Intravesicular Space Research Articles

Electrogenic Antiport Activities of the Gram-positive Tet Proteins Include a Na+(K+)/K+ Mode That Mediates Net K+ Uptake

Two Gram-positive Tet proteins, TetA(L) from Bacillus subtilis and TetK from a Staphylococcus aureus plasmid, have previously been suggested to have multiple catalytic modes and roles. These include: tetracycline (Tc)-metal/H+ antiport for both proteins (Yamaguchi, A., Shiina, Y., Fujihira, E., Sawai, T., Noguchi, N., and Sasatsu, M. (1995) FEBS Lett. 365, 193-197; Cheng, J. Guffanti, A. A., Wang, W., Krulwich, T. A., and Bechhofer, D. H. (1996) J. Bacteriol. 178, 2853-2860); Na+(K+)/H+ antiport for both proteins (Cheng et al. (1996)); and an electrical potential-dependent K+ leak mode for TetK and highly truncated segments thereof that can facilitate net K+ uptake (Guay, G. G., Tuckman, M., McNicholas, P., and Rothstein, D. M. (1993) J. Bacteriol. 175, 4927-4929). Studies of membrane vesicles from Escherichia coli expressing low levels of complete and 3'-truncated versions of tetA(L) or tetK, now show that the full-length versions of both transporters catalyze electrogenic antiport and that demonstration of electrogenicity depends upon use of a low chloride buffer for the assay. The K+ uptake mode, assayed via 86Rb+ uptake, was also catalyzed by both full-length TetA(L) and TetK. This mode does not represent a potential-dependent leak. Such a leak was not demonstrable in energized membrane vesicles. Rather, Rb+ uptake occurred in right-side-out vesicles when the intravesicular space contained either Na+ or K+ but not choline. If an outwardly directed gradient of Na+ or K+ was present, Rb+ uptake occurred without energization in vesicles from cells transformed with a plasmid containing tetA(L) or tetK but not a control plasmid. Experiments in which a comparable exchange was carried out in low chloride buffers to which oxonol was added confirmed that the exchange was electrogenic. Thus, the K+ uptake mode is proposed to be a mode of the electrogenic monovalent cation/H+ antiport activity of TetA(L) and TetK in which K+ takes the place of the external protons. Truncated TetK and TetA(L) failed to catalyze either Tc-metal/H+ or Na+/H+ antiport in energized everted vesicles. Truncated TetK, but not TetA(L), did, however, exhibit modest, electrogenic Na+(K+)/Rb+ exchange as well as a small, potential-dependent leak of Rb+. The C-terminal halves of the TetA(L) and TetK proteins are thus required both for proton-coupled active transport activities of the multifunctional transporter and, perhaps, for minimizing cation leakiness.

Superoxide anion radical-triggered Ca2+ release from cardiac sarcoplasmic reticulum through ryanodine receptor Ca2+ channel.

The ryanodine receptor Ca2+ channel (RyRC) constitutes the Ca2+-release pathway in sarcoplasmic reticulum (SR) of cardiac muscle. A direct mechanical and a Ca2+-triggered mechanism (Ca2+-induced Ca2+ release) have been proposed to explain the in situ activation of Ca2+ release in cardiac muscle. A variety of chemical oxidants have been shown to activate RyRC; however, the role of modification induced by oxygen-derived free radicals in pathological states of the muscle remains to be elucidated. It has been hypothesized that oxygen-derived free radicals initiate Ca2+-mediated functional changes in or damage to cardiac muscle by acting on the SR and promoting an increase in Ca2+ release. We confirmed that superoxide anion radical (O2-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases 45Ca2+ efflux from the heavy fraction of canine cardiac SR vesicles; hypoxanthine-xanthine oxidase also decreases Ca2+ free within the intravesicular space of the SR with no effect on Ca2+-ATPase activity. Current fluctuations through single Ca2+-release channels have been monitored after incorporation into planar phospholipid bilayers. We demonstrate that activation of the channel by O2- is dependent of the presence of calmodulin and identified calmodulin as a functional mediator of O2--triggered Ca2+ release through the RyRC. For the first time, we show that O2- stimulates Ca2+ release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2-. The decreased calmodulin content induced by oxygen-derived free radicals, especially O2-, is a likely mechanism of accumulation of cytosolic Ca2+ (due to increased Ca2+ release from SR) after reperfusion of the ischemic heart.

Glycosphingolipids of skeletal muscle: II. Modulation of Ca 2+-flux in triad membranes by gangliosides

Membrane vesicles of rabbit skeletal muscle were prepared and separated by sucrose density gradient centrifugation. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M) as characterized by their specific marker enzymes, ligand binding, and ion flux activities. The distribution of neutral glycosphingolipids and gangliosides in these membrane preparations has been documented in the preceding paper (J. Müthing, U. Maurer, U. Neumann, B. Kniep, and S. Weber-Schürholz, Carbohydr. Res., (1988) 135–145). G M3(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderately expressed and ganglio-series gangliosides were found in minor quantities, however, all showing different qualitative and quantitative membrane-type specific patterns. The voltage dependent Ca 2+-channels of skeletal muscle reside prevalently in the triad enriched membrane fractions deduced from highest binding capacity of 1,4-dihydropyridines. Calcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active 45Ca 2+-uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in parallel to control liposomes without gangliosides. G M3(Neu5Ac) strongly increased the uptake of 45Ca 2+ (+285%) whereas G M3(Neu5Gc) severely inhibited the ion flux (−61%). Neolacto-series gangliosides evoked miscellaneous effects upon 45Ca 2+-flux depending on isomeric sialic acid configuration, oligosaccharide size and fatty acid chain length of the ceramide portion. VI 3Neu5Ac– nLcOse 6Cer (C 24-fatty acid), IV 3Neu5Ac– nLcOse 4Cer (C 16-fatty acid) and IV 6Neu5Ac– nLcOse 4Cer (C 16-fatty acid) strongly enhanced the 45Ca 2+-flux (+208, +162, and +120%, respectively), whereas IV 3Neu5Ac– nLcOse 4Cer (C 24-fatty acid), VI 3Neu5Ac– nLcOse 6Cer (C 16-fatty acid) and IV 6Neu5Ac– nLcOse 4Cer (C 24-fatty acid) slightly reduced 45Ca 2+-flux (−3, −6, and −17%, respectively). Out of all gangliosides tested in this study, G M1 showed the strongest stimulatory effect (+327%). G D1a and G T1b gave rise to remarkable flux-stimulation of +283 and +255%, respectively, whereas G D1b exhibited only a slightly positive effect (+38%). This data suggest a functional role of gangliosides in subcellular muscle membranes giving strong evidence that gangliosides are capable of modulating the cytosolic calcium level of muscle, which regulates muscle contraction.

内視鏡生検による十二指腸HCO3-測定系の確立について

This study was designed to characterize the mechanisms available for bicarbonate transport across the apical membrane using duodenal brush border membrane vesicles (BBMVs) , including the morphological findings. BBMVs were purified using a double Mg2+ precipitation technique. Electron microscopy of the final pellet showed 11.7±2.9 fold in Al-p and 12.6±1.8 fold in LAP. The time course of 36Cl uptake into BBMVs demonstrated a good response curve and equilibrated to the intravesicular space over 120 min. In this present study, these BBMVs were purified and showed a stabilized structure. Therefore, these results suggest that this model was useful for studying HCO3- secretion and ulcer etiology.

Sparfloxacin binds to rabbit intestinal brush-border membrane vesicles by ionic interactions

The intestinal transport and binding of sparfloxacin has been investigated using rabbit intestinal brush-border membrane vesicles. The apparent uptake of sparfloxacin by these vesicles was very rapid. The plateau was reached after 20 min in NaCl-free buffer with a membrane binding of this drug accounting for about two-thirds of the apparent accumulation. Addition of NaCl did not affect the transport of this fluoroquinolone into the intravesicular space. However, the binding of sparfloxacin to membrane vesicles was sharply reduced by NaCl, suggesting that the binding of sparfloxacin to epithelial cells involves ionic interactions.

Riboflavin transport by rabbit renal brush border membrane vesicles

The present study examined riboflavin (RF) uptake by isolated rabbit renal brush border membrane (BBM). RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfaces. The process of RF uptake was saturable as a function of substrate concentration with an apparent K mof 25.7 ± 7.6 μM and V maxof 75.6 ± 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and its structural analogues, lumiflavin and lumichrome, inhibited the uptake of [ 3H]RF significantly, indicating the involvement of a carrier-mediated process in RF uptake by renal BBM. RF uptake by renal BBM was partly Na +-dependent so that when Na + was replaced by potassium, choline, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60% of the control. This Na +-dependency was unlikely to be due to Na +-cotransport mechanism because RF uptake occurred without the characteristic ‘overshoot’ phenomenon as for other Na +-cotransport systems and the elimination of transmembrane Na +-gradient by preloading Na + to the intravesicular space did not affect RF uptake. In contrast, removal of Na + eliminated the binding component of RF uptake, suggesting the requirement of Na + for RF binding to BBM. The RF uptake was not affected when extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [ 3H]RF uptake was found when the transmembrane electrical potential was altered by either the presence of anions with different membrane permeability ( Cl − = NO 3 − SO 4 −gluconate −) or by using nigericin (10 μg/mg protein) with an outwardly or inwardly directed transmembrane K + gradient. The uptake of RF by BBM vesicles was, however, inhibited by probenecid and organic anion transport inhibitors, 4,4-diiso-thiocyanatostilbene-2,2-disulfonic acid (DIDS, 1 mM) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mM). In summary, these results demonstrate the existence of a membrane-associated, and organic anion inhibitor-sensitive, carrier system for RF uptake by renal BBM.

Sorbitol uptake in plasma membrane vesicles isolated from immortalized rabbit TALH cells: activation by a Ca2+/calmodulin-dependent protein kinase.

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg2+, Ca2+, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37 degrees C to 4 degrees C. A lower level of uptake was also observed in the presence of 100 microM quinidine and when Ca2+ or ATP were omitted from the medium. Membranes preincubated with Mg2+, Ca2+, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 microM trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca2+/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca2+/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells.

Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

The vesicular gastric H,K-ATPase catalyzes an electroneutral H for K exchange allowing acidification of the intravesicular space. There is a total of 28 cysteines present in the alpha subunit of the gastric H,K-ATPase, of which 10 are found in the predicted transmembrane segments and their connecting loop, and 9 are present in the beta subunit, of which 6 are disulfide-linked. To determine which of these was accessible to extracytoplasmic attack, the enzyme was inhibited by four different substituted 2-pyridylmethylsulfinyl benzimidazoles, 5-methoxy-2-[(4-methoxy-3, 5-dimethyl-2-pyridyl)methylsulfinyl]-1H-benzimidazole (omeprazole), 2-[(4-trifluoroethoxy-3-methyl-2-pyridyl)methylsulfinyl]-1H-ben zimida zole (lansoprazole), 5-difluoromethoxy-2-[3, 4-methoxy-2-pyridyl)methylsulfinyl]-1H-benzimidazole (pantoprazole), and 2-[(4-(3-methoxypropoxy)-3-methyl)-2-pyridyl)methylsulfinyl]-1H-++ +benzi midazole (rabeprazole), under acid transporting conditions. All of these compounds are weak bases that accumulate in the acidic space generated by the pump and undergo an acid catalyzed rearrangement to a cationic sulfenamide, which forms disulfides with accessible cysteines. The relative rates of acid activation of these compounds corresponded to the relative rates of inhibition of ATPase activity and acid transport. Fragmentation of the enzyme by trypsin followed by SDS-polyacrylamide gel electrophoresis showed that omeprazole bound covalently to one of the two cysteines in the domains containing the fifth and sixth transmembrane segments and their extracytoplasmic loop and to cysteine 892 in the loop between the seventh and eighth transmembrane segments, but inhibition correlated with the reaction with cysteines in the fifth and sixth domain. Lansoprazole bound to the cysteines in these two domains as well as to cysteine 321 toward the extracytoplasmic end of the third transmembrane segments. Pantoprazole bound only to either cysteine 813 or 822 in the fifth and sixth transmembrane region. The inhibition of Rabeprazole correlated also with its binding to this part of the protein, but this compound continued to bind after full inhibition, eventually binding also to cysteines 321 and 892. No binding was found to any of the cysteines in the seventh to tenth transmembrane segments. Thermolysin digestion of the isolated omeprazole-labeled fifth and sixth transmembrane pair showed that cysteine 813 was the site of labeling. It is concluded that binding of these sided reagents to cysteine 813 in the loop between transmembrane (TM)5 and TM6 is sufficient for inhibition of ATPase activity and acid transport by the gastric acid pump. Of the 10 cysteines present in the membrane and extracytoplasmic domain, only three are exposed sufficiently to allow reactivity with these cationic thiol reagents. The binding to cysteine 813 defines the location of the extracytoplasmic loop between TM5 and TM6 and places the carboxylic acids 820 and 824 conserved between the gastric H,K- and the Na,K-ATPases in TM6, consistent with their assumed role in cation binding.

A Yeast ATP-binding Cassette-type Protein Mediating ATP-dependent Bile Acid Transport

ATP-dependent transport of bile acids is a key determinant of bile flow in mammalian liver and is associated with cholesterol excretion, gallstone formation, and numerous inherited and acquired hepatobiliary diseases. Secretory vesicles and a vacuole enriched fraction purified from Saccharomyces cerevisiae also exhibit ATP-dependent bile acid transport. ATP-dependent transport of bile acids by the vacuolar fraction was independent of the vacuolar proton ATPase, responded to changes in the osmotically sensitive intravesicular space, and was saturable, exhibiting a Km of 63 microM for taurocholate. The BAT1 (bile acid transporter) gene was isolated from yeast DNA by polymerase chain reaction amplification using degenerate oligonucleotides hybridizing to conserved regions of ABC-type proteins. ATP-dependent bile acid transport was abolished when the BAT1 coding region was deleted from the genome and restored upon reintroduction of the gene. The deduced amino acid sequence predicts that Bat1p is an ABC-type protein 1661 amino acids in length, similar to mammalian cMOAT/cMRP1 and MRP1 transporters, yeast Ycf1p, and two yeast proteins of unknown function. Information obtained from the yeast BAT1 gene may aid identification of the gene encoding the mammalian bile acid transporter.

Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.

Effects of zinc and copper on cadmium uptake by brush border membrane vesicles

The effects of essential metals, zinc (Zn) and copper (Cu), on cadmium (Cd) uptake were investigated in brush border membrane vesicles (BBMV) isolated from the rat renal cortex and LLC-PK 1 cells. BBMV were incubated with Cd in the presence or absence of Zn or Cu, and then washed with a chelating agent, EGTA, to remove Cd bound to the outer surface of BBMV. Co-incubation with Zn or Cu decreased Cd accumulation in these BBMV in a concentration-dependent manner. Kinetic analysis of the initial accumulation of Cd suggested that Cd is taken up into rat BBMV via an unsaturable component and a saturable component ( K m = 13.8 μM, V max = 1.44 nmol/mg protein/min), and co-incubation with Zn significantly increased the K m of the saturable component without affecting the V max, whereas Cu significantly increased the K m-value and decreased the V max-value. Increasing the osmolarity of the incubation medium slightly decreased Cd accumulation in the absence of Zn or Cu, whereas it did not decrease Cd accumulation in the presence of these metals. These results suggest the possibility that, in addition to passive diffusion, Cd is also taken up from the renal brush border membrane via carrier-mediated mechanisms that are inhibited by Zn competitively and by Cu non-competitively. Furthermore, these results suggest that: (1) Cd binds externally and internally to BBMV, (2) little Cd is transported into the intravesicular space, and (3) both Zn and Cu decrease the binding and transport of Cd.

Protein-facilitated Export of Arachidonic Acid from Pig Neutrophils

Activated neutrophils release a variety of eicosanoids into the extracellular medium including arachidonic acid, 5-hydroxyicosatetraenoic acid, and leukotriene A4 and B4. In this study, the mechanism of arachidonic acid export has been examined using inside-out plasma membrane vesicles from pig polymorphonuclear leukocytes. Tritiated arachidonic acid associated rapidly with the membrane vesicles and crossed the membrane into the intravesicular space in a time-dependent and saturable manner. Half the maximal influx rate was measured at an arachidonate concentration of 5.7 microM, and a maximal influx velocity of 3.0 nmol/mg x min was determined at pH 6.8. Influx into vesicles was sensitive to a number of common anion transport inhibitors including pentachlorophenol, phloretin, diiodosalicylic acid, and quercetin as well as to the proteases trypsin and Pronase, suggesting a protein-dependent process. Furthermore, influx was temperature-sensitive with an energy of activation of 11.6 kcal/mol. Varying extravesicular concentration of ATP, Na+, or K+ had no impact on arachidonate influx, whereas changes in pH had a profound effect; optimum transport activity was observed at an extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a novel protein-facilitated arachidonate export mechanism in pig neutrophils.

Uptake of methylchlorpromazine by brush-border membrane vesicles from rat small intestine.

The uptake of methylchlorpromazine (MCP), a quaternary derivative of chlorpromazine, was investigated using brush-border membrane vesicles isolated from rat small intestine. MCP was taken up rapidly by the vesicles, a major part of the uptake being due to binding to the membrane. Saturable MCP binding to the brush-border membrane was inhibited strongly by chlorpromazine, moderately by propantheline and imipramine, and slightly but significantly by methylbenactyzine and mepenzolate. However, choline and tetramethylammonium failed to exhibit any such inhibitory effect. The movement of MCP into the Intravesicular space was driven by an inside-negative transmembrane electrical potential difference (TEPD) induced by NaSCN or valinomycin. There was no effect of TEPD on MCP binding to the brush-border membrane. The data suggested that both rapid binding to the brush-border membrane and inside-negative TEPD, which is present physiologically across the membrane, play a significant role in the absorptive movements of MCP across intestinal epithelium.

Interaction and translocation of cysteamine (mercaptoethylamine) with model membranes: a 15N-NMR and 1H-NMR study

We investigated by 15N-NMR (nuclear magnetic resonance) spectroscopy the interactions of 15N-labeled cysteamine ([ 15N]mercaptoethylamine (MEA)), a radioprotecting aminothiol, with model membranes of egg yolk phosphatidylcholine (EPC) and phosphatidic acid (EPA). We prepared large unilamellar vesicles (LUVs) with a pH gradient between the intravesicular space and the bulk medium. Over the pH range from 4.8 to 8.1, the observations show a MEA incorporation into the vesicles. This result is consistent with a specific dissociation balance at the lipid-water interface. We carried out another 1H-NMR experiment with unlabeled MEA in the presence of small unilamellar vesicles (SUVs). It revealed that the interactions of MEA within the hydrophobic core of the phospholipidic bilayer vary with external pH value over the range 4.0–7.1. Through these experiments, and others compiled from the literature, it was concluded that 15N-NMR spectroscopy is particularly successful for transmembrane transport analysis.

Kinetic characterization of zinc binding to brush border membranes from rat kidney cortex: interaction with cadmium

Extravesicular and intravesicular zinc bindings were evaluated in brush border membrane vesicles isolated from rat kidney cortex. The process was found to be time-, temperature- and substrate concentration-dependent and displayed saturability. Zn 2+ influx measurements revealed a progressive uptake and massive accumulation at equilibrium which was 50 times higher than the amount that could have been accommodated by the intravesicular space calculated from the equilibrium uptake of d-glucose. Initial (5 s) and equilibrium uptakes (2 h) were found not to be osmotically sensitive as modified by adding mannitol to the medium. It was concluded from these results that the uptake involved massive binding of the Zn 2+ to the brush border membranes components. The ionophore A23187 enhanced the rates of uptake and efflux of Zn 2+ without affecting equilibrium values, suggesting binding of Zn 2+ to interior sites of the membranes. Zn 2+ flux measurements led to the conclusion that two vesicular pools of Zn 2+ bindings existed: a small external pool, accessible to cation chelator (EGTA) or competitive cation cadmium and large intravesicular pool. Accumulated 65Zn was quickly removed from its internal sites only after the membrane had been permeabilized by the cation ionophore A23187 in association with exchangeable ions like zinc and cadmium. Scatchard plot analysis revealed two distinct types of extravesicular binding sites. High affinity extravesicular zinc binding sites reached saturation at 1.6 mM zinc, had a K d of 137 μM and the number of binding sites were 12 nmol/mg protein. Low affinity extravesicular zinc binding sites could not be saturated under experimental conditions up to 3.2 mM zinc. It had a K d of 526 μM and the number of binding sites 28 nmol/mg protein. Interestingly intravesicular binding of zinc revealed only one type of high affinity binding sites ( K d of 104 μM and number of maximal binding sites 400 nmol/mg protein). Furthermore, kinetic analysis of inhibitory effect of Cd 2+ on extravesicular zinc bindings showed an increase in K d of both types of binding sites but there was no significant change in number of maximal binding sites. Extravesicular zinc binding was temperature-sensitive. Arrhenius plot showed the break point at 30°C. The apparent energies of activation were 13.36 Kcal/mol and 3.1 Kcal/mol below and above the break points respectively. The inhibitory effect of sulfhydryl blocking agents on extravesicular zinc binding suggest the involvement of -SH groups in zinc translocation. An increase in initial zinc uptake was observed in the presence of outwardly directed proton gradient. Intravesicular pool of 65Zn was displaced by unlabelled 2 mM Zn 2+ or 2 mM Cd 2+ but not by calcium present in the bathing medium. It is inferred that intravesicular binding sites have a high affinity and are specific for zinc. It is concluded from the present study that in the first instance the binding of zinc to the exofacial zinc binding component and concomitantly its translocation across the membrane, and subsequently massive binding of zinc to interior sites of brush border membranes occurs.

Hibernation enhances D-glucose uptake by intestinal brush border membrane vesicles in ground squirrels.

The ability to actively transport nutrients is maintained in intestinal tissues of hibernating ground squirrels compared with their active counterparts, and shows apparent upregulation in hibernators when transport rates are normalized to tissue mass. To identify the mechanisms responsible for the preservation of transport function during the extended fast of hibernation, we studied D-glucose uptake into jejunal brush border membrane vesicles prepared from active and hibernating 13-lined ground squirrels. Hibernators were without food and showing regular bouts of torpor for at least 6 weeks before sacrifice. Electron micrographs indicated similar microvillus heights of jejunal enterocytes in the two activity states, whereas microvillus density was slightly greater in the hibernators. Glucose uptake into brush border membrane vesicles was inversely related to medium osmolarity, indicating negligible binding of substrate to brush border membrane vesicles surfaces, and intravesicular spaces were similar in hibernating and active squirrels. Glucose uptake showed strong Na+ dependency in both groups, with equivalent overshoot values in the presence of Na+. Kinetic analysis revealed a significant increase in the maximal velocity of transport (Jmax) in hibernators (55.9 +/- 5.6 nmol.min-1. mg-1) compared with active squirrels (36.7 +/- 5.1 nmol.min-1. mg-1, P < 0.05), with no change in K(m). Thus, the structure and absorptive capacity of the intestinal brush border persists in fasted hibernators, and the increase in Jmax for glucose uptake during hibernation likely contributes to the enhanced Na(+)-dependent glucose absorption previously observed at the tissue level.

Evidence for a Membrane Exchangeable Glucose Pool in the Functioning of Rat Liver Glucose-6-phosphatase

We have investigated the kinetics of tracer uptake into rat liver microsomes in relation to [14C]glucose 6-phosphate (Glu-6-P) hydrolysis by glucose 6-phosphatase (Glu-6-Pase). 1) The steady-state levels of intravesicular tracer accumulated during the rapid (AMP1) and slow (AMP2) phases of uptake both demonstrate Michaelis-Menten kinetics relative to outside Glu-6-P concentrations with Km values similar to those observed for the initial burst (Vi) and steady-state (VSS) rates of Glu-6-P hydrolysis. 2) The AMP1/AMP2 ratio is constant (mean value = 0.105 +/- 0.018) over the whole range of outside Glu-6-P concentrations and is equal to the AMP1max/AMP2max ratio (0.109 +/- 0.032). 3) Linear relationships are observed between the initial rates of glucose transport during the slow uptake phase (V alpha 2) and [AMP1], and between [VSS] and [AMP2]. 4) The value of Vss max exceeds by more than 10-fold that of V alpha 2 max. 5) It is concluded that the substrate transport model is incompatible with those results and that AMP1 represents a membrane exchangeable glucose pool. 6) We propose a new version of the conformational model in which the catalytic site lies deep within a hydrophilic pocket of an intrinsic membrane protein and communicates with the extra- and intravesicular spaces through channels with different glucose permeabilities.

Calcium transport mechanism in human colonic apical membrane vesicles

Background & Aims : Recent studies have shown the role of human colon in the absorption of calcium, especially in the presence of severe disease or resection of the small bowel. The aim of the current study was to explore the mechanism(s) of calcium uptake by the purified human colonic apical membrane vesicles. Methods : Apical membrane vesicles were purified from mucosal scrapings of colons from organ donors, and 45Ca uptake was measured using a rapid filtration technique. Results : The majority of the 45Ca associated with vesicles represented uptake into closed intravesicular space, whereas the remaining 45Ca uptake represented binding to the vesicles. Ca 2+ uptake was found to be dependent on time, pH, temperature, and ionic strength of the incubation medium and inhibitable by ruthenium red, La 3+, and ethylene glycol-bis(β-amino-ethyl ether)- N,N,N′,N′-tetraacetic acid. Experiments of the effects of membrane potential generated by K +/valinomycin or anion substitutions on Ca 2+ uptake showed that the Ca 2+ uptake process was potential insensitive. Calcium uptake was unaffected by outwardly directed H +, K +, and Na + gradients. Ca 2+ uptake showed saturation kinetics with no significant differences in Michaelis constant and maximum velocity values of this transporter between proximal and distal colonic segments. Conclusions : The uptake of Ca 2+ by human colonic apical membranes involves predominantly a carrier-mediated transport mechanism.

Anion dependence of bumetanide binding and ion transport by the rabbit parotid Na(+)-K(+)-2Cl- co-transporter: evidence for an intracellular anion modifier site.

The anion dependence of [3H]bumetanide binding and 22Na+ transport by the rabbit parotid Na(+)-K(+)-2Cl- co-transporter was studied in acinar basolateral membrane vesicles (BLMVs). Cl-, Br- and NO3- have a biphasic effect on binding consistent with the presence of two anion sites associated with the bumetanide binding event, a high-affinity stimulatory site and a lower-affinity inhibitory site. We show that formate shares only the stimulatory site and SO4(2-) only the inhibitory site. The initial rate of [3H]bumetanide binding was stimulated by formate or low [Cl-] and inhibited by SO4(2-) or high [Cl-], but the rate of [3H]bumetanide dissociation was not affected by the presence of these anions in the dissociation medium. However, when [3H]bumetanide was bound to BLMVs in the presence of formate its rate of dissociation was more than four times faster than when binding took place in the presence of Cl-. These observations indicate that the binding of bumetanide and the stimulatory anion are ordered such that the anion must necessarily bind first and subsequently cannot dissociate until after bumetanide dissociates. In zero-trans-flux experiments, extravesicular SO4(2-) and formate had no effect on 22Na+ transport via the co-transporter [Turner and George (1988) J. Membr. Biol. 102, 71-77]. Thus neither of the anion sites associated with bumetanide binding is a Cl- transport site. However, we show here that SO4(2-) inhibits transport when present in the intravesicular space. Since the BLMV preparation is predominantly oriented cytosolic-side-in, this observation indicates the existence of an inhibitory cytosolic anion modifier site. Our data suggest that this site is identical to the inhibitory anion site associated with bumetanide binding.

Riboflavin uptake by rat liver basolateral membrane vesicles

The present study examined riboflavin (RF) uptake by purified rat liver basolateral membrane vesicle (BLMV). Uptake of RF was found to be Na +-and pH-independent in nature. Studies on RF uptake by BLMV as a function of incubation medium osmolarity have indicated that the uptake is the result of transport (66.5%) into the intravesicular space as well as binding (33.5%) to membrane surfaces. The process of RF uptake by BLMV was saturable as a function of substrate concentration with an apparent K m of 3.55 ± 0.70 μM and V maxof 39.89 ± 3.24 pmol/mg protein/5 s, respectively. cis-Addition of unlabeled RF and its structural analogs lumaflavin and lumichrome inhibited the uptake of [ 3H]RF while trans-addition of unlabeled RF stimulated the efflux of [ 3H]RF from preloaded vesicles. No effect on RF uptake was found by the membrane transport inhibitors probenecid, 4,4-diisothiocyanotostilbene-2,2-disulfonic acid (DIDS) and 4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). Induction of a transient positive intravesicular space led to a slight stimulation of RF uptake, while induction of a negative intravesicular space led to a slight inhibition in RF uptake. These results demonstrate the existence of a membrane-associated carrier system for RF uptake by liver BLMV. This system appears to be Na +- and pH-independent and is influenced to a certain degree by changes in transmembrane electrical potential.