Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.     

A spectroscopic assay for the analysis of carbohydrate transport reactions.

A carbohydrate-transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or systems with high-affinity constants for sugars.     

Carrier-mediated transport system for cephalexin in human placental brush-border membrane vesicles

The uptake of cephalosporin antibiotics, cephalexin, was studied with brush-border microvillous plasma membrane vesicles prepared and purified from human full-term placental syncytiotrophoblasts. The uptake of cephalexin by the membrane vesicles was not stimulated in the presence of an Na+ gradient from the outside to the inside of the vesicles, whereas alpha-(methylamino)isobutyrate uptake into the vesicles of the same preparation was stimulated by an Na+ gradient. The equilibrium level of cephalexin uptake decreased with increasing osmolarity of the medium, which indicates that cephalexin is transported into the membrane vesicles. When cephalexin concentrations were varied, the initial rate of uptake obeyed Michaelis-Menten kinetics with Km and Vmax values of 2.29 mM and 2.98 nmol/mg of protein per 60 s, respectively. The uptake of cephalexin was inhibited by structural analogues and sulfhydryl modifying reagents. These results indicate the existence of a carrier-mediated transport system for cephalexin in the human placental brush-border membranes.     

Leucine transport in plasma membrane vesicles of Saccharomyces cerevisiae

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

Human placental brush-border membrane Na(+)-pantothenate cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin pantothenate were investigated by assessing the possible presence of a Na(+)-pantothenate cotransport mechanism in the maternal facing membrane of human placental epithelial cells. The presence of Na(+)-pantothenate cotransport was determined from radiolabeled tracer flux measurements of pantothenate uptake using preparations of purified brush-border membrane vesicles. Compared with other cations the imposition of an inward Na+ gradient stimulated vesicle uptake of pantothenate to levels approximately 40-fold greater than those observed at equilibrium. The observed stimulation of pantothenate uptake was not the result of indirect electrostatic coupling to an inside positive Na+ diffusion potential. In the absence of Na+ and pantothenate concentration gradients an inside negative voltage difference induced a Na(+)-dependent net influx of pantothenate, suggesting the presence of an electrogenic Na(+)-pantothenate cotransport mechanism. The effect of biotin on the kinetics of Na(+)-dependent pantothenate uptake and the effect of pantothenate on the kinetics of Na(+)-dependent biotin uptake suggested that placental absorption of biotin and pantothenate from the maternal circulation occurs by a common Na+ cotransport mechanism in apical brush-border membrane.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

Folate Transport by Prawn Hepatopancreas Brush-Border Membrane Vesicles

The transport system of folic acid (Pte-Glu) by brush-border membrane vesicles (BBMV) isolated from prawn (Penaeus japonicm) hepatopancreas, was studied by measuring the uptake of Pte-Glu. This uptake was found to have two components, intravesicular transport and membrane binding. Membrane binding was not affected by the presence of a transmembrane pH-gradient at a short incubation period. However, a transmembrane pH-gradient increased membrane binding at 60 min. The transport of Pte-Glu appeared to be carrier-mediated, was stimulated by an inwardly proton gradient (pH 5.5 outside, 7.4 inside) and was unaffected by a sodium-gradient. The relationship between pH gradient-driven Pte-Glu uptake and medium Pte-Glu concentration followed saturating Michaelis–Menten kinetics. Eadie–Hofstee representation of the pH gradient-driven Pte-Glu uptake indicated a single transport system with a Km of 0.37 M and Vmax of 1.06 pmol/mg protein/15 s. These findings indicate that BBMV isolated from prawn hepatopancreas possesses a Pte-Glu transport system similar to that described in mammalian intestine.     

Transport of l-arginine in brush border vesicles derived from rabbit kidney cortex

The uptake of l-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37 °C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K_m values of 0.07 and 3.5 mm and V_max values of 1.5 and 8 nmol/mg protein × 15 s, respectively. For the inhibitors of l-[¹⁴C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mm in a mannitol medium) the following sequence of inhibitory strength was established: l-arginine, l-ornithine, l-cystine, l-lysine, d-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K⁺ in the presence of gramicidin D, an overshoot of l-arginine uptake was observed. Initial uptake of l-arginine was slightly faster in the presence of a Na⁺ gradient (outside to inside) than under a K⁺ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, l-arginine uptake in the first 30 s was faster in the NaCl than in the KCl medium. These experiments indicate, beside a major ion-independent l-arginine transport, the presence of a transport stimulated by Na⁺ in isolated brush border vesicles.     

Taurine transport across hepatocyte plasma membranes: analysis in isolated rat liver sinusoidal plasma membrane vesicles

To elucidate the mechanism of taurine transport across the hepatic plasma membranes, rat liver sinusoidal plasma membrane vesicles were isolated and the transport process was analyzed. In the presence of a sodium gradient across the membranes (vesicle inside less than vesicle outside), an overshooting uptake of taurine occurred. In the presence of other ion gradients (K+, Li+, and choline+), taurine uptake was very small and no such overshoot was observed. Sodium-dependent uptake of taurine occurred into an osmotically active intravesicular space. Taurine uptake was stimulated by preloading vesicles with unlabeled taurine (transstimulation) in the presence of NaCl, but not in the presence of KCl. Sodium-dependent transport followed saturation kinetics with respect to taurine concentration; double-reciprocal plots of uptake versus taurine concentration gave a straight line from which an apparent Km value of 0.38 mM and Vmax of 0.27 nmol/20 s x mg of protein were obtained. Valinomycin-induced K+-diffusion potential failed to enhance the rate of taurine uptake, suggesting that taurine transport does not depend on membrane potential. Taurine transport was inhibited by structurally related omega-amino acids, such as beta-alanine and gamma-aminobutyric acid, but not by glycine, epsilon-aminocaproic acid, or other alpha-amino acids, such as L-alanine. These results suggest that Na+-dependent uptake of taurine might occur across the hepatic sinusoidal plasma membranes via a transport system that is specific for omega-amino acids having 2-3 carbon chain length.     

Leucine transport coupled to proton movement in membrane vesicles from Chang liver cells

Leucine transport into membrane vesicles obtained from Chang liver cells was stimulated by an inward H+ gradient. The stimulatory effect of the proton gradient on the rate of leucine uptake (1 min) was inhibited by the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. When the vesicles had been preloaded with a high concentration of KCl, addition of valinomycin stimulated leucine uptake by the vesicles, showing that the leucine transport is dependent on potential gradient. Leucine-coupled H+ accumulation inside the vesicles was confirmed by measuring leucine dependent quenching of the fluorescence of 9-aminoacridine added to medium. These results imply that electrochemical gradient of proton can serve as a driving force for leucine transport across the cell membrane and proton movement is coupled to leucine transport.     

Cyclic AMP-dependent protein phosphorylation in canine renal brush-border membrane vesicles is associated with decreased phosphate transport

It is known that the administration of parathyroid hormone to dogs results in phosphaturia and decreased phosphate transport in brush-border vesicles isolated from the kidneys of those dogs. Parathyroid hormone has been shown to activate adenylate cyclase at the basal-lateral membrane of the renal proximal tubular cell. It has been postulated that parathyroid hormone-induced phosphaturia is effected through phosphorylation of brush-border protein by membrane-bound cAMP-dependent protein kinase. An experimental system was designed such that phosphorylation of brush-border vesicles and Na+-stimulated solute transport could be studied in the same preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane vesicles revealed cAMP-dependent phosphorylation of 2 protein bands (Mr = 96,000 and 62,000), which was enhanced by exposure of the inside of the membrane vesicles to ATP and cAMP. Cyclic AMP-dependent phosphorylation of brush-border vesicles was accompanied by inhibition of Na+-stimulated Pi but not D-glucose transport or 22Na+ uptake. When renal brush-border vesicles from parathyroidectomized and normal dogs were phosphorylated in vitro in the presence and absence of cAMP, both the cAMP-dependent phosphorylation and inhibition of Na+-stimulated Pi transport were greater in vesicles isolated from kidneys of parathyroidectomized dogs relative to control animals. We conclude that the cAMP-dependent phosphorylation of brush-border membrane-vesicle proteins is associated with specific inhibition of Na+-stimulated Pi transport. The phosphaturic action of parathyroid hormone (PTH) could be mediated through the cAMP-dependent phosphorylation of specific brush-border membrane proteins.     

Solubilization and reconstitution of the renal phosphate transporter

Proteins from brush-border membrane vesicles of rabbit kidney cortex were solubilized with 1% octylglucoside (protein to detergent ratio, 1:4 (w/w). The solubilized proteins (80.2 +/- 2.3% of the original brush-border proteins, n = 10, mean +/- S.E.) were reconstituted into artificial lipid vesicles or liposomes prepared from purified egg yolk phosphatidylcholine (80%) and cholesterol (20%). Transport of Pi into the proteoliposomes was measured by rapid filtration in the presence of a Na+ or a K+ gradient (out greater than in). In the presence of a Na+ gradient, the uptake of Pi was significantly faster than in the presence of a K+ gradient. Na+ dependency of Pi uptake was not observed when the liposomes were reconstituted with proteins extracted from brush-border membrane vesicles which had been previously treated with papain, a procedure that destroys Pi transport activity. Measurement of Pi uptake in media containing increasing amounts of sucrose indicated that Pi was transported into an intravesicular (osmotically sensitive) space, although about 70% of the Pi uptake appeared to be the result of adsorption or binding of Pi. However, this binding of Pi was not dependent upon the presence of Na+. Both Na+-dependent transport and the Na+-independent binding of Pi were inhibited by arsenate. The initial Na+-dependent Pi transport rate in control liposomes of 0.354 nmol Pi/mg protein per min was reduced to 0.108 and 0 nmol Pi/mg protein per min in the presence of 1 and 10 mM arsenate, respectively. Future studies on reconstitution of Pi transport systems must analyze and correct for the binding of Pi by the lipids used in the formation of the proteoliposomes.     

Na<Superscript>+</Superscript> gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles

The properties of hypoxanthine transport were investigated in purified brush border membrane vesicles isolated from calf proximal and distal jejunum. Hypoxanthine uptake in the vesicles was stimulated by a transmembrane Na(+) gradient and an inside negative potential resulting in a transient accumulation of intravesicular hypoxanthine, especially in the proximal jejunum. Na(+)-dependent hypoxanthine uptake at this site seemed to occur by two saturable transport systems, a high affinity (K(m)=0.33 micromol/l) and a low affinity (K(m)=165 micromol/l) transporter. Guanine, hypoxanthine, thymine and uracil inhibited intravesicular hypoxanthine uptake, whereas adenine and the nucleosides inosine and thymidine were without effect. These findings represent the first demonstration of active Na(+) gradient-dependent nucleobase transport in intestinal brush border membrane vesicles.     

Evidence for an organic cation-proton antiport system in brush-border membranes isolated from the human term placenta

Uptake of guanidine, an endogenous organic cation, into brush-border membrane vesicles isolated from human term placentas was investigated. Initial uptake rates were manyfold greater in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i) than in the absence of a H+ gradient ([pH]o = [pH]i). Guanidine was transiently accumulated inside the vesicles against a concentration gradient in the presence of the H+ gradient. The H+ gradient-dependent stimulation of guanidine uptake was not due to a H+-diffusion potential because an ionophore (valinomycin or carbonylcyanide p-trifluoromethoxyphenylhydrazone)-induced inside-negative membrane potential failed to stimulate the uptake. In addition, uphill transport of guanidine could be demonstrated even in voltage-clamped membrane vesicles. The H+ gradient-dependent uptake of guanidine was inhibited by many exogenous as well as endogenous organic cations (cis-inhibition) but not by cationic amino acids. The presence of unlabeled guanidine inside the vesicles stimulated the uptake of labeled guanidine (trans-stimulation). These data provide evidence for the presence of an organic cation-proton antiporter in human placental brush-border membranes. Kinetic analysis of guanidine uptake demonstrated that the uptake occurred via two saturable, carrier-mediated transport systems, one being a high affinity, low capacity type and the other a low affinity, high capacity type. Studies on the effects of various cations on the organic cation-proton antiporter and the Na+-H+ exchanger revealed that these two transport systems are distinct.     

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.     

Acute effects of cycloheximide on the translocation of glucose transporters in rat adipose cells

Insulin's rapid action to increase glucose transport is believed to occur primarily through the translocation of glucose transporters from an intracellular pool to the plasma membrane. To better understand the mechanism involved, we studied the role of protein synthesis in glucose transporter translocation by using the protein synthesis inhibitor, cycloheximide. Isolated rat epididymal adipose cells were incubated in the presence or absence of cycloheximide (10 micrograms/ml) for a total of 120 min. Insulin (7 nM) was added to half of the cells from both groups for the final 30 min. Protein synthesis was inhibited by approximately 90%, as measured by [14C]leucine incorporation, in the cells exposed to cycloheximide. The 3-O-methylglucose uptake in intact cells was slightly increased in the basal state with cycloheximide treatment, but the insulin-stimulated 3-O-methylglucose uptake was unchanged by cycloheximide. The distribution of glucose transporters in the different subcellular membrane fractions, as measured by the cytochalasin B binding assay, was unchanged by cycloheximide. These results suggest that insulin's stimulation of glucose transport and translocation of glucose transporters can occur without acute protein synthesis.     

Resolution and reconstitution of active transport of calcium by a protein(s) from Mycobacterium phlei.

Membrane protein(s) responsible for the active transport of calcium in membrane vesicles from Mycobacterium phlei have been solubilized from membranes by sodium cholate treatment and partially purified using a hydrophobic resin. Reconstitution of calcium transport was demonstrated by reconstitution of detergent extracted membranes with the partially purified protein. The uptake of calcium in the reconstituted system was sensitive to proton-conducting uncouplers. Liposomes prepared with partially purified calcium translocating protein were capable of accumulating calcium. The uptake of calcium in this system occurred as a result of an artificial proton gradient generated by the reduction of entrapped ferricyanide with ascorbate-benzoquinone serving as a hydrogen carrier. The addition of the ionophore A23187 caused efflux of accumulated calcium in both native and proteoliposomal-reconstituted system.     

Soluble and Membrane-bound Quinoprotein D-Glucose Dehydrogenases of the Acinetobacter calcoaceticus: The Binding Process of PQQ to the Apoenzymes

Acinetohacter calcoaceticus LMD 79.41 is a unique bacterium containing a soluble quinoprotein D-glucose dehydrogenase (sGDH) in addition to the membrane-bound quinoprotein D-glucose dehydrogenase (mGDH) which is distributed extensively in Gram-negative bacteria. sGDH has been shown to be a distinct enzyme from mGDH, though both enzymes contain a tightly bound pyrroloquinoline quinone (PQQ) as their prosthetic group. In this study, sGDH was detectable in all strains tested of A. calcoaceticus but not in other Gram-negative bacteria tested, indicating that sGDH can be useful as a taxonomic marker for A. calcoaceticus.

The binding process of PQQ with both enzymes was examined by using the apoenzymes purified from a PQQ-deficient mutant strain of A. calcoaceticus. sGDH was able to bind two moles of PQQ in one mole of the homodimer with a fairly high affinity. The binding reaction was much faster at alkaline pH than at acidic pH, and required the presence of some divalent cations such as Cd²⁺, Ca²⁺, Sr²⁺, or Mn²⁺. On the other hand, mGDH bound one mol of PQQ in the monomeric enzyme with a relatively slow reacting process, which was optimum at acidic pH and in the presence of different types of divalent cations such as Mg²⁺, Ca²⁺, Zn²⁺, or Sr²⁺. Thus, it is suggested that sGDH and mGDH have distinct structures around their PQQ binding site. Furthermore, binding of PQQ affects the conformation of both enzymes, which can be shown from the diminishing intrinsic fluorescence of the enzymes and the increase in resistance against proteolysis upon PQQ binding. Data also suggest that the conformational changes caused by PQQ-binding are more dramatic in sGDH than in mGDH. Based on the results obtained, the differences in PQQ-binding mode between the enzymes and the physiological meanings of sGDH are discussed.     

A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver. Characterization, validation, and bile acid transport studies

Basolateral plasma membrane vesicles were prepared from rat liver by a new technique using self-generating Percoll gradients. The method is rapid (total spin time of 2.5 h) and protein yields were high (0.64 mg/g of liver). Transmission electron microscopy studies and measurements of marker enzyme activities indicated that the preparation was highly enriched in basolateral membranes and substantially free of contamination by canalicular membranes or subcellular organelles. High total recoveries for protein yield and marker enzyme activities during the fractionation procedure indicated that enzymatic activity was neither lost (inactivation) nor increased (activation). Thus, the pattern of marker enzyme activities found in the membrane preparation truly reflected substantial enrichment in membranes from the basolateral surface. Analysis of freeze-fracture electron micrographs suggested that approximately 75% of the vesicles were oriented "right-side-out." In order to assess the functional properties of the vesicles, the uptake of [3H]taurocholate was studied. In the presence of a Na+ gradient, taurocholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot"). In the absence of a gradient but in the presence of equimolar Na+ inside and outside of the vesicle, taurocholate uptake was faster than in the absence of Na+. These findings support a direct co-transport mechanism for the uptake of taurocholate and Na+. Kinetic studies demonstrated that Na+-dependent taurocholate uptake was saturable with a Km of 36.5 microM and a Vmax of 5.36 nmol mg-1 protein min-1. The high yield, enzymatic profile and retention of transport properties suggest that this membrane preparation is well suited for studies of basolateral transport.