Transport In Membrane Vesicles Research Articles

Neutral amino acid transport in placental plasma membrane vesicles in the late pregnant rat: Evidence for a B 0-like transport system

Rat placental plasma membrane vesicles have been used to study both alanine and leucine transport at late gestation. The results presented are consistent with the presence of more than two separate transport systems for neutral amino acids in the rat placenta. One system is clearly Na +-independent and transports alanine ( K M ≅ 2 mM; V max ≅ 360 pmol ala/mg prot × 5 s), leucine ( K M ≅ 0.07 mM; V max ≅ 100 pmol leu/mg prot × 5 s), serine, cysteine and 2-amino-2 norbornane carboxylic acid (BCH) showing similar properties to the L system, present in many cell types. The other systems are Na +-dependent and transport alanine (a, K M ≅ 5 mM; V max ≅ 3761 pmol ala/mg prot × 5 s; b, K M ≅ 0.07 mM; V max ≅ 376 pmol ala/mg prot × 5 s), cysteine, serine and leucine ( K M ≅ 0.2 mM; V max ≅ 112 pmol leu/mg prot × 5 s) with different kinetic behaviour referring affinity and capacity. While one of them is sensitive to inhibition by methylaminoisobutyric acid (MeAIB), the other is a B 0-like system similar to that characterized in bovine brush-border enterocyte membrane vesicles.

37] Measurements and characteristics of intestinal riboflavin transport

Publisher Summary The transport of labeled riboflavin (RF) in intestinal intact tissue and in cellular preparations from differing animal species, at low substrate concentrations, was found to involve a carrier-mediated process that could be inhibited by metabolic inhibitors, by unlabeled RF, and by some of its structural analogs. Intestinal absorption includes transport of RF across both enterocyte membranes (brush border and basolateral membranes) and cytoplasm, where RF can be enzymatically esterified. Each of these steps can be examined separately by means of appropriate intestinal preparations. This chapter describes certain characteristics of RF transport both in rat brush border membrane vesicles, which enable investigation into the pure membrane process of cellular entry, and in isolated enterocytes, which allow intracellular RF metabolism to be investigated. This chapter also describes a high-performance liquid chromatography (HPLC) method that is suitable for an investigation of RF intracellular biotransformation.

Characterization of guanidine transport in human renal brush border membranes.

Organic cation transporters in the renal proximal tubule are important in the secretion of many clinically used drugs and their metabolites. The goal of this study was to determine the mechanisms of guanidine transport in human kidney. Brush-border membrane vesicles were prepared from donor human kidneys deemed unsuitable for renal transplantation. Uptake of [14C]-guanidine (50 microM) in the vesicles, as determined by rapid filtration, was significantly greater in the presence of an outwardly-directed proton gradient, at all early time points, than in the absence of the gradient. Proton-stimulated uptake of [14C]-guanidine at 30 sec (32.0 +/- 1.24 pmol/mg protein) was significantly inhibited by a number of organic cations including 5 mM unlabeled guanidine (14.8 +/- 1.84 pmol/mg protein) and 5 mM MIBA (9.14 +/- 3.80 pmol/ mg protein), but not by 5 mM TEA (28.4 +/- 5.67 pmol/mg protein). Guanidine, but not TEA, trans-stimulated [14C]-guanidine uptake. Conversely, TEA, but not guanidine, trans-stimulated [14C]-TEA uptake in the vesicles. The proton-dependent transport of guanidine was characterized by a Km of 3.52 +/- 0.42 mM (SE) and a Vmax of 34.6 +/- 8.64 pmol/mg protein/sec (SE). These results demonstrate that guanidine transport in human renal brush border membrane vesicles is stimulated by a proton gradient. Evidence was obtained suggesting that the transporter for guanidine is distinct from the previously described organic cation proton antiporter for TEA.

Role of membrane potential in hypoxic inhibition of L-arginine uptake by lung endothelial cells.

System y+ accounts for the majority of L-arginine transport by pulmonary artery endothelial cells (PAEC). Given that membrane potential is a driving force for transport via system y+, we examined the hypothesis that hypoxia inhibits this transport by decreasing membrane potential. Porcine PAEC or plasma membrane vesicles derived from these cells were exposed to normoxia (room air-5% CO2) or hypoxia (0% O2-95% N2-5% CO2). After exposure, L-[3H]arginine transport and/or accumulation of the lipophilic cation [3H]tetraphenylphosphonium, a quantitative sensor of changes in cell membrane potential, were measured. Hypoxia caused reversible time-dependent decrease in L-arginine transport and membrane potential in PAEC and in plasma membrane vesicles. Comparable decreases in membrane potential and L-arginine transport by PAEC were also observed after depolarization induced by KCl or ouabain. Hyperpolarization, induced by valinomycin, increased membrane potential and L-arginine transport in PAEC and plasma membrane vesicles. Valinomycin also prevented the hypoxia-mediated decreases in membrane potential and L-arginine transport in PAEC. These results indicate that hypoxia-induced plasma membrane depolarization is responsible for reduced L-arginine transport by system y+ in hypoxic porcine PAEC.

Effects of endothelin 1 on phosphate transport in brush border membrane vesicles.

Endothelin is the most potent vasoconstrictive agent released from endothelial and many other types of cells. It has been reported that endothelin has physiological effects on the rat kidney, especially on the renal proximal tubules. We investigated the role of endothelin 1 in the renal brush border membrane. The V(max) of P(i) uptake by brush border membrane vesicles (BBMV) in the endothelin-1-treated group was significantly greater than that in the control group. There were no significant changes in apparent K(m) values between the two groups. To define the direct effect of endothelin 1 on P(i) transport of BBMV, BBMV from normal rats were incubated with endothelin 1 in vitro. The P(i) uptake by BBMV incubated with endothelin 1 did not differ from that by BBMV incubated with a salt solution as a control. These results suggested the probability of the presence of endothelin 1 receptor in the proximal tubules. Administration of the endothelin 1 might increase the P(i) reabsorption by BBMV according to changes in the capacity of the transporter.

Sodium-driven, osmotically activated glycine betaine transport in Listeria monocytogenes membrane vesicles.

Transport of the osmoprotectant and cryoprotectant glycine betaine was investigated in membrane vesicles of Listeria monocytogenes. Uptake-driving transmembrane potentials ranging from 111 to 122 mV within the pH range of 5.5 to 7.5 could be generated by the electron donor system ascorbate-phenazine methosulfate but not by the electron donor system ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. Transport was dependent on both high concentrations of sodium ion and the presence of a hypertonic solute gradient. Arrhenius-type temperature activation was observed. Lineweaver-Burk plots indicated a Km of 4.4 microM for glycine betaine and a Vmax of 700 pmol/min x mg of protein. The Michaelis constant for NaCl depended on the solute used to maintain a constant hyperosmotic pressure, and the Km values were 200 and 75 mM when KCl and sucrose were employed, respectively. Transport was 65% lower in vesicles derived from cells grown under stress provided by KCI rather than NaCl and approximately 94% lower in vesicles derived from cells that were not grown under osmotic stress. This porter appears to be specific for glycine betaine, since neither proline, carnitine, nor choline inhibited uptake effectively. Kinetic studies using ionophores and artificial gradients indicate that glycine betaine is cotransported with sodium ion.

Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles.

2-Chloropropionate (2-CP) is a halogenated monocarboxylic acid generally used to decrease blood lactate concentration in various metabolic states. To investigate whether it has an inhibitory effect on sarcolemmal lactate transport, we compared the initial rate of lactate transport in sarcolemmal membrane vesicles purified from 20 male Wistar rats with and without 2-CP. Transport by these vesicles was measured as uptake of L-(+)-[U-14C]lactate under pH gradient-stimulated cis inhibition. The time courses of 1 mM L-(+)-lactate uptake into vesicles both with and without 10 mM 2-CP (L- or D-) displayed saturation kinetics. Lactate uptake values were lower with 10 mM L-2-CP and 10 mM D-2-CP in comparison to the control values. Both 10 mM L-2-CP and 10 mM D-2-CP significantly inhibited 1 mM L-(+)-lactate uptake (55.8 +/- 9.1 and 53.5 +/- 12.1%, respectively; P < 0.001), whereas a smaller inhibition was observed with a higher lactate concentration of 50 mM (40.2 +/- 11.2 and 38.7 +/- 12.4%; P < 0.001 and P < 0.05, respectively). However, a higher D-2-CP concentration (50 mM) increased the inhibition of pH-stimulated 1 mM L-(+)-lactate uptake (77.0 +/- 9.4%; P < 0.001). D-2-CP had a trans-stimulation effect on the initial rate of lactate efflux of 1 mM L-(+)-lactate compared with baseline efflux (9.5 +/- 0.8 vs. 5.1 +/- 0.4 nmol.min-1.mg protein-1; P < 0.05). 2-CP significantly inhibited the initial rate of lactate uptake in skeletal muscle sarcolemmal membrane vesicles. This result suggests that 2-CP is a nonstereoselective substrate of the lactate muscle carrier that impairs lactate transport.

The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase.

Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown. The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts. Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption. Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 > or = 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 microM) and inhibited the activity of purified yeast V-ATPase. The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme. It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPase than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase.

Monoclonal antibody against conductive chloride transport in pig ileal apical membrane vesicles.

Conductive chloride transport in the small intestine is an important factor controlling fluid movement from the blood to the lumen of the gut. Several proteins with potential conductive chloride ion channel activity are expressed in the enterocyte cell population. However, it is not clear whether one or more than one protein species is normally responsible for mediating conductive chloride transport. We have raised monoclonal antibodies that inhibit conductive chloride transport in apical membrane vesicles prepared from porcine ileal enterocytes. These monoclonal antibodies have been used to identify a unique protein involved with this conductive chloride transport. Here, we report that anti-chloride conductance monoclonal antibodies did not detect any antigen in Western blots of enterocyte apical membrane protein. Dot blotting and immunoprecipitation experiments indicated that the antigen recognized by these monoclonal antibodies was not the cystic fibrosis transmembrane conductance regulator. The antigen was localized to both villus and crypt regions of ileum on immunohistochemistry. A 90-kDa protein species was immunoprecipitated from a primary enterocyte cell line by these monoclonal antibodies. This 90-kDa protein may be a chloride ion channel or may play some regulatory role in conductive chloride transport in enterocyte apical membrane vesicles.

L-lysine transport in chicken jejunal brush border membrane vesicles.

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (KmA = 2.4 +/- 0.7 micromol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na+ (l-methionine inhibition constant KiA, NaSCN = 21.0 +/- 8.7 micromol/L and KSCN = 55.0 +/- 8. 4 micromol/L); second, a low-affinity transport mechanism (KmB = 164. 0 +/- 13.0 micromol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na+ (KiB for L-methionine, NaSCN = 1.7 +/- 0.3 and KSCN = 3.4 +/- 0.9 mmol/L). L-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b0,+ and y+, respectively.

Glucose ingestion causes GLUT4 translocation in human skeletal muscle.

In humans, ingestion of carbohydrates causes an increase in blood glucose concentration, pancreatic insulin release, and increased glucose disposal into skeletal muscle. The underlying molecular mechanism for the increase in glucose disposal in human skeletal muscle after carbohydrate ingestion is not known. We determined whether glucose ingestion increases glucose uptake in human skeletal muscle by increasing the number of glucose transporter proteins at the cell surface and/or by increasing the activity of the glucose transporter proteins in the plasma membrane. Under local anesthesia, approximately 1 g of vastus lateralis muscle was obtained from six healthy subjects before and 60 min after ingestion of a 75-g glucose load. Plasma membranes were isolated from the skeletal muscle and used to measure GLUT4 and GLUT1 content and glucose transport in plasma membrane vesicles. Glucose ingestion increased the plasma membrane content of GLUT4 per gram muscle (3,524 +/- 729 vs. 4,473 +/- 952 arbitrary units for basal and 60 min, respectively; P < 0.005). Transporter-mediated glucose transport into plasma membrane vesicles was also significantly increased (130 +/- 11 vs. 224 +/- 38 pmol.mg-1.s-1; P < 0.017), whereas the calculated ratio of glucose transport to GLUT4, an indication of transporter functional activity, was not significantly increased 60 min after glucose ingestion (2.3 +/- 0.4 vs. 3.0 +/- 0.5 pmol.GLUT4 arbitrary units-1.s-1; P < 0.17). These results demonstrate that oral ingestion of glucose increases the rate of glucose transport across the plasma membrane and causes GLUT4 translocation in human skeletal muscle. These findings suggest that under physiological conditions the translocation of GLUT4 is an important mechanism for the stimulation of glucose uptake in human skeletal muscle.

Pathways for Oxalate Transport in Rabbit Renal Microvillus Membrane Vesicles

Recent evidence suggests that apical membrane Cl--oxalate exchange plays a major role in mediating Cl- absorption in the renal proximal tubule. To sustain steady-state Cl- absorption by a mechanism of exchange for intracellular oxalate requires the presence of one or more pathways for recycling oxalate from lumen to cell. Accordingly, we evaluated the mechanisms of oxalate transport in luminal membrane vesicles isolated from the rabbit renal cortex. We found that transport of oxalate by Na+ cotransport is negligible compared to the transport of sulfate. In contrast, we demonstrated that oxalate shares the electroneutral pathway mediating Na+-independent sulfate-carbonate exchange. We also demonstrated the presence of OH--oxalate exchange (indistinguishable from H+-oxalate cotransport). The process of OH--oxalate exchange was electrogenic and partially inhibited by Cl-, indicating that it occurs, at least in part, as a mode of the Cl--oxalate exchanger described previously. An additional component of OH--oxalate exchange was insensitive to inhibition by either Cl- or sulfate, suggesting that it takes place by neither the Cl--oxalate exchanger nor the sulfate-carbonate exchanger. We conclude that multiple anion exchange mechanisms exist by which oxalate can recycle from lumen to cell to sustain Cl- absorption occurring via apical membrane Cl--oxalate exchange in the renal proximal tubule.

Sulfate transport in human placental brush-border membrane vesicles

Membrane transport pathways for transplacental transfer of sulfate were investigated by assessing the possible presence of a bicarbonate-coupled anion exchange mechanism for sulfate in the maternal facing membrane of human placental epithelial cells. The presence of a SO 4 2−/HCO 3 − exchange mechanism was determined from 35S0 4 2− tracer flux measurements in preparations of purified brush-border membrane vesicles. Under 10% CO 2/90% N 2 the imposition of an outwardly directed bicarbonate gradient (pH o 6/pH i 7.5) stimulated sulfate uptake to levels approximately 4-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, [ K +] o = [ K +] i ) significantly reduced bicarbonate gradient-induced sulfate uptake but concentrative accumulation of sulfate persisted. Early time point determinations performed in the presumed absence of membrane potential suggest the reduced level of bicarbonate gradient-induced sulfate uptake resulted from a more rapid dissipation of the imposed bicarbonate gradient. Concentrative accumulation of sulfate was not observed in the presence of a pH gradient alone under 100% N 2, suggesting a preference of bicarbonate over hydroxyl ions as substrates for exchange. Static head determinations of opposing sulfate and bicarbonate gradients resulting in zero net flux of sulfate suggests the anion exchange mechanism mediates the electroneutral exchange of 2 bicarbonate or 1 carbonate for each sulfate. Sulfate uptake was increased with increasing intravesicular concentrations of carbonate at constant bicarbonate but was constant with increasing intravesicular concentrations of bicarbonate at constant carbonate suggesting carbonate as a substrate for anion exchange. The mechanism mediating bicarbonate gradient-induced sulfate uptake was sensitive to inhibition by stilbene derivatives, furosemide, bumetanide and probenecid. Substrate specificity studies suggest possible interactions of the anion exchange mechanism with salicylate, butyrate, thiosulfate, sulfite, selenate, chromate and oxalate. The results of this study provide evidence for the presence of a bicarbonate-coupled anion exchange mechanism as an electroneutral pathway for sulfate transport across the maternal-facing membrane of human placental epithelial cells.

Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles).

The effect of controlled trypsin digestion of a calmodulin-stimulated Ca2+-ATPase in low-density intracellular membranes from cauliflower (Brassica oleracea L.) inflorescences was investigated. Ca2+ uptake into vesicles was measured either continuously with the fluorescent Ca2+ indicator Calcium Green-5N or with a radio-active filter technique. Trypsin treatment of vesicles resulted in a 3-fold activation of Ca2+ uptake and loss of calmodulin sensitivity. Immunoblotting experiments with an antiserum raised against the Ca2+-ATPase showed that the trypsin activation was accompanied by a decrease in the amount of intact Ca2+-ATPase (111 kD) and by successive appearances of polypeptides of 102 and 99 to 84 kD. 125I-Calmodulin overlays showed that only the intact Ca2+-ATPase bound calmodulin. Removal of the calmodulin-binding domain (about 9 kD) was not enough to obtain full activation. Trypsin proteolysis resulted in a Ca2+ concentration necessary for half-maximal activity of 0.5 [mu]M, whereas a value of about 2 [mu]M was obtained with untreated membranes in the presence of calmodulin. Without trypsin treatment or calmodulin the activity was not saturated even at 57 [mu]M free Ca2+. The data suggest that trypsin digestion and calmodulin activate the cauliflower Ca2+-ATPase by at least partly different mechanisms.

Sequential ordered mechanism for the sodium-glutamate transport in intestinal brush border membrane vesicles

The glutamate/aspartate carrier localized in the brush-border membrane vesicles from enterocytes is known as a transport system catalyzing a sodium-substrate cotransport driven by the sodium gradient across the membrane. The kinetics of this transport system is studied by analogy with an enzymatic bi-substrate reaction. The results of this approach can be summarized as follows: (1) The dependence of the l-glutamate transport rate on the sodium concentration is sigmoidal, and the stoichiometry of the transport is 2 Na +/1 glutamate/1 carrier molecule. (2) The mechanism is sequential ordered, with l-glutamate binding after both the sodium cations. In addition, there is a very high degree of cooperativity between the two sodium binding sites.

Dipeptide transport in brush-border membrane vesicles (BBMV) prepared from human full-term placentae

The uptakes of the tritiated, hydrolysis-resistant cationic (d-Phe-L-Lys), neutral (D-Phe-L-Ala) and anionic (D-Phe-L-Glu) peptides into human full-term placental brush-border membrane vesicles (BBMV) were time-dependent and into an osmotically-active space. Uptakes of D-Phe-L-Lys and D-Phe-L-Glu were temperature-dependent. Uptake of D-Phe-L-Lys was electroneutral (either cation exchange or anion co-transport), whereas D-Phe-L-Ala and D-Phe-L-Glu were both stimulated by an increasingly inside-positive membrane potential (explained by either cation exchange or anion co-transport, or translocation alone, respectively). Uptake of D-Phe-L-Ala was stimulated (approximately 50 per cent) by an inwardly-directed proton gradient (pHin = 7.4, pHout = 5.5), whereas D-Phe-L-Glu was unaffected, and D-Phe-L-Lys uptake was inhibited (approximately 50 per cent) but was unaffected by the organic cation-exchange inhibitors 1,1-diethyl-2,2-cyanine (decynium22) and 5-(N-methyl-N-isobutyl)amiloride (MIBA). Over the concentration range studies, the peptides did not self-inhibit, and the only cross-inhibition was by D-Phe-L-Glu on D-Phe-L-Lys uptake (estimated K(I) 24.2 +/- 1.36 mM), suggesting very low affinity transporter(s). Under conditions favouring its transport by PepT1, D-Phe-L-Glu uptake was unaffected by diethylpyrocarbonate (DEPC); neither D-Phe-L-Ala nor D-Phe-L-Lys was inhibited by DEPC under maximally proton-stimulated conditions of uptake. We conclude that Pep-T-like transporters are not responsible for peptide uptake into human placental BBMV; while the molecular identity of the transporter(s) involved remains unclear, we hypothesize that they could be similar to the as yet unidentified epithelial basolateral peptide transporter(s).

Aluminum Interactions with Voltage-Dependent Calcium Transport in Plasma Membrane Vesicles Isolated from Roots of Aluminum-Sensitive and -Resistant Wheat Cultivars.

The role of Al interactions with root-cell plasma membrane (PM) Ca2+ channels in Al toxicity and resistance was studied. The experimental approach involved the imposition of a transmembrane electrical potential (via K+ diffusion) in right-side-out PM vesicles derived from roots of two wheat (Triticum aestivum L.) cultivars (Al-sensitive Scout 66 and Al-resistant Atlas 66). We previously used this technique to characterize a voltage-dependent Ca2+ channel in the wheat root PM (J.W. Huang, D.L. Grunes, L.V. Kochian [1994] Proc Natl Acad Sci USA 91: 3473-3477). We found that Al3+ effectively blocked this PM Ca2+ channel; however, Al3+ blocked this Ca2+ channel equally well in both the Al-sensitive and -resistant cultivars. It was found that the differential genotypic sensitivity of this Ca2+ transport system to Al in intact roots versus isolated PM vesicles was due to Al-induced malate exudation localized to the root apex in Al-resistant Atlas but not in Al-sensitive Scout. Because malate can effectively chelate Al3+ in the rhizosphere and exclude it from the root apex, the differential sensitivity of Ca2+ influx to Al in intact roots of Al-resistant versus Al-sensitive wheat cultivars is probably due to the maintenance of lower Al3+ activities in the root apical rhizosphere of the resistant cultivar.

Purification of the Tn10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein.

The bacterial tetracycline-resistance determinant from Tn10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/lac operator in a plasmid that provided fusion of TetA to a polyhistidine-carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10-40% that of the wild-type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3-13-fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an alpha-helix content of 54-64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane-spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13-(cyclopentylthio)-5-hydroxy-6-alpha-deoxytetracycline. These findings suggested that the purified protein was in a native state.

Modulation of putrescine transport in rat intestinal brush-border membrane vesicles by fasting and refeeding.

Fasting and refeeding dramatically alter small intestinal mucosal growth which is greatly dependent on polyamine biosynthesis and transport. The aim of this study was therefore to examine the uptake of the diamine putrescine by brush-border membrane vesicles from the small intestine of rats fasted for 3 days or refed a standard diet after a PERIOD OF FASTING. WHILE THE MICHAELIS-MENTEN CONSTANT KM WAS essentially unaltered, the maximum velocity (Vmax) for putrescine uptake was 1.85-fold higher in fasted animals than in ad libitum-fed controls. Refeeding fasted rats for 24 h caused a 31% decrease in the Vmax value that, however, remained 1.27-fold higher than in control RATS, WHILE THE KM VALUE WAS STILL UNCHANGED. FASTING RATS OR refeeding rats after a period of fasting caused only a 13 or 17% increase, respectively, in the value of the constant for the nonsaturable component (P) of putrescine transport relative to the corresponding control condition. Our study also confirms that both the mucosal polyamine biosynthesis and intestinal content are altered by fasting. We suggest that an increased uptake activity may have a conservative role by preventing a substantial loss of tissue polyamines during fasting.

The Inhibition of Maltose Transport by the Unliganded Form of the Maltose-binding Protein ofEscherichia coli: Experimental Findings and Mathematical Treatment

Binding protein-dependent transport systems in Gram-negative enteric bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site. Usually, there are two integral membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur. Transport is against the concentration gradient and needs energization by an ATP hydrolizing polypeptide. Overall transport is considered mainly unidirectional due to the high energy of ATP hydrolysis coupled to transport. In the study reported here, maltose transport in membrane vesicles in the presence of varying concentrations of unliganded maltose-binding protein but with constant amounts of maltose was measured. The conditions were chosen such that the concentration of maltose was always smaller than that of the binding protein and the initial concentration of the liganded binding protein was essentially constant. It was found that the initial rate of transport went through a maximum with increasing amounts of binding protein and declined thereafter. This finding strongly supports the conclusion that both the liganded and the unliganded forms of the binding protein interact with the membrane components of the transport system. The mathematical treatment of the experimental data allowed the ratio of the affinities for the membrane components of the substrate loaded and unloaded binding protein to be estimated. Published data on the binding protein-dependent transport of histidine in membrane vesicles ofSalmonella typhimuriumwere also used. The data allowed the ratio of the binding affinity of the membrane components to the substrate-loaded and free binding protein to be determined. In addition, theKMof transport to theKDof binding protein was approximated.