Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Proton potential-dependent polyamine transport by vacuolar membrane vesicles of Saccharomyces cerevisiae.

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulated spermine and spermidine in the presence of ATP, not in the presence of ADP. Spermine and spermidine transport at pH 7.4 showed saturation kinetics with Km values of 0.2 mM and 0.7 mM, respectively. Spermine uptake was competitively inhibited by spermidine and putrescine, but was not affected by seven amino acids, substrates of active transport systems of vacuolar membrane. Spermine transport was inhibited by the H(+)-ATPase-specific inhibitors bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, but not by vanadate. It was also sensitive to Cu2+ or Zn2+ ions, inhibitors of vacuolar H(+)-ATPase. Both 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847) and nigericin blocked completely the spermine uptake, but valinomycin did not. [14C]Spermine accumulated in the vesicles was exchangeable with unlabeled spermine and spermidine. However, it was released by a protonophore only in the presence of a counterion such as Ca2+. These results indicate that a polyamine-specific transport system depending on a proton potential functions in the vacuolar membrane of this organism.     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

Kinetics of the intestinal brush border proline (Imino) carrier

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.     

Characterization of Vacuolar Arginine Uptake and Amino Acid Efflux inNeurospora crassaUsing Cupric Ion to Permeabilize the Plasma Membrane

Treatment ofNeurospora crassamycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K⁺-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: theK_mfor arginine uptake was found to be 4.2–4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.     

Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles

The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.     

Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulate Ca2+ ion in the presence of ATP, not in the presence of ADP or adenyl-5'-yl imidodiphosphate. Calcium transport showed saturation kinetics with a Km value of 0.1 mM and optimal pH of 6.4. Ca2+ ion incorporated in the vesicles was exchangeable and released completely by a protonophore uncoupler, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847), or calcium-specific ionophore, A23187. The transport required Mg2+ ion but was inhibited by Cu2+ or Zn2+ ions, inhibitors of H+-ATPase of the vacuolar membrane. The transport activity was sensitive to the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked Ca2+ uptake completely, but valinomycin stimulated it 1.35-fold. These results indicate that an electrochemical potential difference of protons is a driving force for this Ca2+ transport. The ATP-dependent formation of the deltapH in the vesicles and its partial dissipation by CaCl2 were demonstrated by fluorescence quenching of quinacrine. This Ca2+ uptake by vacuolar membrane vesicles is suggested to be catalyzed by a Ca2+/H+ antiport system.     

N-acetyl-D-glucosamine countertransport in lysosomal membrane vesicles.

Countertransport of GlcNAc was examined in membrane vesicles prepared from rat liver lysosomes which had been lysed by exposure to 5 mM-methionine methyl ester. These vesicles have a random orientation, have intact ATP-dependent acidification and are largely free of soluble hydrolases. Vesicular volume (24.69 +/- 4.51 microliters/mg of protein) was greater than that of lysosomes (3.02 +/- 0.56 microliters/mg of protein), corresponding to a doubling of diameter. Characteristics of GlcNAc transport in vesicles (Km = 1.3 mM) were similar to those observed in intact lysosomes (Km = 4.4 mM). Sulphation or phosphorylation of the substrate resulted in loss of recognition by the carrier. Hydroxyl group orientation at multiple positions did not appear to be critical, whereas orientation of the acetyl group appeared to have a fundamental role in recognition by the carrier. Based on these criteria, phenyl isothiocyanate-GlcNAc (PITC-GlcNAc) was identified as a possible substrate for transport. Under mild conditions, PITC-GlcNAc reversibly inhibited GlcNAc countertransport in lysosomes and vesicles. This and other modified substrates may be of value in identification of the GlcNAc/GalNAc lysosomal transporter. Lysosomal membrane vesicle preparation is a technique that should be useful for the study of other lysosomal transport systems.     

Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean

Inorganic pyrophosphatase was purified from the vacuolar membrane of mung bean hypocotyl tissue by solubilization with lysophosphatidylcholine and QAE-Toyopearl chromatography. The molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 73,000 daltons. Among the amino-terminal first 30 amino acids are 25 nonpolar hydrophobic residues. For maximum activity, the purified pyrophosphatase required 1 mM Mg2+ and 50 mM K+. The enzyme reaction was stimulated by exogenous phospholipid in the presence of detergent. Excess pyrophosphate as well as excess magnesium inhibited the pyrophosphatase. The enzyme reaction was strongly inhibited by ATP, GTP, and CTP at 2 mM, and the inhibition was reversed by increasing the Mg2+ concentration. An antibody preparation raised in a rabbit against the purified enzyme inhibited both the reactions of pyrophosphate hydrolysis of the purified preparation and the pyrophosphate-dependent H+ translocation in the tonoplast vesicles. N,N'-Dicyclohexylcarbodiimide became bound to the purified pyrophosphatase and inhibited the reaction of pyrophosphate hydrolysis. It is concluded that the 73-kDa protein in vacuolar membrane functions as an H+-translocating inorganic pyrophosphatase.     

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA³ was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.     

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K(+), a low-affinity saturable transport of arginine in both AM- and P-BBMV (K(m) 1.01 mM, V(max) 4.07 nmol/7s/mg protein and K(m) 1.38 mM, V(max) 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Deltapsi) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N(G)-monomethylarginine, N(G)-nitroarginine>lysine>ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K(+), the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (K(m) 0.035 mM, V(max) 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Deltapsi and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N(G)-monomethylarginine>cysteine, lysine>N(G)-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K(+)/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH(out)) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.     

Arginine residues are involved in the transport function of bilitranslocase

Specific guanido group reagents inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles. Their reaction is shown to be affected by sulfobromophthalein, Thymol blue and bilirubin, which are translocated by bilitranslocase across the plasma membrane. It is concluded that the transport function of bilitranslocase depends on arginine residues, which are involved in the interaction with the molecules to be translocated.     

Isolation, purification, and reconstitution of a proline carrier protein from Mycobacterium phlei.

Membrane vesicles from Mycobacterium phlei contain carrier proteins for proline, glutamine, and glutamic acid. The transport of proline is Na+ dependent and required substrate oxidation. A proline carrier protein was solubilized from the membrane vesicles by treatment with cholate and Triton X-100. Electron microscopic observation of the detergent-treated membrane vesicles showed that they are closed structures. The detergent-extracted proteins were purified by means of sucrose density gradient centrifugation, followed by gel filtration and isoelectric focusing. A single protein with a molecular weight of 20,000 +/- 1000 was found on polyacrylamide gel electrophoresis. Reconstitution of proline transport was demonstrated when the purified protein was incubated with the detergent-extracted membrane vesicles. This reconstituted transport system was specific for proline and required substrate oxidation and Na+. The purified protein was also incorporated into liposomes, and proline uptake was demonstrated when energy was supplied as a membrane potential introduced by K+ diffusion via valinomycin. The uptake of proline was Na+ dependent and was inhibited by uncoupler or by sulfhydryl reagents.     

Dinitrophenyl glutathione efflux from human erythrocytes is primary active ATP-dependent transport.

Dinitrophenyl S-glutathione is accumulated by inside-out vesicles made from human erythrocytes in a process totally dependent on ATP and Mg2+. The vesicles were shown to accumulate dinitrophenyl S-glutathione against a concentration gradient. The vesicles were able to concentrate this glutathione derivative even in the absence of membrane potential. This indicated that the ATP-dependent uptake of dinitrophenyl S-glutathione by inside-out vesicles represented an active transport process. Neither extravesicular EGTA nor intravesicular ouabain inhibited the transport process, indicating that neither the Ca2+-ATPase nor the Na+, K+-ATPase were involved. These results indicated that dinitrophenyl S-glutathione uptake by inside-out vesicles probably represented primary active transport. The uptake of dinitrophenyl S-glutathione was a linear function of time (up to 5 h) and vesicle protein. The rate of uptake was optimal between pH 7.0 and 8.0 and at 37 degrees C. The Km values determined for dinitrophenyl S-glutathione and ATP were 0.29 mM and 1 mM, respectively. The transport process was completely inhibited by vanadate and by p-hydroxymercuribenzene sulphonate and inhibited to a lesser extent by N-ethylmaleimide. GTP could efficiently substitute for ATP as an energy source for the transport process, but CTP and UTP were comparatively much less effective.     

Reconstitution of the uncoupling protein of brown adipose tissue mitochondria. Demonstration of GDP-sensitive halide anion uniport

The fluorescent anion indicator 6-methoxy-N-(3-sulfopropyl)quinolinium was trapped in proteoliposomes reconstituted with purified 32-kDa uncoupling protein and used to detect GDP-sensitive uniports of Cl-, Br-, and I-. Transport of these halide anions was rapid and potential-dependent. F- and nitrate were found to inhibit Cl- uptake competitively, suggesting that these anions are also substrates for transport. This preparation also exhibited H+(OH-) transport, showing that the reconstituted uncoupling protein possesses both halide and H+ transport functions, as is observed in intact brown adipose tissue mitochondria. Cl- transport was inhibited to the residual level observed in liposomes without protein when GDP was present on both sides of the membrane. Cl- transport was inhibited by about 50% when GDP was present only on one side of the membrane. We infer that uncoupling protein reconstitutes into proteoliposomes with a 1:1 ratio of sidedness orientation. The Km values for Cl- uniport were 100 and 65 mM, respectively, in GDP-loaded and non-GDP-loaded vesicles. Participation of the inner membrane anion channel in the observed transport is rendered unlikely by the fact that this carrier is insensitive to GDP. A variety of additional experiments probing for inner membrane anion channel yielded uniformly negative results, confirming the absence of contamination by this protein. Our results therefore demonstrate that the uncoupling protein mediates anion translocation, a function previously reported as lacking in the reconstituted system.     

Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase

The uptake of beta-lactam antibiotics into small intestinal enterocytes occurs by the transport system for small peptides. The role of membrane-bound peptidases in the brush border membrane of enterocytes from rabbit and pig small intestine for the uptake of small peptides and beta-lactam antibiotics was investigated using brush border membrane vesicles. The enzymatic activity of aminopeptidase N was inhibited by beta-lactam antibiotics in a non-competitive manner whereas dipeptidylpeptidase IV was not affected. The peptidase inhibitor bestatin led to a strong competitive inhibition of aminopeptidase N whereas the uptake of cephalexin into brush border membrane vesicles was only slightly inhibited at high bestatin concentrations (greater than 1 mM). Modification of brush border membrane vesicles with the histidine-modifying reagent diethyl pyrocarbonate led to a strong irreversible inhibition of cephalexin uptake whereas the activity of aminopeptidase N remained unchanged. A modification of serine residues with diisopropyl fluorophosphate completely inactivated dipeptidylpeptidase IV whereas the transport activity for cephalexin and the enzymatic activity of aminopeptidase N were not influenced. With polyclonal antibodies raised against aminopeptidase N from pig renal microsomes the aminopeptidase N from solubilized brush border membranes from pig small intestine could be completely precipitated; the binding protein for beta-lactam antibiotics and oligopeptides of apparent Mr 127,000 identified by direct photoaffinity labeling with [3H]benzylpenicillin showed no crossreactivity with the aminopeptidase N anti serum and was not precipitated by the anti serum. These results clearly demonstrate that peptidases of the brush border membrane like aminopeptidase N and dipeptidylpeptidase IV are not directly involved in the intestinal uptake process for small peptides and beta-lactam antibiotics and are not a constituent of this transport system. This suggests that a membrane protein of Mr 127,000 is (a part of) the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of small intestinal enterocytes.     

Arginine transport in mitochondria of Neurospora crassa.

Transport of arginine into mitochondria of Neurospora crassa has been studied. Arginine transport was found to be saturable (Km = 6.5 mM) and to have a pH optimum of pH 7.5. Mitochondrial arginine transport appeared to be facilitated transport rather than active transport because: (i) the arginine concentration within the mitochondrial matrix after transport was similar to that of the reaction medium, and (ii) uncouplers and substrates of oxidative phosphorylation did not affect the transport rate. The basic amino acids ornithine, lysine, and D-arginine inhibited arginine transport. The arginine transport system could be irreversibly blocked by treating mitochondria with the reactive arginine derivative, N-nitrobenzyloxycarbonyl-arginyl diazomethane.     

Transport of cyclic adenosine 3'',5''-monophosphate across Escherichia coli vesicle membranes.

The uptake and efflux of cyclic adenosine 3',5'-monophosphate (3',5'-cAMP) by Escherichia coli membrane vesicles were studied. Metabolic energy was not required for the uptake process and was found to actually decrease the amount of 3',5'-cAMP found in the vesicles. 3',5'-cAMP uptake exhibits saturation kinetics (Km = 10 mM, Vmax = 2.8 nmol/mg of protein per min) and was competitively inhibited by a number of 3',5'-cAMP analogs. The uptake of 3',5'-cAMP was found to be sharply affected by a membrane phase transition. The excretion of 3',5'-cAMP was studied by using everted membrane vesicles. Efflux in this system was dependent upon metabolic energy and was reduced or abolished by uncouplers. Different energy sources powered efflux at different rates, showing a relationship between the degree of membrane energization and rate of excretion of 3',5'-cAMP. The efflux process also displayed saturation kinetics (Km = 10.0 mM, Vmax = 0.98 nmol/mg of protein per min) and was competitively inhibited by the same 3',5'-cAMP analogs and to the same degree as was the uptake process. 3',5'-cAMP was found to be chemically unaltered by both the uptake and excretion processes. These data are interpreted as showing that the uptake and excretion of 3',5'-cAMP in E. coli membrane vesicles are carrier-mediated phenomena, possibly employing the same carrier system. Uptake is by facilitated diffusion whereas efflux is via an energy-dependent, active transport process. Evidence is presented showing that cells can regulate the number of 3',5'-cAMP transport carriers. The rate of 3',5'-cAMP excretion is possibly regulated by both the degree of membrane energization and the number of carriers present per cells.     

Transport of sugars and amino acids in bacteria. XVIII. Properties of an isoleucine carrier in the cytoplasmic membrane vesicles of Escherichia coli.

The properties of the carrier for isoleucine in Escherichia coli were studied using cytoplasmic membrane vesicles (IM vesicles) prepared by the method of Yamato, Anraku, and Hirosawa (J. Biochem. 77, 705 (1975)). The IM vesicles exhibited respiration-dependent isoleucine transport activity which was more than 30-fold higher than that of "Kaback vesicles" prepared by our hand from the same strains of E. coli K12. The isoleucine carrier activity of IM vesicles was inhibited by norleucine but not by threonine. The carrier was driven by proton motive force. Mutants were isolated which had lost the carrier activity for isoleucine, as judged by assay with IM vesicles. Using these mutants, the effects of binding proteins specific for branched chain amino acids on the translocation of substrate in IM vesicles were studied. Leucine-isoleucine-valine-threonine-binding protein (LIVT-binding protein) stimulated the initial rate of isoleucine uptake by IM vesicles only when the vesicles possessed carrier activity and it did not affect the Kt value for entry of substrate. This evidence suggests the partial reconstitution of the osmotic shock-sensitive transport reaction in which the binding protein seems to affect the carrier activity with turnover ability.