Inside-out Membrane Vesicles Research Articles

Reconstitution of periplasmic transport in inside-out membrane vesicles: Energization by ATP

Abstract The periplasmic histidine permease of Salmonella typhimurium has been reconstituted in inside-out vesicles (IOV) of Escherichia coli by disrupting the cells with a French press in the presence of a high concentration of the periplasmic histidine-binding protein, HisJ. Efflux from IOV, which is equivalent to uptake in whole cells, is induced by ATP. The reconstituted system depends on the presence of the membrane-bound permease proteins, HisQ, HisM, and HisP, and does not function if reconstitution is performed in the presence of a mutant HisJ protein, HisJ5625, that can bind histidine normally but can't interact properly with the membrane complex. Efflux is not induced by the nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate, supporting the contention that ATP hydrolysis is necessary. 8-Azido ATP inactivates IOV, indicating that the ATP effect occurs through the HisP protein, which has previously been shown to be modified by 8-azido ATP (Hobson, A., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 733-7337). The estimated Km of the vesicles for ATP is about 200 microM. Vanadate, an inhibitor of phosphohydrolase enzymes, inhibits ATP-induced efflux. We conclude that ATP is likely to be the proximal energy source for periplasmic permeases.

The effect of [formula omitted], [formula omitted] and [formula omitted], [formula omitted] cellular aging on the active calcium transport in human inside-out red cell membrane vesicles

Modelling of the in , vivo and in , vitro aging processes in the human red cell has stressed the following features of the active calcium uptake by inside-out vesicles: 1) it is higher in the outdated, in , vitro aged, than in the fresh red cell (p<0.0005), and in the densest, in , vivo aged fraction than in the lightest, young fraction (p=0.08); 2) it increases following stimulation by excess calmodulin to values that are not significantly different; 3) it decreases to the same value in the absence of endogenous calmodulin and inhibitor, with and without exogenous calmodulin; 4) it is the target of a non-competitive inhibition, that is stronger in the fresh than in the outdated red cell. We conclude that the aging process does not involve neither membrane Ca-ATPase nor calmodulin, but rather the interaction of the calcium pump with the inhibitor of Ca-ATPase.

Proton efflux from rat intestinal basal-lateral membrane vesicles is stimlated by ATP and NA +

1. . ATP-stimulated 22Na uptake and 14C-methylamine efflux were studied in inside-out rat intestinal basal-lateral membrane vesicles (BLMV). 2. 2. Uptake of 22Na by basal-lateral membrane vesicles was stimulated by addition of ATP and by an acidic vesicle interior. 3. 3. Efflux of 14 C-methylamine was stimulated by ATP and Na + 4. 4. 14C-methylamine efflux was not influenced by vanadate or amiloride by themselves but was inhibited by the presence of both agents. 5. 5. These data are consistent with a basal-lateral proton translocation mechanism which may be responsible for alkalinization of the lateral intercellular space and implicates the Na +-pump in this mechanism.

Inhibition of Na,K-ATPase and sodium pump by anticancer ether lipids and protein kinase C inhibitors ET-18-OCH 3 and BM 41.440

The anticancer ether lipid analogs ET-18-OCH3 and BM 41.440 inhibited Na, K-ATPase in the purified rat brain membrane fragments, with a potency comparable to that of their inhibition of protein kinase C. They also inhibited Na,K-ATPase in the crude membrane fraction of HL60 cells. Kinetic analysis indicated that the lipids had a mode of action different from that of ouabain, a classic inhibitor of the ATPase. The lipids also blocked 22Na uptake in the inside-out membrane vesicles of human erythrocytes. It is suggested that Na,K-ATPase might represent an additional site with which certain protein kinase C inhibitors can interact to alter cellular activities.

Protons as substitutes for sodium and potassium in the sodium pump reaction.

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.

Effects of neomycin on K+ transport into inside-out erythrocyte membrane vesicles

Effects of neomycin on K+ transport into inside-out erythrocyte membrane vesicles

Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein.

An NAD:cysteine ADP-ribosyltransferase designated ADP-ribosyltransferase C was purified approximately 35,000-fold from human erythrocytes with an 11% yield. The purified ADP-ribosyltransferase C exhibited one predominant protein band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight (Mr) of 28,500. The Km values for NAD and cysteine methyl ester were determined to be 65 and 4,400 microM, respectively. By using human erythrocyte inside-out membrane vesicles, the transferase C was found to ADP-ribosylate the alpha subunit (Mr = 41,000) of Gi, which is a substrate for pertussis toxin. The ADP-ribosylation of Gi alpha catalyzed by ADP-ribosyltransferase C was inhibited by pre-ADP-ribosylation with pertussis toxin. The linkage of ADP-ribose-Gi alpha in the membranes formed by ADP-ribosyltransferase C was as stable to hydroxylamine as that formed by pertussis toxin. These data represent the first demonstration that eukaryotic cells contain an ADP-ribosyltransferase which can catalyze the ADP-ribosylation of a cysteine residue in Gi alpha.

Inside-out plant plasma membrane vesicles of high purity obtained by aqueous two-phase partitioning

Highly purified plasma membranes obtained from leaves of sugar beet ( Beta vulgaris L.) by aqueous two-phase partitioning were separated into two fractions by further phase partition steps. The vesicles partitioning to the interface showed an ATP-dependent H +-uptake (measured using the pH probe acridine orange) and a negligible K +,Mg 2+-ATPase latency, while the vesicles partitioning in the upper phase showed only slow H +-uptake and a high ATPase latency on addition of Triton X-100. Based on these results the material at the interface is estimated to contain ∼90% sealed, inside-out vesicles, and the material in the upper phase ∼90% sealed, right-side-out vesicles

Interactions of spectrin in hereditary elliptocytes containing truncated spectrin beta-chains.

An abnormal spectrin, in which one subunit is truncated, has been detected in a large German family. The inheritance is autosomal dominant. The affected members of the family suffer in widely varying degree from a microcytic hemolytic anemia. The red cell morphology varies correspondingly from smooth elliptocytes to predominantly poikilocytes. The abnormal spectrin makes up approximately 30% of the total and is almost entirely present as the dimer. The truncated chain is not phosphorylated by the endogenous cAMP-independent kinase, and it has been identified as a chain of beta-type, using monoclonal antibodies. Because a univalent terminal spectrin alpha-chain fragment will bind to normal dimers with an association constant lower by only a factor of two than that for the self-association of the dimers, it would be expected that the mutant dimers (alpha beta') would readily enter into an association with normal (alpha beta) dimers to give alpha 2 beta beta' tetramers (though not with each other). In dilute solution this is indeed observed, and the diminution in tetramer concentration when 30% of normal spectrin is replaced by alpha beta' dimers, amounts to only a small proportion. Moreover, in the membrane skeleton, if there is pairwise apposition of dimer units, only 9% of pairings will be between units that cannot associate. We have shown that the failure of alpha beta' dimers to enter into heterologous associations in situ is not due to the elimination of the ankyrin binding site near the truncated end of the beta-chain: this site is fully functional, as judged by rebinding to spectrin-depleted vesicles. When the spectrin is extracted from the membrane in the cold, the material released initially consists almost entirely of alpha beta' dimers; when the spectrin of normal membranes is partly dissociated to dimers in situ by warming at low ionic strength, extraction in the cold then leads similarly to much more rapid release of the dimer than of the tetramer. The similar rates of liberation of normal and abnormal dimer make it unlikely that the interaction of the latter with the membrane is in any way defective. When mixtures of alpha beta and alpha beta' dimers are bound to spectrin-depleted inside-out membrane vesicles from normal cells and tetramers are allowed to form by equilibration at 30 degrees C, the proportion of the abnormal species appearing in the tetramer is much lower than would be expected on a statistical basis. The relation of the self-association equilibrium on the membrane to that of spectrin in dilute solution is analyzed.

NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.

Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.

Procalpain is activated on the plasma membrane and the calpain acts on the membrane

The mechanism of activation of human erythrocyte calpain was investigated using the immunoblotting technique with anticalpain monoclonal antibody. The purified calpain underwent a Ca 2+-induced fragmentation of the 80 kDa subunit to 76 kDa and 36 kDa fragments. The behavior of the 76 kDa fragment in electrophoresis corresponded to the proteinase activity of calpain, whereas the behavior of the 80 kDa subunit and the 36 kDa fragment did not. When inside-out membrane vesicles were added to the reaction mixture of calpain and Ca 2+ and the vesicles were separated from the supernatant solution by centrifugation, the 80 kDa subunit and 76 kDa fragment were found in the vesicle fraction. No other fragments were found in this fraction. On the other hand, the 80 kDa subunit and 36 kDa fragment were found in the supernatant fraction. When right-side-out membrane vesicles were added to the reaction mixture and the vesicles were separated from the supernatant fraction, no fragment was found in the vesicle fraction, while only the 36 kDa fragment was found in the supernatant fraction. These results indicate that the 80 kDa subunit of procalpain was bound in a Ca 2+-dependent manner to the cytosolic surface of the plasma membrane and then underwent fragmentation to produce the 76 kDa fragment (active form) and that it expressed its proteinase activity at the surface of the membrane.

Effect of lysophosphatidylcholine on K + transport in rat heavy gastric membranes enriched with (H +-K +)-ATPase

K +-and ATP-dependent H +-accumulation in rat heavy gastric membrane vesicles enriched with (H +-K +)-ATPase was markedly stimulated by amphiphiles like lysophosphatidylcholine and Zwittergent 3–14 at concentrations of 10 −5 M. Their stimulatory effect was dependent on K +-concentration in the medium and was abolished by SCH 28,080, a specific inhibitor of (H +-K +)-ATPase. Lysophosphatidylcholine at the optimal dose (3 × 10 −5 M) showed dual effects on K +-dependent membrane functions; it stimulated the rate of K +-uptake by nearly 60%, but partially inhibited SCH 28,080-sensitive and K +-dependent ATP-hydrolysis (about 20% reduction). These data indicate that H +-pumping through (H +-K +)-ATPase in the inside-out gastric membrane vesicles was facilitated by the stimulatory effect of lysophosphatidylcholine on membrane K +-transport in spite of its partial inhibition of ATP-hydrolysis. It appears that the rate limitingstep for operation of the ATPase is the availability of K + ions in the luminal side of the pump. We propose that ionic amphiphiles may modulate K +-transport in rat heavy gastric membranes through specific interactions with the putative K +-transporter

Conformational changes of the in situ red cell membrane calcium pump affect its proteolysis

In inside-out red cell membrane vesicles trypsin digestion reduces the molecular mass of the 32P-labeled acyl-phosphate intermediate of the calcium pump from the original 140 kDa to about 80 kDa with a simultaneous activation of the calcium uptake. This process is slightly stimulated by the presence of calcium, as compared to EGTA, or EGTA + vanadate, but the proteolytic pattern is similar under all these conditions. However, trypsin degradation of the 80 kDa polypeptide, resulting in the loss of calcium transport activity and 32P-phosphoenzyme formation, is rapid in the presence of calcium, inhibited by EGTA and almost fully blocked by EGTA + vanadate. In the presence of these latter ligands, probably locking the calcium pump in an E 2 conformation, the 80 kDa protein becomes insensitive even to excessive digestion by the non-specific protease, pronase. The data indicate major changes in the molecular arrangement of the calcium pump protein when transformed from a calcium-liganded (E 1) to an E 2 conformation.

The maximal velocity and the calcium affinity of the red cell calcium pump may be regulated independently.

The kinetics of active Ca2+ transport in inside-out red cell membrane vesicles and the Ca2+-ATPase activity of the purified Ca2+ pump were studied and the effects of calmodulin, acidic phospholipids, and controlled trypsinization were compared. In the presence of calmodulin the maximal rate and the apparent affinity of the pump for Ca2+ were greatly increased in both preparations. The lowest value of Km(Ca) was between 0.5 and 0.7 microM depending on the concentration of calmodulin and on the enzyme preparation. Positive cooperativity for Ca2+ activation with a Hill coefficient of 1.6-1.7 was observed in all cases. When acidic phospholipids (phosphatidylinositol 4-phosphate was routinely used) were added to the inside-out vesicles or to the purified enzyme, maximal transport rates equal to those obtained with calmodulin were measured but the Km(Ca) decreased to 0.25 microM and the positive cooperativity disappeared (the Hill coefficient approached 1). Highly active, calmodulin-independent proteolytic fragments of molecular mass of 81 and 76 kDa were produced with controlled trypsinization. When the trypsin treatment was directed to obtain primarily the 81-kDa fragment, the preparation showed characteristics similar to those of the intact Ca2+ pump in the presence of calmodulin; that is, the same Vmax was obtained, the Km(Ca2+) was 0.5-0.6 microM, and the Hill coefficient was about 1.6. Addition of phosphatidylinositol 4-phosphate or allowing further proteolysis to produce the 76-kDa fragment, shifted the Km(Ca) to 0.25 and reduced the Hill coefficient to 1, without changes in the maximal rate. Based on these results it is suggested that the maximal velocity and the Ca2+ affinity on the erythrocyte Ca2+ pump may be regulated independently and that independent polypeptide regions of the enzyme are involved in the regulations.

Synaptosomes and synaptosome membrane vesicles from the brain of Mamestra configurata: Application to voltage-dependent and ATP-dependent Ca 2+ ion transport studies

High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded 10 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulum and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy. Voltage-dependent Ca 2+ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive 45Ca 2+ from the incubation medium. The rate of uptake was increased up to three-fold when the synaptosomes were incubated in a depolarizing, high [K +] medium. Time course studies indicated that voltage-dependent Ca 2+ uptake was composed of an early (<2 sec) fast phase and a late (>10 sec) slow phase. ATP-dependent Ca 2+ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported 45Ca 2+ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated 45Ca 2+ from the vesicles. The maximal rate of ATP-dependent Ca 2+ transport occurred at a [Ca 2+ free] of 0.1 to 0.2 nM, indicating that the transport process has a very high affinity for Ca 2+ ions.

Immunological evidence for two types of PQQ-dependent d-glucose dehydrogenase in bacterial membranes and the location of the enzyme inEscherichia coli

Abstract Using an antibody raised against d -glucose dehydrogenase (EC 1.1.99.17) purified from Pseudomonas fluorescens, immuno-cross-reactivity with the enzymes from several bacterial strains and localization of the enzyme in Escherichia coli were examined. The antibody cross-reacted with glucose dehydrogenases from various Gram-negative bacteria examined. As a result, it became apparent that the enzymes from Gluconobacter, Acetobacter, Pseudomonas and Acinetobacter, which existed as holoenzymes in the membranes, had lower molecular weights than those from E. coli and Klebsiella, which were apoenzymes. Treatment with trypsin of right-side out and inside-out membrane vesicles from E. coli clearly demonstrated that d -glucose dehydrogenase was located on the outer surface of the cytoplasmic membrane of E. coli, as had been suggested for Pseudomonas .

Effect of a high NaCl diet on the active mechanisms of Na+, extrusion in rat kidney

Healthy male rats were exposed to a chronic ingesta of an isotonic NaCl solution during a period of four months. Two different preparations were utilized to study the effect of this treatment on the active mechanisms of Na+ reabsorption of kidney proximal tubular cells: outermost kidney cortex slices (rich in proximal tubules) and inside-out basolateral plasma membrane vesicles prepared from the same tissue. It was found that the activity of the ouabain-insensitive, Na-pump of basolateral plasma membranes of kidney proximal tubular cells was increased in about 70%, whereas the ouabain-sensitive, Na,K-pump activity did not change with the experimental treatment. These results represent a strong support to the two Na-pump's hypothesis since the treatment affected the Na-pump differentially without affecting the Na,K-pump.

Inhibition of A Chloride Pump By Acetazolamide In The Intestine of Aplysia Californica

ABSTRACT The isolated intestine of Aplysia californica, bathed in a substrate-free NaCl seawater bathing medium, generates a spontaneous transepithelial potential difference such that the serosal surface is negative relative to the mucosal surface (Gerencser, 1978b). The short-circuit current (Isc) is accounted for by active absorptive mechanisms for both Na+ and Cl−, the Cl− transport mechanism being more vigorous than that for Na+ (Gerencser, 1978a). However, Cl− transport appeared to be independent of Na+ transport, for the Isc measured in an Na+-free seawater bathing medium was shown to be identical to a net active absorptive flux of Cl− (Gerencser, 1984a). It was hypothesized that active Cl− absorption in Aplysia enterocytes was mediated by a primary active transport process, because it had been demonstrated that the intracellular Cl− electrochemical potential was less than that measured in the extracellular medium (Gerencser &amp; White, 1980), even in the absence of extracellular Na+ (Gerencser, 1983). Lending strength to this hypothesis, Gerencser &amp; Lee (1983, 1985a) demonstrated the existence of a CU-stimulated ATPase activity in Aplysia enterocyte plasma membranes, suggesting a cause-and-effect relationship between ATPase activity and Cl− transport. The ATPase activity stimulated by Cl− was strongly inhibited by acetazolamide. In addition, Gerencser (1984b) and Gerencser &amp; Lee (1985b) have demonstrated an ATP-dependent Cl− uptake in Aplysia inside-out enterocyte plasma membrane vesicles (EPMV). Therefore, the present study was undertaken to assess the effect of acetazolamide on the ATP-driven Cl− uptake mechanism in EPMV.. Seahares (Aplysia californica) were obtained from Marinus Inc. (Westchester, CA) and were maintained at 25 °C in circulating filtered seawater. Adult Aplysia (600-1000 g) were used in these experiments. The plasma membrane vesicles were prepared from Aplysia intestinal enterocytes by homogenization and differential and discontinuous sucrose density-gradient centrifugation techniques as described previously (Gerencser &amp; Lee, 1985a). Vesicle transport experiments were also performed as previously described (Gerencser &amp; Lee, 1985b).

Somatostatin inhibits fusion of pituitary secretion vesicles with the plasma membranes

Somatostatin has been found to inhibit secretion vesicle fusion with the iodinated (125-I) inside-out plasma membrane vesicles (both were isolated from the anterior pituitaries). This effect of somatostatin was specific and dose-dependent (half-maximal effect at 10(-9) M). Calmodulin (10 microM), but not cyclic AMP, enhanced the fusion process between the two organelles and somatostatin inhibited calmodulin-stimulated fusion. These observations suggest that one facet of somatostatin action on hormone secretion may be its inhibition of secretion vesicle fusion with the plasma membrane.