Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases

Purified preparations of D-amino acid dehydrogenase [Olsiewski, P.J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 225, 4487] and D-lactate dehydrogenase [Kohn, L.D., & Kaback, H.R. (1973) J. Biol. Chem. 248, 7012] bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition. The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport. The enzymes do not share common sites of association on either face of the E. coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors. Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation. The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.     

Monoclonal antibodies against the lac carrier protein from Escherichia coli. 2. Binding studies with membrane vesicles and proteoliposomes reconstituted with purified lac carrier protein

Monoclonal antibodies 4B1 and 5F7 bind to distinct, nonoverlapping epitopes in the lac carrier protein. By use of immunofluorescence microscopy and radiolabeled monoclonal antibodies and Fab fragments, it is shown that both 4B1 and 5F7 bind to spheroplasts and to right-side-out vesicles, but only to a small extent to inside-out vesicles. Clearly, therefore, the lac carrier protein has an asymmetric orientation within the cytoplasmic membrane of Escherichia coli, and both epitopes are located on the periplasmic surface. In right-side-out vesicles, radiolabeled 4B1 binds with a stoichiometry of 1 mol of antibody per 2 mol of lac carrier protein, while radiolabeled 4B1 Fab fragments bind 1:1. Importantly, the intact antibody and its Fab fragments bind to proteoliposomes reconstituted with purified lac carrier protein with a stoichiometry very similar to that observed in right-side-out membrane vesicles. Thus, it seems highly likely that the orientation of the lac carrier protein in the reconstituted system is similar to that in the bacterial cytoplasmic membrane, at least with respect to 4B1 epitope.     

Acetobacter aceti possesses a proton motive force-dependent efflux system for acetic acid

Acetic acid bacteria are obligate aerobes able to oxidize ethanol, sugar alcohols, and sugars into their corresponding acids. Among them, Acetobacter and Gluconacetobacter species have very high ethanol oxidation capacity, leading to accumulation of vast amounts of acetic acid outside the cell. Since these bacteria are able to grow in media with high concentrations of acetic acid, they must possess a specific mechanism such as an efflux pump by which they can resist the toxic effects of acetic acid. In this study, the efflux pump of Acetobacter aceti IFO 3283 was examined using intact cells and membrane vesicles. The accumulation of acetic acid/acetate in intact cells was increased by the addition of a proton uncoupler and/or cyanide, suggesting the presence of an energy-dependent efflux system. To confirm this, right-side-out and inside-out membrane vesicles were prepared from A. aceti IFO 3283, and the accumulation of acetic acid/acetate in the vesicles was examined. Upon the addition of a respiratory substrate, the accumulation of acetic acid/acetate in the right-side-out vesicles was largely decreased, while its accumulation was very much increased in the inside-out vesicles. These respiration-dependent phenomena observed in both types of membrane vesicles were all sensitive to a proton uncoupler. Acetic acid/acetate uptake in the inside-out membrane vesicles was dependent not on ATP but on the proton motive force. Furthermore, uptake was shown to be rather specific for acetic acid and to be pH dependent, because higher uptake was observed at lower pH. Thus, A. aceti IFO 3283 possesses a proton motive force-dependent efflux pump for acetic acid.     

Mechanics of solute translocation catalyzed by enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The kinetics of binding of mannitol to enzyme IImtl embedded in the membrane of vesicles with an inside-out or a right-side-out orientation were analyzed at 4 degrees C in the absence of the phosphoryl group donor, P-HPr. The binding to the right-side-out oriented vesicles equilibrated too fast to be monitored by the flow dialysis technique. On the other hand, with the inside-out oriented membrane vesicles two conformational changes of the enzyme could be detected kinetically. One change involved a recruitment of binding sites from a state of the enzyme where the binding sites were inaccessible from the cytoplasmic volume. The second change involved a conformational change of the enzyme that followed upon the initial binding to the cytoplasmic-facing binding site leading to a state with a higher affinity for mannitol. Equilibrium binding to the inside-out and right-side-out oriented membrane vesicles at 4 degrees C indicated that the two transitions did not represent the translocation of the binding site, free and with mannitol bound to it, to the other side of the membrane. Instead, a model is proposed in which the conformational changes represent transitions from states with the binding pocket opened to the cytoplasmic side of the membrane to occluded states of the enzyme in which the binding sites, with or without mannitol bound, are not accessible to either side of the membrane.     

D-lactate oxidation and generation of the proton electrochemical gradient in membrane vesicles from Escherichia coli GR19N and in proteoliposomes reconstituted with purified D-lactate dehydrogenase and cytochrome o oxidase

The respiratory chain in the cytochrome d deficient mutant Escherichia coli GR19N is a relatively simple, linear system consisting of primary dehydrogenases, ubiquinone 8, cytochrome b-556, and cytochrome o oxidase. By use of right-side-out and inside-out membrane vesicles from this strain, various oxidase activities and the generation of the H+ electrochemical gradient were studied. Oxidation of ubiquinol 1 or N,N,-N',N'-tetramethyl-p-phenylenediamine, which donate electrons directly to the terminal oxidase, generates a H+ electrochemical gradient comparable to that observed during D-lactate oxidation. In contrast, D-lactate/ubiquinone 1 or D-lactate/ferricyanide oxidoreductase activity does not appear to generate a membrane potential, suggesting that electron flow from D-lactate dehydrogenase to ubiquinone is not electrogenic. Moreover, proteoliposomes reconstituted with purified D-lactate dehydrogenase, ubiquinone 8, and purified cytochrome o catalyze D-lactate and ubiquinol 1 oxidation and generate a H+ electrochemical gradient similar to that observed in membrane vesicles. Strikingly, in inside-out vesicles, NADH oxidation generates a H+ electrochemical gradient that is very significantly greater than that produced by either D-lactate or ubiquinol 1; furthermore, NADH/ubiquinone 1 and NADH/ferricyanide oxidoreductase activities are electrogenic. It is suggested that the only component between D-lactate dehydrogenase or ubiquinol and oxygen in GR19N membranes that is directly involved in the generation of the H+ electrochemical gradient is cytochrome o, which functions as a "half-loop" (i.e., the oxidase catalyzes the scalar release of 2 H+ from ubiquinol on the outer surface of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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.     

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.     

Conversion of everted thylakoids into vesicles of normal sidedness exposing the outer grana partition membrane surface

Inside-out spinach thylakoid vesicles can be isolated by aqueous polymer two-phase partition following mechanical disruption of spinach chloroplast lamellae (Andersson, B and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472) and a mechanism for their formation has been experimentally supported (Andersson B., Sundby, C. and Albertsson, P.-Å. (1980) Biochim. Biophys. Acta 599, 391–402). Upon disruption, inside-out vesicles may form under stacking conditions, e.g., in 5 mM MgCl₂ or 150 mM NaCl, while disruption under destacking conditions, i.e., low concentrations of monovalent cations, gives only right-side-out vesicles. This study deals with the sidedness stability of the isolated inside-out thylakoid vesicles when stored or disrupted by sonication in various ionic environments. The sidedness of thylakoid vesicles was determined by their partition behaviour in an aqueous polymer phase system, direction of proton translocation and aggregation response (stacking) upon addition of MgCl₂. The results show that no spontaneous change from everted to normal sidedness occurs upon storage of the inside-out thylakoids. In contrast, sonication of these vesicles under destacking conditions (5 mM NaCl) results in a nearly complete transformation to right-side-out orientation. Also, in the presence of 5 mM MgCl₂ or 150 mM NaCl, sonication induced a change in sidedness of the inside-out vesicles but to a lesser extent. The stabilizing effect on the everted sidedness by cations was shown to be a result of preventing vesicle fragmentation by maintaining internal thylakoid appresions rather than by influencing the membrane curvature during resealing. Once released from an appressed state by overcoming the stacking forces, an opened thylakoid membrane shows an absolute preference for turning right-side-out in all media tested. These results strongly support the proposed formation mechanism, in which pairs of neighbouring grana membranes after disruption reseal with each other promoted by their close proximity. Since the inside-out vesicles derive from the grana appressions, their transformation back to normal sidedness exposes the outer membrane surface of appressed thylakoids. This region of the thylakoid membrane is normally hidden in the grana appressions and removal of grana leads concomitantly to lateral intermixing with non-appressed thylakoid components. Thus the current isolation of right-sided vesicles derived from the grana appressions should be a new tool for studies on the molecular organization of the thylakoid membrane.     

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes

A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.     

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment

Summary The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1 4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.Abbreviations Brij 58 polyoxyethylene 20 cetyl ether - DPH 1,6-diphenyl-1,3,5-hexatriene - HCF III hexacyanoferrate (III) - ISO inside-out - PM plasma membrane - RSO right-side-out - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene     

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.     

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.     

荧光假单胞菌TM5-2中D-型氨基酸脱氢酶的鉴定和表达

从荧光假单胞菌TM5-2中得到一个含丙氨酸消旋酶基因的DNA片段(8.8kb),相邻的一个开读框(ORF)与甘氨酸/D-型氨基酸氧化酶基因相似。该ORF经过克隆、表达,并没有检测到甘氨酸/D-型氨基酸氧化酶的活性,推导而得的氨基酸序列与D-型氨基酸脱氢酶序列比较发现,ORF含有D-型氨基酸脱氢酶的所有重要的保守序列。经TTC培养基鉴定,其具有D-型氨基酸脱氢酶的活性,并对一系列D-型氨基酸有作用,最佳作用底物是D-组氨酸。     

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 pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.     

Solubilisation of D-amino acid dehydrogenase of Escherichia coli K12 and its re-binding to envelope preparations

D-amino acid dehydrogenase was found to be solubilised from envelope preparations of Escherichia coli by treatment with detergents but not with aqueous buffer solutions. Triton X-100-solubilised dehydrogenase was found to rebind to envelope preparations from the wild strain (AB 259) and its D-amino acid dehydrogenase-less mutant (DAD 13), and activities higher than those of native envelopes could be obtained. Re-binding was stimulated by magnesium. D-alanine stimulated cytochrome reduction and oxygen uptake were reconstituted when the solubilised dehydrogenase rebound to AB 259 envelopes. Re-binding of solubilised dehydrogenase to DAD 13 envelopes was independent of the growth medium used for DAD 13.     

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.     

Immunological evidence that plasma-membrane 5'-nucleotidase is a transmembrane protein

Antibodies inhibiting specifically plasma membrane-bound 5'nucleotidase were used to determine the disposition of the enzyme in various lymphoma cell lines. Fluorescent Fab fragments of the inhibiting antibodies bound to MF2s and MOPC 173 plasmacytoma cells, whereas no fluorescence was observed with P 1798 thymoma cells in which the enzyme was absent. The relative potency of the antiserum in inhibiting the enzyme in right-side-out and inside-out plasma membrane vesicles prepared from the above cell lines indicated that various group of antigenic determinants were present. One group of antigenic determinants was present at the external face of the cell, and a second was associated with the inner surface of the membrane. A third group of antigenic determinants was located in the vicinity of the active site of the enzyme and it is the group that varied in the various plasmacytoma cells studied. The results are interpreted as immunological evidence that 5'nucleotidase is a transmembrane glycoprotein.     

Inside-out membrane vesicles isolated from spinach thylakoids.

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The sepration was obtained by partitionin an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.