicles is usually attributed to the requirement of such systems for a periplasmic protein. This cannot explain the failure to demonstrate the TrkA system in vesicles, since this system does not require a periplasmic protein. Our findings indicate that membrane vesicles cannot couple energy to ATP-driven transport systems. Since vesicles can generate a proton motive force, the inability of vesicles to generate ATP or couple ATP to transport (or both) must be invoked to explain the absence of TrkA in vesicles. The TrkF system should function in vesicles, but its very low rate may make it difficult to identify.     

Renal transport of neutral amino acids. Cation-dependent uptake of L-alanine by luminal-membrane vesicles.

The characteristics of L-alanine transport in luminal-membrane vesicles isolated either from whole cortex or from pars convoluta or pars recta of rabbit proximal tubules were studied by a rapid filtration technique and by a spectrophotometric method. Uptake of L-alanine by vesicles from whole cortex was mediated by both Na+-dependent and Na+-independent, but electrogenic, processes. The nature, mechanism and tubular localization of the transport systems were studied by the use of vesicles derived from pars convoluta and pars recta. In vesicles from pars recta transport of L-alanine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.14 mM) and a low-affinity system (half-saturation 9.6 mM). The cation-dependent but Na+-unspecific transport system for L-alanine was exclusively localized to the pars convoluta, which also contained an Na+-preferring system of intermediate affinity (half saturation 2.1 mM). A closer examination of the mechanism of transport of L-alanine in vesicles from pars convoluta revealed that an H+ gradient (extravesicular greater than intravesicular) can drive the transport of L-alanine into the vesicles both in the presence and in the absence of Na+. The physiological importance of various L-alanine transporters is briefly discussed.     

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts was shown to be stereospecific, activated by glucose starvation and occurred by both high and low affinity systems. Transport by the high affinity system was shown to occur by an active transport process. Furthermore, the high affinity system was shown to be defective in vesicles prepared from F72 cells (hexose transport mutant). These results indicate that the high affinity hexose transport system is retained in the plasma membrane vesicles. Thus plasma membrane vesicles could be of value in further characterization of the L6 high affinity hexose transport system, without interference from the various metabolic events occurring in whole cells.     

Uptake of proline by brushborder vesicles isolated from human kidney cortex

Proline transport into renal brushborder membrane vesicles isolated from human kidney is mediated by two uptake systems. The high-affinity system is stimulated by a Na gradient and appears to be shared with glycine while the low-affinity system is not. Uptake curves of low concentrations of proline exhibit a Na-gradient-dependent overshoot indicative of electrogenic transport. The proline transport systems observed in isolated human renal brushborder membrane vesicles appear to have characteristics similar to those in rat kidney membranes.     

Amino acid transport in whole cells and membrane vesicles of Arthrobacter pyridinolis

Arginine, and several other amino acids, can only support growth of Arthrobacter pyridinolis if malate is also present in the medium. Arginine is transported by a high affinity lysine-arginine-ornithine-type transport system which is stimulated by malate in both whole cells and vesicles, is respiration-coupled, and appears to depend upon a respiration-generated membrane potential but not on a ΔpH. Arginine is also transported by a low-affinity system which transports canavanine. Studies of an arginine auxotroph suggest that the lysine-arginine-ornithine system may be the system of major physiological significance for arginine transport. Phenylalanine is one of a few amino acids which can act as sole source of carbon for A. pyridinolis. Transport of phenylalanine occurs by two kinetically distinct systems. Both of these transport systems are respiration-coupled, are not appreciably stimulated by malate either in cells or vesicles, but are markedly stimulated by ascorbate-phenazine methosulfate. Studies with inhibitors indicate that the transport systems for phenylalanine utilize both a ΔpH and a membrane potential.     

Renal transport of monocarboxylic acids. Heterogeneity of lactate-transport systems along the proximal tubule.

The characteristics of D- and L-lactate transport in luminal-membrane vesicles derived from whole cortex, from the pars convoluta and from the pars recta of rabbit kidney proximal tubule were studied. It was found that uptake of both isomers in vesicles from whole cortex occurred by means of dual electrogenic transport systems, namely a low-affinity system and a high-affinity system. Uptake of both isomers in vesicles from the pars recta was strictly Na+-dependent and is mediated via a single high-affinity common transport system. Vesicles from the pars convoluta contained a cation-dependent but Na+-unspecific low-affinity common transport system for these compounds. The physiological importance of this system is briefly discussed.     

Relationship between amino acid transport and electron transport by membrane vesicles of Micrococcus denitrificans

Membrane vesicles were prepared from Micrococcus denitrificans by osmotic shock of lysozyme spheroplasts. These vesicles concentrated 4 amino acids via two systems; one for glycine-alanine and the other for asparagine-glutamine. Amino acid transport was coupled to the membrane-bound electron transport system and involved interactions of the primary dehydrogenases, cytochromes, cytochrome oxidase and oxygen. After transport the amino acids were recovered unchanged from the vesicles. The substrates of the membrane-bound electron transport system d-lactate, l-lactate, formate, succinate, NADH, glucose-6-phosphate and α-glycerolphosphate all stimulated transport at least 2-fold. Both oxygen and nitrate could serve as terminal electron acceptors with vesicles prepared from cells grown anaerobically with nitrate. Anaerobic transport in the presence of nitrate was not inhibited by cyanide but was inhibited by nitrite. A system stimulated by substrates of the electron transport system but independent of added terminal electron acceptors was found also in the vesicles prepared from anaerobically grown cells. Addition of one combination of two substrates for electron transport produced an amino acid uptake 12 to 15% greater than the sum of the rates for each substrate added singly. Additions of other combinations gave rates of transport less than the sum of the rates of each added alone. Both the dehydrogenase activities and the coupling of electron transport to amino acid uptake were modified by changing the growth conditions and differences between the effectiveness of each substrate for each of the two transport systems could be detected. The efficiency of the vesicles per protoheme, the prosthetic group of the membrane-bound cytochrome b, with d-lactate as substrate was 27% for glutamine and 6% for glycine of the rates of transport of these two amino acids in intact cells when driven by endogenous respiration. Assuming one amino acid transported per electron, the transport of glycine utilized 1% of the respiratory capacity with glucose-6-phosphate as substrate. The coupling to the electron transport with the other substrates was less efficient. It appeared that a small portion of the total capacity of the electron transport system was coupled to amino acid transport and the coupling to respiration, as well as the primary dehydrogenase activities and terminal cytochrome oxidase, were modified in response to the conditions of growth.     

Modification of system A amino acid carrier by diethyl pyrocarbonate

Sodium-dependent alanine transport in plasma membrane vesicles from rat liver was inactivated in a time- and concentration-dependent fashion by prior treatment of membranes with the acylating reagent diethyl pyrocarbonate (DEPC). Both components of Na+/alanine cotransport (systems A and ASC) were inhibited. Exposure of vesicles to p-bromophenacyl bromide and methyl p-nitrobenzenesulfonate, which share with DEPC reactivity against histidine residues, also led to inhibition of alanine transport through systems A and ASC. The presence of Na+ (100 mM NaCl) and L-alanine (10 mM) during exposure to vesicles to DEPC protected against inactivation of system A (but not system ASC) transport activity. This protective effect was specific and required the presence of L-alanine since the presence of L-phenylalanine alone (10 mM) or L-phenylalanine plus Na+ (100 mM NaCl) did not cause any detectable protection. This overall pattern of protection is opposite to that previously found against specific sulfhydryl reagents (i.e. N-ethylmaleimide), where protection of system ASC was nearly maximal. The pH profile for DEPC-dependent inhibition of system A transport activity suggests modification of amino acid residue(s) with a pKr of approximately 7, most likely histidine(s), in close parallel with the pH dependence of system A transport activity. Our results suggest the presence of critical histidine residues on the system A carrier that may be responsible for the pH dependence of system A transport activity.     

Intracellular transport based on actin polymerization

In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called “comet-like tail”, is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

The use of membrane vesicles in transport studies

Transport-competent plasma membrane vesicles isolated from mammalian cells provide a system to investigate mechanisms and regulation of nutrient and ion transport systems. The characteristics of membrane vesicle systems to study transport in erythrocytes, renal and epithelial membranes, Ehrlich ascites cells, and mouse fibroblasts are discussed. Studies of Na+-stimulated and Na+-independent amino acid and glucose transport in these systems are evaluated, with emphasis on experimental verification of concepts stated in the Na+ gradient hypothesis. Nucleoside, phosphate, and calcium transport systems in plasma membrane vesicles from mouse fibroblast cultures are discussed. Also, current biochemical approaches to investigate mechanisms of regulation of nutrient transport systems by hormones or cellular proliferative state are described.     

Transport of alpha-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens.

The transport of alpha-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent Km values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for alpha-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting alpha-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using 137Cs in the presence of valinomycin. Direct attempts to demonstrate a protonsymport mechanism for alpha-aminoisobutyrate transport were negative.     

Characteristics of D-galactose transport systems by luminal membrane vesicles from rabbit kidney

The characteristics of renal transport of D-galactose by luminal membrane vesicles from either whole cortex, pars recta or pars convoluta of rabbit proximal tubule were investigated by a spectrophotometric method using a potential-sensitive carbocyanine dye. Uptake of D-galactose by luminal membrane vesicles prepared from whole cortex was carried out by an Na+-dependent and electrogenic process. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for the uptake of D-galactose. Tubular localization of the transport systems was studied by the use of vesicles derived from pars recta and pars convoluta. In pars recta, Na+-dependent transport of D-galactose and D-glucose occurred by means of a high-affinity system (half-saturation: D-galactose, 0.15 +/- 0.02 mM; D-glucose, 0.13 +/- 0.02 mM). These results indicated that the "carrier' responsible for the uptake of these hexoses does not discriminate between the steric position of the C-4 hydroxyl group of these two isomers. This is further confirmed by competition experiments, which showed that D-galactose and D-glucose are taken up by the same and equal affinity transport system by these vesicle preparations. Uptake of D-galactose and D-glucose by luminal membrane vesicles isolated from pars convoluta was mediated by a low-affinity common transport system (half-saturation: D-galactose, 15 +/- 2 mM; D-glucose, 2.5 +/- 0.5 mM). These findings strongly suggested that the "carrier' involved in the transport of monosaccharides in vesicles from pars convoluta is specific for the steric position of the C-4 hydroxyl group of these sugars and presumably interacts only with D-glucose at normal physiological concentration.     

Carrier-mediated transport of cephalexin via the dipeptide transport system in rat renal brush-border membrane vesicles

Carrier-mediated transport of aminocephalosporin antibiotics by renal brush-border membrane vesicles has been studied in relation to the transport systems for dipeptides and amino acids. Dipeptides such as L-carnosine (beta-alanyl-L-histidine) and L-phenylalanylglycine competitively inhibited the uptake of cephalexin, but amino acids did not. Cephalexin uptake was stimulated by the countertransport effect of L-carnosine in the normal and papain-treated vesicles, and by the effect of L-phenylalanylglycine only in the papain-treated vesicles. In the papain-treated vesicles, the hydrolysis of dipeptides was markedly decreased, and the specific activity for cephalexin transport was increased approx. 2-fold because of the partial removal of membrane proteins. These results suggest that carrier-mediated transport of cephalexin can be transported by the system for dipeptides in renal brush-border membranes.     

Mechanisms of uptake of ketone bodies by luminal-membrane vesicles

The energetics and location of renal transport of acetoacetate, beta-hydroxybutyrate, alpha-hydroxybutyrate and gamma-hydroxybutyrate by luminal-membrane vesicles from either whole cortex or pars convoluta or pars recta of rabbit proximal tubule were studied. Addition of either acetoacetate or beta-hydroxybutyrate or its analogues to dye-membrane-vesicle suspensions in the presence of Na+ gradient (extravesicular greater than intravesicular) resulted in absorbance changes indicative of depolarizing event(s). Valinomycin enhanced the Na+-dependent uptake of monocarboxylic acids, provided a K+ gradient (intravesicular greater than extravesicular) was present. By contrast, Na+-dependent uptake of these compounds was nearly abolished by ionophores that permit Na+ to pass through the luminal-membrane via another channel, either electrogenically (e.g. gramicidin D) or electroneutrally (e.g. nigericin). These results established that the Na+-dependent transport of ketone bodies and analogues by luminal-membrane vesicles is an electrogenic process. Eadie-Hofstee analysis of saturation kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for these compounds. Tubular localization of the transport systems was studied by the use of vesicles derived from pars convoluta and pars recta. In pars recta uptake of all these compounds was mediated by means of a single high affinity common transport system. Uptake of these compounds by vesicles from pars convoluta was carried out via a relatively low affinity but common transport system. The physiological importance of the transport systems is discussed.     

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.     

Distinction of three types of D-glucose transport systems in animal cells

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.     

Effect of parathyrin on the transport properties of isolated renal brush-border vesicles.

The transport properties of brush-border membrane vesicles isolated by a calcium-precipitation method from the renal cortex of normal and parathyrin (parathyroid hormone)-treated rats were studied by a rapid-filtration technique. Parathyrin elicited a dose-dependent decrease in the Na+-dependent phosphate uptake by the brush-border membrane vesicles, but the uptake of D-glucose, Na+ and mannitol was not affected. A maximum inhibition of 30% was observed after the application of 30 U.S.P. units intramuscularly 1 h before the animals were killed. Intravenous infusion of dibutyryl cyclic AMP (0.5-1.5 MG) also decreased the phosphate uptake by the brush-border vesicles. Both dibutyryl cyclic AMP and parathyrin were ineffective when added in vitro to brush-border membrane vesicles isolated from normal rats. These data suggest that parathyrin exerts its action on the phosphate reabsorption in the renal proximal tubule by affecting the Na+/phosphate co-transport system in the brush-border membrane. The effects of parathyrin on Na+ and glucose transport, however, seem to be due to alterations to the driving forces for transport and not to the brush-border transport systems.     

Na+-Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.