The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane

The mannitol permease (EII(Mtl)) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EII(Mtl) contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EII(Mtl), all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EII(Mtl) undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path.     

The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.     

The glucose-specific carrier of the Escherichia coli phosphotransferase system.

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS). 2',3'-Epoxypropyl beta-d-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates. They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter. 3a is the only analogue that inhibits EIIGlc by binding to the high-affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7-anhydro-d,l-glycero-alpha-d-gluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl alpha-d-glucopyranoside (1a) and methyl alpha-d-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-alpha-d-glucopyranoside (1e), beta-d-glucopyranosyl isothiocyanate (3c) and beta-d-glucopyranosyl phenyl isothiocyanate (3d). Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter. Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.     

Orientation of ferrochelatase in bovine liver mitochondria

The orientation of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, was examined in bovine liver mitochondria. The ability of a membrane-impermeable sulfhydryl reagent, 4,4'-dimaleimidylstilbene-2,2'-disulfonic acid, to inactivate ferrochelatase in intact or disrupted mitochondria and mitoplasts was examined. Using succinate dehydrogenase as an internal marker, it was found that ferrochelatase was inactivated only in disrupted mitochondria and mitoplasts, suggesting an internal location for the active site of the enzyme. In addition, antibodies raised against purified ferrochelatase were found to inhibit activity only in disrupted but not in intact mitoplasts. These data demonstrate that in bovine liver mitochondria ferrochelatase is located on the matrix side of the inner mitochondrial membrane. Data obtained with the membrane-impermeable amino reagent isethionyl acetimidate indicate that ferrochelatase physically spans the inner mitochondrial membrane with portions of the protein exposed on both sides of the membrane.     

Location of the catalytic site of the respiratory fumarate reductase of Escherichia coli

The location of the catalytic site of the membrane-bound respiratory fumarate reductase of Escherichia coli was investigated using mutants and inhibitors of dicarboxylic acid transport. Comparison of apparent Km and Vmax values for fumarate in intact cells and in inverted membrane vesicles showed that externally added fumarate was required to be transported across the cytoplasmic membrane prior to reduction. The catalytic site of fumarate reductase must therefore be located on the cytoplasmic face of the membrane.     

The importance of carboxyl groups on the lumenal side of the membrane for the function of the Ca(2+)-ATPase of sarcoplasmic reticulum

The conventional model for transport of Ca(2+) by the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) involves a pair of binding sites for Ca(2+) that change upon phosphorylation of the ATPase from being high affinity and exposed to the cytoplasm to being low affinity and exposed to the lumen. However, a number of recent experiments suggest that in fact transport involves two separate pairs of binding sites for Ca(2+), one pair exposed to the cytoplasmic side and the other pair exposed to the lumenal side. Here we show that the carbodiimide 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC) is membrane-impermeable, and we use EDC to distinguish between cytoplasmic and lumenal sites of reaction. Modification of the Ca(2+)-ATPase in sealed SR vesicles with EDC leads to loss of ATPase activity without modification of the pair of high affinity Ca(2+)-binding sites. Modification of the purified ATPase in unsealed membrane fragments was faster than modification in SR vesicles, suggesting the presence of more quickly reacting lumenal sites. This was confirmed in experiments measuring EDC modification of the ATPase reconstituted randomly into sealed lipid vesicles. Modification of sites on the lumenal face of the ATPase led to loss of the Ca(2+)-induced increase in phosphorylation by P(i). It is concluded that carboxyl groups on the lumenal side of the ATPase are involved in Ca(2+) binding to the lumenal side of the ATPase and that modification of these sites leads to loss of ATPase activity. The presence of MgATP or MgADP leads to faster inhibition of the ATPase by EDC in unsealed membrane fragments than in sealed vesicles, suggesting that binding of MgATP or MgADP to the ATPase leads to a conformational change on the lumenal side of the membrane.     

Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex.

Incubation of [3H]palmitic acid, ATP, and CoA with inside-out membrane vesicles prepared from human or other mammalian red cells resulted in nearly exclusive 3H-palmitoylation of the Mr = 32,000 Rh polypeptides. [3H]Palmitic, [3H]myristic, and [3H]oleic acids were comparably esterified onto Rh polypeptides in inside-out membrane vesicles in the presence of ATP and CoA, although [3H]palmitic acid was preferentially incorporated by intact human red cells. Experiments using sulfhydryl reagents or tryptic digestions suggested that multiple sulfhydryl groups on the Rh polypeptides located near the cytoplasmic leaflet of the lipid bilayer were 3H-palmitoylated; the exofacial sulfhydryl group essential for Rh antigenic reactivity was not 3H-palmitoylated. Transfer of fatty acid from [14C]palmitoyl-CoA to sites on the Rh polypeptides occurred even after previous incubation of inside-out membrane vesicles at 95 degrees C or after solubilization of inside-out membrane vesicles in Triton X-100. Hydrodynamic analyses of Triton X-100-solubilized membranes surprisingly demonstrated that 3H-palmitoylated Rh polypeptides behaved as a protein of apparent Mr = 170,000. These in vitro studies suggest that palmitoylation of Rh polypeptides occurs within a macromolecular complex by a highly selective but possibly nonenzymatic mechanism.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2.

The melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of melibiose. Starting with the cysteine-less melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis. We suggest that these residues line the path of melibiose and its associated cation through the carrier.     

Topology of the anion exchange protein AE1: the controversial sidedness of lysine 743

The topology of the band 3 (AE1) polypeptide of the erythrocyte membrane is not fully established despite extensive study. Residues near lysine 743 (K743) have been reported to be extracellular in some studies and cytoplasmic in others. In the work presented here, we have attempted to establish the sidedness of K743 using in situ proteolysis. Trypsin, papain, and proteinase K do not cleave band 3 at or near K743 in intact red cells, even under conditions that cause cleavage on the C-terminal side of the glycosylation site (N642) in extracellular loop 4. In contrast, trypsin sealed inside red cell ghosts cleaves at K743, as does trypsin treatment of inside-out vesicles (IOVs). The transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H(2)DIDS), acting from the extracellular side, blocks trypsin cleavage at K743 in unsealed membranes by inducing a protease-resistant conformation. H(2)DIDS added to IOVs does not prevent cleavage at K743; therefore, trypsin cleavage at K743 in IOVs is not a consequence of cleavage of right-side-out or leaky vesicles. Finally, microsomes were prepared from HEK293 cells expressing the membrane domain of AE1 lacking the normal glycosylation site. This polypeptide does not traffic to the surface membrane; trypsin treatment of microsomes containing this polypeptide produces the 20 kDa fragment, providing further evidence that K743 is exposed at the cytoplasmic surface. Therefore, the actions of trypsin on intact cells, resealed ghosts, unsealed ghosts, inside-out vesicles, and microsomes from HEK293 cells all indicate that K743 is cytoplasmic and not extracellular.     

Comparative inhibition studies of the phosphotransferase and glycerophosphate acylation systems in membrane vesicles of Escherichia coli

Purified membrane vesicles were treated with various reagents specific for different amino acid side-chains. Titration of sulfhydryl groups with specific reagents shows that the sulfhydryl content of membrane vesicles as estimated directly is similar to that found by treating spheroplasts or cells and then isolating the membrane vesicles. The blocking of sulfhydryl groups specifically inhibits the α-methylglucoside transport system (phosphotransferase system), whereas the glycerophosphate acylation system is not affected. The kinetics of inhibition of the first system show that a high reactivity of the sulfhydryl groups is involved. Inhibition of the acyltransferase activity by sulfhydryl reagents occurs only on partial denaturation of the membranes induced by mild sonication, heat or toluene treatment. The Inhibition is at the level of the glycerol 3-phosphate:acyl thioester acyltransferase.The effects of sonication and/or sulfhydryl reagents were measured by sulfhydryl titration, by assays of NADH oxidase and d-lactate dehydrogenase activities, as well as by 1-anilino-8-naphthalene sulfonate binding. The results support the hypothesis that the acyltransferase system is embedded within the membrane and that the readily accessible permease system is closer to (or at) the surface of the membrane.     

Spheroplast-derived membrane vesicles from Rhodobacter capsulatus cells catalyzing nucleotide transport

Rodobacter capsulatus cells, which were cultured anaerobically in high light intensity, had fewer foldings in the cytoplasmic membrane than those which were grown in lower light intensities. Spheroplast-derived membrane fractions obtained from cells cultured under high light intensity contained a high yield of large right-side-out membrane vesicles. The right-side-out vesicles catalyzed reversible light-induced proton efflux as did intact cells. Nucleotide transport activity was also catalyzed by these membrane vesicles. This activity was indirectly monitored by measurement of photophosphorylation or hydrolysis of externally added diphospho- and triphosphonucleosides. These enzymatic activities occur inside the cytoplasmic membrane of spheroplasts and membrane vesicles and therefore require the transport of the externally added reagents. The indirect measurements of transport were complemented by the demonstration of direct uptake of radiolabeled nucleotides into the membrane vesicles. These data support the suggestion that a nucleotide transporter located in the cytoplasmic membrane of R. capsulatus bacteria mediates these activities.     

Passive membrane permeability to small molecules and ions in transformed mammalian cells: probable role of surface phosphorylation

Addition of ATP to medium surrounding intact, transformed 3T3 cells causes the formation of aqueous channels in the plasma membrane. This effect of extracellular ATP is sharply dependent on the pH and temperature of the incubation medium, and is inhibited by low levels of La3+ or ruthenium red; inhibition is also obtained with concentrations of Mg2+ ions that exceed a ratio of Mg/ATP of one. The effect of ATP on membrane channel formation is unaffected by chelators of metal ions or by prior modification of the cell surface with various surface-active enzymes or sulfhydryl reagents. Under conditions which favor aqueous channel formation, incubation of intact 3T6 cells with ATP (gamma-32P) leads to phosphorylation of two membrane components with apparent molecular weight of 40,000 (40K) and 110,000 (110K) daltons; the 110K component which is unaffected by trypsin under normal conditions is rendered trypsin-sensitive by the phosphorylation reaction, probably as a result of a conformational change. Conditions which inhibit aqueous channel formation also inhibit phosphorylation of the 110K protein and decrease the labeling of the 40K component. These results indicate the probable role of cell surface phosphorylation, involving one or both of these components, in the formation of aqueous channels in transformed 3T3 cells. Aqueous channel formation by extracellular ATP is not associated with gross unfolding of the cell surface as revealed by lactoperoxidase-catalyzed iodination of the 3T6 cell surface.     

A method to investigate protein association with intact sealed mycobacterial membrane vesicles

In mycobacteria, probing the association of cytoplasmic proteins with the membrane itself, as well as with integral or peripheral membrane proteins, is limited by the difficulty in extracting intact sealed membrane vesicles due to the complex cell wall structure. Here we tested the association of Mycobacterium tuberculosis SecA1 and SecA2 proteins with intact membrane vesicles by a flotation assay using iodixanol density gradients. These protocols have wide applications for studying the association of other mycobacterial cytoplasmic proteins with the membrane and membrane-associated proteins.     

Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands

The topology of ceramide glucosyltransferase and de novo synthesized glucosylceramide was studied in sealed and 'right-side-out' vesicles of porcine submaxillary glands derived from Golgi apparatus. Pronase treatment which did not cause any breakdown of the luminal glycoprotein galactosyltransferase activity, inhibited the ceramide glucosyltransferase to more than 50% at a ratio proteinase to Golgi protein 1:100. Trypsin at the same concentration, while producing no inactivation of luminal galactosyltransferase, caused a complete loss of ceramide glucosyltransferase activity. The membrane-impermeable compound, DIDS, which did not cause any inhibition of the galactosyltransferase, inhibited the ceramide glucosyltransferase (70% reduction at 80 microM DIDS). Thus, the enzyme ceramide glucosyltransferase is accessible from the cytoplasmic side of the Golgi vesicles. The orientation of the newly synthesized glucosylceramide is studied by the ability of the enzyme glucosylceramidase to hydrolyse this compound both on intact and on disrupted vesicles. The same percentage (respectively, 36 and 30%) of hydrolysis was obtained during an incubation of 3 h, showing that glucosylceramide is not at all protected from external hydrolysis. Pronase-treated vesicles revealed an increase in glucosylceramidase hydrolysis (up to 45%), which indicates that glucosylceramide that glucosylceramide may be cryptic. All these results indicate that the ceramide glucosyltransferase, as well as related glucosylceramide, are cytoplasmically oriented in Golgi vesicles from porcine submaxillary glands.     

Organization of mitochondrial UDP-glucose: Dolichylmonophosphate glucosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.     

Inhibition of Na+-dependent phosphate transport by group-specific covalent reagents in rat kidney brush border membrane vesicles. Evidence for the involvement of tyrosine and sulfhydryl groups on the interior of the membrane

The effects of tyrosine- and sulfhydryl-specific reagents on the Na+-dependent transport of phosphate in brush border membrane vesicles prepared from rat renal cortex were investigated. This study is the first to show that the tyrosine-specific reagents 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane inactivate the transporter in a concentration- and time-dependent fashion while the membrane impermeant tyrosine reagent, N-acetylimidazole, has no effect on phosphate uptake. The membrane permeant sulfhydryl reagent N-ethylmaleimide also caused a time- and concentration-dependent inactivation of this transport process but the membrane impermeant reagents 7-chloro-4-sulfobenzo-2-oxa-1,3-diazole and eosin-5-maleimide had little effect on phosphate uptake. The inhibitory effects of both tyrosine- and sulfhydryl-specific reagents were additive, but no protection from inactivation by tyrosine-specific reagents could be achieved by preincubation of the vesicles with the substrates of the transporter or with competitive inhibitors of the transport process. These results suggest that the amino acids modified by these agents are located either within the membrane or on the cytosolic surface of the transporter. These residues may not participate in substrate binding, but may be important for the conformational change of the transporter necessary for the translocation of phosphate across these membranes. This study also shows that Na+-dependent phosphate transport can be inactivated by other reagents which covalently modify histidine, carboxyl, and amino groups on proteins.     

An essential role for vicinal dithiol groups in the catalytic activity of the human placental Na(+)-H+ exchanger

We examined the effects of phenylarsine oxide, a reagent specific for vicinal dithiol groups, on the catalytic activities, Na+ influx and H+ efflux, of the human placental Na(+)-H+ exchanger. Treatment of the placental brush-border membrane vesicles with the reagent markedly inhibited both the activities. The inhibition was partially reversible by dithiols. The effect of phenylarsine oxide was to reduce the maximal velocity of the exchanger without influencing its affinity for Na+. The exchanger was partially protected from this inhibition by amiloride but not by cimetidine even though both these compounds interacted with the Na(+)-binding site. The data demonstrate that vicinal dithiol groups are essential for the catalytic function of the placental Na(+)-H+ exchanger and that the critical dithiol groups are located at a site distinct from the Na(+)-binding site.     

Localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes

The location of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in human erythrocyte membranes was determined. This was accomplished by comparing the enzyme's accessibility with that of glyceraldehyde-3-phosphate dehydrogenase (cytoplasmic surface marker) and acetylcholinesterase (external marker) in sealed and unsealed ghosts and normal and inverted membrane vesicles. The results showed that 2′,3′-cyclic nucleotide 3′-phosphodiesterase, like glyceraldehyde-3-phosphate dehydrogenase, meets several criteria for an inner (cytoplasmic) membrane location: (1) the enzyme was accessible to substrate in unsealed ghosts and inside-out vesicles but not in sealed or right-side-out vesicles, (2) latent activity in sealed ghosts could be exposed with detergent (Triton X-100), (3) activity in unsealed ghosts was gradually sequestered during resealing and could be re-exposed with detergent, and (4) the enzyme was susceptible to trypsin proteolysis only in unsealed ghosts. These results demonstrate that the active site of 2′,3′-cyclic nucleotide 3′-phosphodiesterase faces the cytoplasm of erythrocytes and that the enzyme may not span the lipid bilayer of the membrane. The localization of the phosphodiesterase on the inner membrane surface of erythrocytes suggests that the similar enzyme of myelin may be embedded within the major dense line of the compact lamellae.