Solute Symporter Research Articles

Abnormal Sodium Stimulation of Carnitine Transport in Primary Carnitine Deficiency

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the sodium-dependent carnitine cotransporter OCTN2. The majority of natural mutations identified in this and other Na(+)/solute symporters introduce premature termination codons or impair insertion of the mutant transporter on the plasma membrane. Here we report that a missense mutation (E452K) identified in one patient with primary carnitine deficiency did not affect membrane targeting, as assessed with confocal microscopy of transporters tagged with the green fluorescent protein, but reduced carnitine transport by impairing sodium stimulation of carnitine transport. The natural mutation increased the concentration of sodium required to half-maximally stimulate carnitine transport (K(Na)) from the physiological value of 11.6 to 187 mm. Substitution of Glu(452) with glutamine (E452Q), aspartate (E452D), or alanine (E452A) caused intermediate increases in the K(Na). Carnitine transport decreased exponentially with increased K(Na). The E452K mutation is the first natural mutation in a mammalian cotransporter affecting sodium-coupled solute transfer and identifies a novel domain of the OCTN2 cotransporter involved in transmembrane sodium/solute transfer.

Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli.

The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters. To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis. Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type). The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered. Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions. Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions. These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport. The observations confirm the functional importance of TM II described in earlier studies [M. Quick and H. Jung (1997) Biochemistry 36, 4631-4636]. In contrast to Arg40, Arg117 is apparently not important for function of the mature protein. The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane. However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments. These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i. e., by stabilizing a folding intermediate.

H(+)/solute-induced intracellular acidification leads to selective activation of apical Na(+)/H(+) exchange in human intestinal epithelial cells.

The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces - transepithelial ion gradients - are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH(i)) regulation in response to H(+)-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na(+)/H(+) exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na(+)/H(+) exchange was determined by a combination of pH(i), (22)Na(+) influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H(+)/solute-induced acidification (using glycylsarcosine or beta-alanine) led to Na(+)-dependent, EIPA-inhibitable pH(i) recovery or EIPA-inhibitable (22)Na(+) influx at the apical membrane only. Selective activation of apical (but not basolateral) Na(+)/H(+) exchange by H(+)/solute cotransport demonstrates that coordinated activity of H(+)/solute symport with apical Na(+)/H(+) exchange optimizes the efficient absorption of nutrients and Na(+), while maintaining pH(i) and the ion gradients involved in driving transport.

Properties of a Na +/galactose(glucose) symport system in Vibrio parahaemolyticus

We have investigated galactose transport in a mutant strain of Vibrio parahaemolyticus that lacks a glucose-PTS (phospho enolpyruvate: carbohydrate phosphotransferase system) and a trehalose-PTS. Cells of the V. parahaemolyticus actively transported d-galactose and Na + greatly stimulated the transport. Maximum stimulation of d-galactose transport activity was observed at 10 mM NaCl, and Na + could be replaced with Li +. Addition of galactose to the cell suspension under anaerobic conditions elicited Na + uptake. Therefore, we conclude that this organism accomplishes galactose transport by a Na +/solute symport mechanism. Judging from inhibition results, d-galactose, d-glucose and to a lesser extent α- d-fucose are substrates of this transport system. The Na +/galactose symport system exhibited a high affinity for d-galactose ( K m: 40 μM)and showed a relatively lower affinity for d-glucose ( K m: 420 μM), but the maximum velocities for galactose and glucose transport were almost same (about 52 nmol/min per mg protein). The Na +/ d-galactose symport system was induced by either d-galactose or α- d-fucose, and repressed by d-glucose.

An overview of membrane transport proteins in Saccharomyces cerevisiae.

All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to approximately 60 transport proteins whose function was at least partially known, approximately 100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K+ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na+-solute symporters, a protein very similar to electroneural cation-chloride cotransporters, and a putative Na+-H+ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.

Mechanism and energetics of a citrate‐transport system of Klebsiella pneumoniae

The citrate-transport determinant of plasmid pES1 from Klebsiella pneumoniae [Schwarz, E. & Oesterhelt, D. (1985) EMBO J. 4, 1599-1603] has been subcloned in Escherichia coli DH1. Uptake of citrate in E. coli membrane vesicles via this uptake system is an electrogenic process, although the pH gradient is the main driving force for citrate uptake. The rate of citrate uptake, driven by artificially imposed ion-gradients, is high in the presence of an artificial delta pH and low in the presence of an artificial delta psi. Citrate transport does not depend on the presence of Na+ or Mg2+ as has been observed for other citrate-transport systems. Citrate has three pK values: 3.14, 4.77 and 5.40. Citrate forms a stable complex with Mg2+ with a stability constant of 3.2. Kinetic parameters and calculations of the different citrate (Cit) species at a given pH, indicate that the HCit2- is the species transported and that transport occurs in symport with three protons. This citrate-transport system is thus a unique example of a 3H solute symport system.

Nucleotide sequence of gltS, the Na+/glutamate symport carrier gene of Escherichia coli B.

The nucleotide sequence of the gltS gene coding for an Na+/glutamate symport carrier of Escherichia coli B has been determined, and the amino acid sequence of the carrier protein was deduced. The predicted glutamate carrier consists of 401 amino acids with a molecular weight of 42,455. A Shine-Dalgarno sequence and putative promoter sequences were found in the 5'-flanking region of the putative gltS gene. The predicted protein is very hydrophobic (73% nonpolar amino acids), and judging from its hydropathy profile, the protein is composed of 12 hydrophobic membrane-spanning segments with a mean length of 21.6 residues/segment. A typical rho-independent transcription termination signal was found downstream of the gltS gene. We found a conserved alignment of 5 amino acid residues (Gly42--Ala82-X-X-X-X-Leu87-X-X-X-Gly91-Arg92 ), which commonly exists in four Na+ symport carrier proteins, the glutamate carrier, and the proline carrier of E. coli, and the Na+/glucose co-transporters of rabbit and human intestines. We propose that this consensus sequence (or motif) may play an important role in cation recognition or binding in the Na+/solute symport reaction.

Strategies of Nutrient Transport by Ruminal Bacteria

The survival of bacteria in natural environments like the rumen depends on the ability of the bacteria to scavenge nutrients. It is now evident that ruminal bacteria use a variety of transport mechanisms. Hydrophobic substances, such as ammonia and acetate, are permeable to the lipid bilayers of cell membranes and can be taken up by passive diffusion. Hydrophilic compounds (e.g., sugars, amino acids, peptides) do not easily pass through lipid bilayers and must be transported across cell membranes on carrier proteins. Facilitated diffusion can display saturable kinetics but does not result in accumulation of solute. Active transport can establish extremely high concentration gradients, and this work may be driven by the hydrolysis of chemical bonds (e.g., ATP) or ion gradients, which are coupled to solute symport. Many solute symports involve protons, but sodium systems also are common in ruminal bacteria. The phosphotransferase system chemically modifies sugars as they pass across the cell membrane, and several ruminal bacteria have this method of group translocation. Many feed additives have either a direct or indirect effect on rumen bacterial transport. For instance, ionophores can inhibit transport by destroying (sometimes even reversing) ion gradients, lowering intracellular pH, or causing excessive ATP hydrolysis.

Energy transduction and amino acid transport in thermophilic aerobic and fermentative bacteria

Comparative studies on energy-transducing enzymes from thermophilic and mesophilic bacilli showed that functional analogues of enzymes from the thermophilic species are more thermostable and possess higher maximal turnover rates at the respective growth temperatures than those of the mesophilic species, Differences in membrane fatty-acid composition of thermophilic and mesophilic bacilli have been found which explain the similar apparent membrane-microviscosities of these bacteria at their respective growth temperatures. At elevated temperatures the efficiency of energy transduction is substantially declined in the thermophilic Bacillus stearothermophilus due to an extreme increase in H+-conductivity. In membrane vesicles of B. Stearothermophilus the acidic amino acids l-gultamate and l-aspartate are transported by a Na+-H+-solute symport mechanisms in a 1:1:1 stoichiometry. This is concluded from the observations that the membrane potential, pH-gradient and Δμ Na+ are equivalent as driving force for uptake. Uptake of neutral (brached chain) amino acids is also facilitated by Na+-solute mechanisms. This is confirmed by the stimulatory effect of monensin on the steady-state level of accumulation of these amino acids and the absence of uptake in the presence of nonactin in membrane vesicles. Furthermore, the initial rate of l-alanine or l-leucine uptake is specifically enchanced by the addition of sodium ions and to some extent by lithium ions. The affinities of both transport systems for sodium ions was rather low (mM-range). In contrast to this the Na+-H+-spl|-glutamate transport system in B. Stearothermophilus possesses an extremely high affinity fro sodium ionds (Kt<5 μM). l-Glutamate and l-alanine trasnport activities in B. stearothermophilus are allosterically influenced by the pH. Detailes mechanistic studies of amino acid transport have also been performed in a hybridmembrane system composed out of membrane vesicles of Clostridium fervidus and proteoliposomes containing the thermostable and thermoactive primary proton pump cytochrome c oxidase from B. stearothermophilus. The effects of ionophores, uptake driven by artificial gradients and the observed sodium-ion dependence of transport of neutral, acidic and aromatic amino acids indicates that these amino acids are transported in symport with one Na+- or Li+ in Cl. fervidus.

Energy transduction and amino acid transport in thermophilic aerobic and fermentative bacteria

Comparative studies on energy-transducing enzymes from thermophilic and mesophilic bacilli showed that functional analogues of enzymes from the thermophilic species are more thermostable and possess higher maximal turnover rates at the respective growth temperatures than those of the mesophilic species, Differences in membrane fatty-acid composition of thermophilic and mesophilic bacilli have been found which explain the similar apparent membrane-microviscosities of these bacteria at their respective growth temperatures. At elevated temperatures the efficiency of energy transduction is substantially declined in the thermophilic Bacillus stearothermophilus due to an extreme increase in H +-conductivity. In membrane vesicles of B. Stearothermophilus the acidic amino acids l-gultamate and l-aspartate are transported by a Na +-H +-solute symport mechanisms in a 1:1:1 stoichiometry. This is concluded from the observations that the membrane potential, pH-gradient and Δ μ Na + are equivalent as driving force for uptake. Uptake of neutral (brached chain) amino acids is also facilitated by Na +-solute mechanisms. This is confirmed by the stimulatory effect of monensin on the steady-state level of accumulation of these amino acids and the absence of uptake in the presence of nonactin in membrane vesicles. Furthermore, the initial rate of l-alanine or l-leucine uptake is specifically enchanced by the addition of sodium ions and to some extent by lithium ions. The affinities of both transport systems for sodium ions was rather low (mM-range). In contrast to this the Na +-H +-spl|-glutamate transport system in B. Stearothermophilus possesses an extremely high affinity fro sodium ionds ( K t<5 μM). l-Glutamate and l-alanine trasnport activities in B. stearothermophilus are allosterically influenced by the pH. Detailes mechanistic studies of amino acid transport have also been performed in a hybridmembrane system composed out of membrane vesicles of Clostridium fervidus and proteoliposomes containing the thermostable and thermoactive primary proton pump cytochrome c oxidase from B. stearothermophilus. The effects of ionophores, uptake driven by artificial gradients and the observed sodium-ion dependence of transport of neutral, acidic and aromatic amino acids indicates that these amino acids are transported in symport with one Na +- or Li + in Cl. fervidus.

The Na+ cycle of extreme alkalophiles: a secondary Na+/H+ antiporter and Na+/solute symporters.

Extremely alkalophilic bacteria that grow optimally at pH 10.5 and above are generally aerobic bacilli that grow at mesophilic temperatures and moderate salt levels. The adaptations to alkalophily in these organisms may be distinguished from responses to combined challenges of high pH together with other stresses such as salinity or anaerobiosis. These alkalophiles all possess a simple and physiologically crucial Na+ cycle that accomplishes the key task of pH homeostasis. An electrogenic, secondary Na+/H+ antiporter is energized by the electrochemical proton gradient formed by the proton-pumping respiratory chain. The antiporter facilitates maintenance of a pHin that is two or more pH units lower than pHout at optimal pH values for growth. It also largely converts the initial electrochemical proton gradient formed by respiration into an electrochemical sodium gradient that energizes motility as well as a plethora of Na+ solute symporters. These symporters catalyze solute accumulation and, importantly, reentry of Na+. The extreme nonmarine alkalophiles exhibit no primary sodium pumping dependent upon either respiration or ATP. ATP synthesis is not part of their Na+ cycle. Rather, the specific details of oxidative phosphorylation in these organisms are an interesting analogue of the same process in mitochondria, and may utilize some common features to optimize energy transduction.

The sodium cycle: a novel type of bacterial energetics.

The progress of bioenergetic studies on the role of Na+ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na+ can, under certain conditions, substitute for H+ as the coupling ion. Various primary Na+ pumps (delta mu Na+ generators) are described, i.e., Na+ -motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The delta mu Na+ formed is shown to be consumed by Na+ driven ATP-synthase, Na+ flagellar motor, numerous Na+, solute symporters, and the methanogenesis-linked reverse electron transfer system. In Vibrio alginolyticus, it was found that delta mu Na+, generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na+, solute symports), and mechanical (rotation of the flagellum). In Propionigenum modestum, circulation of Na+ proved to be the only mechanism of energy coupling. In other species studied, the Na+ cycle seems to coexist with the H+ cycle. For instance, in V. alginolyticus the initial and terminal steps of the respiratory chain are Na+ - and H+ -motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na+ as well as of H+. In the alkalo- and halotolerant Bacillus FTU, there are H+ - and Na+ -motive terminal oxidases. Sometimes, the Na+ -translocating enzyme strongly differs from its H+ -translocating homolog. So, the Na+ -motive and H+ -motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H+ -motive and Na+ -motive terminal oxidases differ in that the former is of aa3-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na+ and H+ can be translocated by one and the same P. modestum ATPase which is of the F0F1-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary delta mu Na+ generator(s) and delta mu Na+ consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera: Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcina, etc. Thus, the "sodium world" seems to occupy a rather extensive area in the biosphere.

Presence of a nonmetabolizable solute that is translocated with Na+ enhances Na+-dependent pH homeostasis in an alkalophilic Bacillus.

The presence of a nonmetabolizable solute whose uptake is coupled to the inward translocation of Na+ has been found to enhance Na+-dependent pH homeostasis and survival of an obligately alkalophilic bacterium. Upon shift of cells of Bacillus firmus RAB from growth medium to buffers at pH 10.5, viability and maintenance of a relatively acidified cytoplasm depended upon the presence of Na+ and was augmented by the inclusion of alpha-aminoisobutyric acid in the buffer. Similarly, when cells were first equilibrated at pH 8.5 and then shifted to buffer at pH 10.5, an extraordinary capacity to maintain a relatively low pHin was exhibited, but only in the presence of Na+. In this protocol, the inclusion of alpha-aminoisobutyric acid actually resulted in an early overshoot of proton influx and also rendered a suboptimal concentration of Na+ efficacious in pH homeostasis. When a protonophoric uncoupler was added to the equilibration and shift buffers, Na+-dependent acidification of the interior was inhibited at early time points. The results support the conclusion drawn from earlier work that a Na+/H+ antiporter plays a critical role in pH homeostasis in the obligately alkalophilic bacilli. Moreover, the current findings indicate that the Na+/solute symporters are a physiologically functional pathway for completing the sodium cycle that controls pHin.

The interaction between electron transfer, proton motive force and solute transport in bacteria.

The properties of proton solute symport have been studied in Streptococcus cremoris, Rhodopseudomonas sphaeroides and Escherichia coli. In the homolactic fermentative organism S. cremoris the efflux of lactate is a membrane protein-mediated process, which can lead to the generation of a proton motive force. These observations support the energy-recycling model that postulates the generation of metabolic energy by end-product efflux. Studies with oxidants and reductants and specific dithiol reagents in E. coli membrane vesicles demonstrated the presence of two redox-sensitive dithiol-disulphide groups in the transport proteins of proline and lactose. The redox state of these groups is controlled by the redox potential of the environment and by the proton motive force. One redox-sensitive group is located at the inner surface, the other at the outer surface of the membrane. In Rps. sphaeroides and E. coli the activity of several transport proteins depends on the activity of the electron transfer systems. On the basis of these results a redox model for proton solute transport coupled in parallel to the electron transfer system is postulated.

A novel type of energetics in a marine alkali-tolerant bacterium: Δ [formula omitted]Na-driven motility and sodium cycle

Motility of a marine alkali-tolerant bacterium, Vibrio alginolyticus, can be observed in the presence of high concentrations of a protonophorous uncoupler, CCCP. Motility in the CCCP-containing media is completely inhibited by decrease in extracellular [Na +] or by monensin-induced increase in intracellular [Na +]. A mutant has been selected that grows only in media supplemented with a substrate such as acetate requiring no Δ μ - Na to be transported into the cell. Motility of the mutant was found to be completely inhibited by CCCP. Cyanide, CCCP and vanadate added separately or in twos inhibit motility only partially. The three poisons added together completely paralyse the cells. In this inhibitor cocktail, arsenate can substitute for CCCP + vanadate; cyanide can be replaced by anaerobiosis. It is concluded that (i) Δ μ - Na rather than Δ μ - NH powers the flagellar motor of V. alginolyticus in the presence of CCCP, and (ii) in addition to the Na +-motive respiratory chain [Tokuda, H. and Unemoto, T. (1982) J. Biol. Chem. 257, 10007–10014] there is a vanadate and arsenate-sensitive oxygen-independent mechanism of Δ μ Na generation, presumably an ion-motive ATPase. A suggestion is put forward that circulation of Na + can replace that of H + in V. alginolyticus, Δ μ - Na being formed by the Na +-motive respiratory chain and utilized by Na +-solute symporters, the Na +-driven flagellar motor and maybe by a reverse ion-motive ATPase.

Bioenergetic properties and viability of alkalophilic Bacillus firmus RAB as a function of pH and Na+ contents of the incubation medium.

The bioenergetic properties and viability of obligately alkalophilic Bacillus firmus RAB have been examined upon incubation in alkaline and neutral buffers in the presence or absence of added Na+. At pH 10.5, cells incubated in the absence of Na+ exhibited an immediate rise in cytoplasmic pH from less than 9.5 to 10.5, and they lost viability very rapidly. Viability experiments in the presence or absence of an energy source further suggested that the Na+-dependent mechanism for pH homeostasis is an energy-requiring function. The Na+/H+ antiporter, which catalyzes the vital proton accumulation at alkaline pH, was only slightly operational at pH 7.0; both whole cells and vesicles exhibited net proton extrusion even in the presence of Na+. Moreover, cells incubated in buffer at pH 7.0 were actually more viable in the presence of Na+ than in its absence. Thus, the inability of B. firmus RAB to grow at neutral pH is not due to excessive acidification of the cytoplasm. Rather, the transmembrane electrical potential, delta psi, generated at pH 7.0 was found to be much lower than at alkaline pH. The very low delta psi compromised several cell functions, e.g., Na+/solute symport and motility, which in this and other alkalophiles specifically depend upon delta psi and Na+.

Physical mechanism for regulation of proton solute symport in Escherichia coli.

The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers. A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity. Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition. These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide. Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline. The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate. The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+). These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.

The eup genetic locus of Escherichia coli and its role in H+/solute symport.

The eup genetic locus of Escherichia coli and its role in H+/solute symport.

ATP-driven active transport in right-side-out bacterial membrane vesicles.

Membrane vesicles from Salmonella typhimurium induced for phosphoglycerate transport, were loaded with pyruvate kinase and ADP by lysing spheroplasts under appropriate conditions. Vesicles so prepared catalyze active transport of proline and serine in the presence of phosphoenolpyruvate; this activity is abolished by the protonophore carbonyl cyanide-m-chlorophenylhydrazone and by the H+-ATPase inhibitor N,N' dicyclohexylcarbodiimide but not by anoxia or cyanide. In contrast, D-lactate-driven active transport is abolished by the hydrazone and by anoxia or cyanide but not by the carbodiimide. Moreover, phosphoenolpyruvate does not drive transport effectively in vesicles that lack the phosphoglycerate transport system. The results are consistent with an overall mechanism in which phosphoenolpyruvate gains access to the interior of the vesicles by means of the phosphoglycerate transporter and is then acted on by pyruvate kinase to phosphorylate ADP. ATP formed inside of the vesicles is then hydrolyzed by the H+-ATPase, leading to the generation of a proton electrochemical gradient that drives H+/solute symport. By using pBR322 as vector and Escherichia coli as host, a fragment of S. typhimurium DNA coding for the phosphoglycerate transport system has been cloned. E. coli membrane vesicles containing the phosphoglycerate transport system also catalyze transport in the presence of phosphoenolpyruvate when they are loaded with pyruvate kinase and ADP.

Biochemical differentiation of the plasma membrane of the intestinal epithelial cell.

The brush border and basolateral regions of the surface of the intestinal epithelial cell (enterocyte) are exposed to environments which differ even more than those which bathe the different functional regions of the surface of the hepatocyte or the renal tubular cell, and therefore the biochemical differentiation of the enterocyte plasma membrane is also more striking. In addition, it has been easier to study, since the highly adapted brush-border membrane of the enterocyte is one of the easiest of plasma membranes to isolate pure and in quantity (see Isselbacher, 1974). From the study of brush-border preparations a picture has emerged of a membrane with a variety of terminal digestive hydrolases (especially aminopeptidase, disaccharidases and alkaline phosphatase) attached to its luminal surface (Louvard et al., 1975). These are large proteins (Maestracci et al., 1975), which are heavily glycosylated (Kelly & Alpers, 1973; Weiser, 1973; Quaroni et al., 1974) and are moored to the membrane by relatively small hydrophobic polypeptide segments, probably at their N-terminal ends (Maroux & Louvard, 1976). Some of the end-products of digestion that these enzymes generate are then passed across the membrane by transport systems which are closely coupled to the hydrolases and are Na+-independent (Ramaswamy et al., 1976). Some enter the cell via one of several electrogenically driven Na+/solute symport systems (Crane, 1965,1974; Schultz & Curran, 1970; Berger et a[., 1972; Murer & Hopfer, 1974; Hopfer et al., 1973, 1976; Sigrist-Nelson et al., 1976; Alvarado, 1976) and others are taken into the cell by specific transport systems that are independent of Na+ (e.g. fructose; Sigrist-Nelson & Hopfer, 1974). At the cytoplasmic surface of the brush-border membrane, and attached to it by aactinin bridges, is an actin array which forms the microvillus core that also interacts with myosin-like proteins in the terminal web (Mooseker & Tilney, 1975). This raises the exciting, but still largely unexplored, possibility that the brush border is motile (Thuneberg & Rostegaard, 1969; Sandstrom, 1971; Mooseker, 1974) and that this somehow facilitates its digestive and absorptive function, perhaps by preventing the formation of unstirred layers of fluids at either surface of the brush-border membrane. The remainder of the enterocyte plasma membrane (the basolateral region) is separated from the brush border by the terminal bar. Thus, in contrast with the brush border, it interacts with the protective and controlled internal environment of the body and might be expected to be much more similar to the plasma membranes of other cells. It is this region of the plasma membrane which plays the key role in such processes as generating * Present address: Department of Biochemistry, University of Bristol, Medical School, Bristol BS8 1TD. U.K.