The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit

The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation. Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate. At equilibrium, bound mannitol is situated at the periplasmic side of the membrane. The apparent binding constant is 40 nM for the intact membranes. Solubilization of the membranes in detergent decreases the affinity by about a factor of 2. Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities. These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain.     

Reconstitution of Escherichia coli membrane vesicles with D-amino acid dehydrogenase

When purified D-amino acid dehydrogenase [Olsiewski, P. J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 255, 4487] is incubated with right-side-out membrane vesicles from Escherichia coli, the enzyme binds to the membrane in a time- and concentration-dependent manner. As a result, the vesicles acquire the ability to oxidize D-alanine and catalyze D-alanine-dependent active transport. Similarly, incubation of D-amino acid dehydrogenase with inside-out vesicles results in binding of enzyme and D-alanine oxidase activity. Antibody inhibition studies indicate that the enzyme is bound exclusively to the inner cytoplasmic surface of the membrane in native vesicles (i.e., membrane vesicles prepared from cells induced for D-amino acid dehydrogenase). In contrast, similar studies with reconstituted vesicles demonstrate that enzyme binds to the surface exposed to the medium regardless of the orientation of the membrane. Thus, enzyme bound to right-side-out vesicles is located on the opposite side of the membrane from where it is normally found. Remarkably, in the presence of D-alanine, reconstituted right-side-out and inside-out vesicles generate electrochemical proton gradients of similar magnitude but opposite polarity, indicating that enzyme bound to either surface of the membrane is physiologically functional. The results suggest that vectorial proton translocation via the respiratory chain occurs at a point distal to the site where electrons enter the respiratory chain from the primary dehydrogenase, a conclusion that is inconsistent with the notion that the dehydrogenase forms part of a proton-translocating loop.     

Adenosine 3',5' monophosphate binds only to the inner surface of human erythrocyte membranes

The binding of adenosine 3′,5′ monophosphate (cyclic AMP) to each surface of the isolated human erythrocyte membrane was measured. Unsealed ghosts, in which both membrane faces are accessible, and sealed inside-out vesicles, which expose only the cytoplasmic side of the membrane, both bound approximately 6,000 cyclic AMP molecules per cell membrane equivalent with a dissociation constant, K ? 2.5 × 10^?9. The binding of this nucleotide by preparations rich in sealed ghosts and right-side-out vesicles, which sequester the inner surface, was limited and could be correlated precisely with small amounts of exposed cytoplasmic surface. We conclude that these binding sites for cyclic AMP are confined to the cytoplasmic side of the erythrocyte membrane.     

Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose: H+ carrier

The orientation of the lactose:H+ carrier of Escherichia coli in various preparations of native and reconstituted vesicles is determined with two impermeant, macromolecular probes: antibodies directed against the C-terminal decapeptide of the carrier and carboxypeptidase A (EC 3.4.17.1). Two methods are employed. Method I is based upon the digestion of all accessible and, therefore, presumably external, C termini of the carrier with carboxypeptidase A and detection of the remaining, internal C termini with 125I-labelled anti-(C-terminus) antibody after electrophoresis of the carrier in the presence of sodium dodecyl sulfate and transfer to nitrocellulose filters. Method II is based upon the binding of 125I-labelled anti-(C-terminus) antibody to the external C termini of the carrier in vesicles and the subsequent isolation of bound antibody by centrifugation. The labelled antibodies are calibrated using a preparation of inside-out vesicles prepared by high-pressure lysis of strain T206. The carrier content is determined by substrate binding. Because the C terminus of the carrier is known to reside on the cytoplasmic side of the membrane, these methods can also be used to determine the sidedness of various preparations of membrane vesicles. Spheroplasts are confirmed to contain carrier molecules of a single orientation, corresponding to that in right-side-out vesicles. In contrast, in purified cytoplasmic membrane vesicles and in crude membrane preparations obtained by sonication or by high-pressure lysis, 96% of the C termini are accessible to carboxypeptidase A, even after repeated sonication. This implies that nearly all carrier molecules in these preparations possess an orientation opposite to that in the cell or in right-side-out vesicles. In proteoliposomes containing carrier reconstituted or purified and reconstituted by two different methods, only 48% of the carrier molecules are oriented in the same way as in the cell. Subjecting such proteoliposomes to cycles of freezing and thawing or to sonication results in a reshuffling of carrier molecules between the inside-out and right-side-out populations while maintaining 41% in the right-side-out orientation. Digestion of the C terminus of the carrier with carboxypeptidase A does not alter either galactoside binding or countertransport. Thus carrier molecules of the inside-out orientation cannot be selectively inactivated. Additionally, an antiserum directed against the purified carrier is demonstrated to contain nearly exclusively anti-(C-terminus) antibodies, which can, in principle, be used in Method I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of enzymes involved in polyphosphoinositids metabolism on the cytoplasmic surface of the human erythrocyte membrane.

1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. Incubation of vesicles with [gamma-32P]ATP at 37 degrees C for periods of up to 1 hr did not change the topography or the permeability of the vesicles. 3. Vesicles incorporated labeled phosphate from [gamma-32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side out vesicles were not as active. 5. Impermeable right-side-out vesicles, treated with 0.01 percent saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane.     

High affinity L-triiodothyronine binding to right-side-out and inside-out vesicles from rat and human erythrocyte membrane

3,5,3'-Triiodo-L-thyronine (L-T3)-binding sites from rat and human red cells were characterized as to their distribution between the two surfaces of the membrane. Analysis of L-T3 binding to sealed right-side-out and inside-out vesicles from erythrocyte membrane revealed that high affinity L-T3-binding sites are located on the external side in rat erythrocytes and on the internal side in human red cells. These results were further confirmed by preincubation of intact red cells with p-chloromercuribenzoate, a slowly permeant reagent that interacts reversibly with SH groups of proteins. Following this treatment only the SH groups of L-T3 sites from rat erythrocytes were found to be blocked. Scatchard analysis of the binding data for rat right-side-out and human inside-out vesicles showed high affinity sites with Kd values of 0.2 x 10(-10) and 2 x 10(-10) M, respectively. The results suggest that the orientation of L-T3-binding sites in the erythrocyte membrane is species-dependent.     

Sealed inside-out and right-side-out plasma membrane vesicles : optimal conditions for formation and separation

Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of inside-out vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H⁺ pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% right-side-out vesicles, respectively, were obtained. ATPase activity and H⁺ pumping were both completely inhibited by vanadate (K_i ≈ 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli

A site-directed antibody was generated against a synthetic polypeptide corresponding to the 14 amino acid residues of the carboxyl terminus of the Tn10 TetA protein. The antibody reacted preferentially with inside-out vesicles, rather than right-side-out vesicles, prepared from Escherichia coli cells harboring transposon Tn10. When inside-out vesicles were treated with trypsin, the TetA protein was completely digested in the vicinity of the carboxyl terminus, as judged on immunoblot analysis using the antibody. In contrast, when right-side-out vesicles were treated with trypsin, the TetA protein was hardly digested. These results indicate that the carboxyl terminus of TetA is exposed to the cytoplasmic side of the membrane.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

Preparation and characterization of unilamellar myelin vesicles

Myelin vesicles have been obtained from isolated rat brain myelin and shown by electron microscopy to consist of single bilayer membranes. The yield of the preparation is approximately 25% of the myelin proteins. The vesicles show a typical myelin protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contain activity for the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase). The preparation consists of both inside-out and right-side-out vesicles, and the proportion in each orientation varies from one preparation to another. The occurrence of two populations is demonstrated by the observation that hypotonically lysed vesicles compete to a greater extent than intact vesicles in a competitive enzyme-linked immunosorbent assay with myelin basic protein antiserum. In addition, only a portion of the CNPase activity of the vesicles is trypsin-sensitive and detectable in the absence of detergent; the remaining, trypsin-insensitive activity is present in detergent-disrupted membranes. Thus, there are vesicle populations in which myelin basic protein and CNPase are accessible and others in which they are inaccessible. A population of uniformly oriented right-side-out vesicles has been obtained by ConA-Agarose affinity column chromatography and elution of the bound fraction with methyl-alpha-D-mannopyranoside. In the absence of detergent, less than 10% of the total CNPase activity of these vesicles can be demonstrated, suggesting that the active site of CNPase is opposite to that of the ConA binding site and, therefore, appears to be on the cytoplasmic face of the myelin membrane.     

Sidedness of yeast plasma membrane vesicles and mechanisms of activation of the ATPase by detergents

The binding of concanavalin A and of fluorescein 5'-isothiocyanate indicate similar amount of right-side-out and inside-out vesicles in plasma membrane vesicles from either glucose-starved or glucose-fermenting yeast cells. These vesicles contain low-activity and high-activity states of the ATPase, respectively. Unmasking of latent active sites can explain the limited ATPase activation (about 2-fold) produced by several detergents on both kinds of vesicles. On the other hand, lysophosphatidic acid (oleoyl) produces a 7-fold activation of the ATPase in vesicles from glucose-starved cells. This effect is accompanied by a change in Km of the enzyme and probably reflects a direct action of the detergent on the ATPase. A similar activation and Km change can be obtained by sonication of the vesicles, although in this case soybean phospholipids are required for maximal activity. Apparently the low-activity state of the yeast plasma membrane ATPase can be activated not only by glucose metabolism 'in vivo' (mechanism unknown) but also by some detergents and physical treatments 'in vitro'. Experiments with purified ATPase from glucose-starved cells also indicate that lysophosphatidic acid (oleoyl) specifically activates the enzyme. These results suggest a note of caution on considering the usual interpretation of the effects of detergents on membrane enzymes, which only take into account the unmasking of latent active sites.     

Sidedness of plant plasma membrane vesicles altered by conditions of preparation

Right-side-out vesicles of plasma membrane from soybean (Glycine max Merr.) were isolated by aqueous two-phase partition. Inside-out vesicles were formed when these preparations were diluted or frozen and thawed. Sidedness (orientation) was determined by preparative free-flow electrophoresis, concanavalin A binding, and ATPase latency. Under usual conditions of aqueous two-phase partition, the bulk of the vesicles were strongly reactive with concanavalin A-peroxidase and showed a high level of structure-linked latency as expected of a right-side-out (cytoplasmic-side-in) orientation. The vesicles migrated as a single electrophoretic peak. When frozen and thawed, vesicle diameters were reduced and a second population of vesicles of increased electrophoretic mobility was obtained. This second population of vesicles was weakly reactive with concanavalin A-peroxidase and showed low latency as expected of an inside-out (cytoplasmic-side-out) orientation. If the plasma membrane vesicles were diluted with water, a mixture of right-side-out and inside-out vesicles again was obtained. However, some of the cytoplasmic-side-out vesicles that were concanavalin A-unreactive and had low ATPase latency migrated more slowly as a second, less electronegative peak, upon free-flow electrophoresis. The results suggest that right-side-out and inside-out plasma membrane vesicles differ in electrophoretic mobility but that both the orientation and the absolute electrophoretic mobility of the differently oriented vesicles may be influenced by the preparative conditions.     

Symmetry properties of the Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles

The Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles can catalyze the exchange of Ca2+ on either side of the sarcolemmal membrane for Na+ on the opposing side. Little is known regarding the relative affinities of Na+ and Ca2+ for exchanger binding sites on the intra- and extracellular membrane surfaces. We have previously reported (Philipson, K.D. and Nishimoto, A.Y. (1982) J. Biol. Chem. 257, 5111-5117) a method for measuring the Na+-Ca2+ exchange of only the inside-out vesicles in a mixed population of sarcolemmal vesicles (predominantly right-side-out). We concluded that the apparent Km(Ca2+) for Na+i-dependent Ca2+ uptake was similar for inside-out and right-side-out vesicles. In the present study, we examine in detail Na+o-dependent Ca2+ efflux from both the inside-out and the total population of vesicles. To load vesicles with Ca2+ prior to measurement of Ca2+ efflux, four methods are used: 1, Na+-Ca2+ exchange; 2, passive Ca2+ diffusion; 3, ATP-dependent Ca2+ uptake; 4, exchange of Ca2+ for Na+ which has been actively transported into vesicles by the Na+ pump. The first two methods load all sarcolemmal vesicles with Ca2+, while the latter two methods selectively load inside-out vesicles with Ca2+. We are able to conclude that the dependence of Ca2+ efflux on the external Na+ concentration is similar in inside-out and right-side-out vesicles. Thus the apparent Km(Na+) values (approximately equal to 30 mM) of the Na+-Ca2+ exchanger are similar on the two surfaces of the sarcolemmal membrane. In other experiments, external Na+ inhibited the Na+i-dependent Ca2+ uptake of the total population of vesicles much more potently than that of the inside-out vesicles. Apparently Na+ can compete for the Ca2+ binding site more effectively on the external surface of right-side-out than on the external surface of inside-out vesicles. Thus, although affinities for Na+ or Ca2+ (in the absence of the other ion) appear symmetrical, the interactions between Na+ and Ca2+ at the two sides of the exchanger are not the same. The Na+-Ca2+ exchanger is not a completely symmetrical transport protein.     

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.     

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.     

Reassociation of ankyrin with band 3 in erythrocyte membranes and in lipid vesicles

The binding of human erythrocyte ankyrin (band 2.1) to the erythrocyte membrane has been characterized by reassociating purified ankyrin with ankyrin-depleted inside-out vesicles. Ankyrin reassociates at high affinity with a limited number of protease-sensitive sites located only on the cytoplasmic side of the erythrocyte membrane. Depleting the vesicles of band 4.2 does not affect their binding capacity. A 45,000-dalton polypeptide derived from the cytoplasmic portion of band 3 competitively inhibits the binding of ankyrin to inside-out vesicles. Although the bulk of band 3 molecules appear to have the potential for binding ankyrin, nly a fraction of the band 3 molecules in native membranes or in reconstituted liposomes actually provides accessible high affinity ankyrin binding sites.     

Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system

Mannitol bound to enzyme IImtl could be trapped specifically by rapid phosphorylation with P-HPr. The assay was used to demonstrate transport of mannitol across the cytoplasmic membrane with and without phosphorylation of mannitol. The latter was 2-3 orders of magnitude slower. The fraction of bound mannitol molecules that was actually phosphorylated, the efficiency of the trap, was less than 50%. The efficiency was not very different for enzyme IImtl embedded in the membrane of vesicles with an inside-out orientation or solubilized in detergent. Subsequently, it is argued that the fraction of the bound mannitol molecules that was not phosphorylated dissociated into the cytoplasmic space. A model for the catalytic mechanism of enzyme IImtl is proposed on the basis of interpretations of the present experiments. The main features of the model are the following: (i) mechanistically, the coupling between transport and phosphorylation is less than 50%; (ii) in the physiological steady state of mannitol transport and metabolism, the coupling is 100%; (iii) phosphorylated enzyme IImtl catalyzes facilitated diffusion at a high rate; (iv) the state of phosphorylation of the cytoplasmic domain modulates the activity of the translocator domain; (v) the enzyme catalyzes phosphorylation of free cytoplasmic mannitol at least as fast as it catalyzes transport plus phosphorylation of free periplasmic mannitol.     

Cation sidedness in the phosphorylation step of Na+/K+-ATPase

Na+/K+ -ATPase, reconstituted into phospholipid vesicles, has been used to study the localisation of binding sites of ligands involved in the phosphorylation reaction. Inside-out oriented Na+/K+ -ATPase molecules are the only population in this system, which can be phosphorylated, as the rightside-out oriented as well as the non-incorporated enzyme molecules are inhibited by ouabain. In addition, the right-side-out oriented Na+/K+ -ATPase molecules have their ATP binding site intravesicularly and are thus not accessible to substrate added to the extravesicular medium. Functional binding sites for the following ligands have been demonstrated: (i) Potassium, acting at the extracellular side with high affinity (stimulating the dephosphorylation rate of the E2P conformation) and low affinity (inducing the non-phosphorylating E2K complex). (ii) Potassium, acting at the cytoplasmic side with both high and low affinity. The latter sites are also responsible for the formation of an E2K complex and complete with Na+ for its binding sites. (iii) Sodium at the cytoplasmic side responsible for stimulation of the phosphorylation reaction. (iv) Sodium (and amine buffers) at the extracellular side enhancing the phosphorylation level of Na+/K+ -ATPase where choline chloride has no effect. (v) Magnesium at the cytoplasmic side, stimulating the phosphorylation reaction and inhibiting it above optimal concentrations.     

The orientation of insulin receptors in the red cell membrane

High-affinity binding of insulin to receptors in human erythrocyte membranes occurred at the external surface, but not at the cytoplasmic surface of the plasma membrane, as assessed by insulin binding to right-side-out and inside-out membrane vesicles. Even after prolonged (3 h) incubation at 22°C, binding at the cytoplasmic membrane aspect remained negligible. The data indicate that the insulin receptor displays its hormone-binding site exclusively toward the extracellular space and that transmembrane mobility (“flip-flop”) of the receptor from one to the other membrane leaflet is severely restricted.