ANTrMICROBuL AGENTS AND CHEMOTHERAPY, May 1975, p. 530-537 Copyright i 1975 American Society for Microbiology
Vol. 7, No. 5 Printed in U.SA.
Inhibition by Levorphanol and Related Drugs of Amino Acid Transport by Isolated Membrane Vesicles from Escherichia coli MARY J. C. HOLLAND* AND ERIC J. SIMON Department of Medicine, New York University Medical Center, New York, New York 10016 Received for publication 27 November 1974
Levorphanol inhibits the transport of the amino acids proline and lysine by cytoplasmic membrane vesicles derived from Escherichia coli. The degree of inhibition increases with increasing levorphanol concentration and ranges from 26% at 10-6 M levorphanol to 92% at 10-3 M levorphanol. The effect is independent of the energy source, since levorphanol inhibits proline uptake to the same extent in the presence of 20 mM D-lactate or 20 mM succinate and in the absence of an exogenous energy source. Levorphanol does not irreversibly alter the ability of membrane vesicles to transport proline, since incubation of membrane vesicles for 15 min in the presence of 0.25 mM levorphanol, a concentration which inhibits proline transport by more than 75%, has no effect on the rate of proline transport by these vesicles once the drug is removed. Both the maximum velocity and the Km of proline transport are modified by levorphanol, hence, the type of inhibition produced by levorphanol is mixed. The inhibitor constant (K) for levorphanol inhibition of proline transport is approximately 3 x 10- 4 M. Membrane vesicles incubated in the presence of levorphanol accumulate much less proline at the steady state than do control vesicles. Furthermore, the addition of levorphanol to membrane vesicles preloaded to the steady state with proline produces a marked net efflux of proline. Levorphanol does not block either temperature-induced efflux or exchange of external proline with ["4C ]proline present in the intravesicular pool. Dextrorphan, the enantiomorph of levorphanol, and levallorphan, the N-allyl analogue of levorphanol, inhibit proline and lysine transport in a similar manner. Possible mechanisms of the effects of these drugs on cell membranes are discussed. Levorphanol, a synthetic analogue of morphine, inhibits the transport of amino acids in intact Escherichia coli and Staphylococcus aureus (4, 17). However, levorphanol produces a number of other metabolic effects in these organisms including growth inhibition (15), decreased ribonucleic acid (RNA) synthesis (18, 19), changes in membrane phospholipid composition (5, 23), and disappearance of cellular adenosine triphosphate (ATP) (6), leaving open the possibility that the inhibition of transport might be a secondary effect. Levallorphan, the N-allyl analogue of levorphanol, which exerts very similar effects on intact bacteria, has been shown to inhibit arginine uptake by a crude preparation of E. coli membranes in the presence of ATP (12). Greene and Magasanik (6) have suggested that the primary effect of levorphanol is on the cellular level of ATP and hence on metabolic energy. Although the mechanism of energy coupling to active transport in bacteria is not completely understood, work with isolated
membrane vesicles from a number of bacterial species provides evidence that ATP is not required for coupling energy to the transport of various amino acids and sugars by these vesicles (1, 7, 22; W. L. Klein, A. S. Dahms, and P. D. Boyer, Fed. Proc. 29:341, 1970). Moreover, the ATP content of such membrane vesicles is below the limits of the luciferin-luciferase assay and does not increase when the vesicles are incubated with electron donors (14). This paper describes a detailed study of the effects of levorphanol and related drugs on the transport of proline and lysine by isolated membrane vesicles prepared from E. coli. MATERIALS AND METHODS Chemicals. The radioactive proline and lysine were purchased from the New England Nuclear Corporation. Unlabeled proline was obtained from Mann Research Laboratories. D-Lactic acid lithitan salt was obtained from Calbiochem. Levorphanol, dextrorphan, and levallorphan were the generous gift of Hoffman-LaRoche, Inc. 530
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LEVORPHANOL AND TRANSPORT IN E. COLI VESICLES
All other materials used in these experiments were of reagent grade and were obtained from commercial sources. Preparation of membrane vesicles. E. coli W 3110, obtained from J. Adler, University of Wisconsin, was grown on medium A (2) containing 0.5% glucose. Membrane vesicles were prepared by the lysozymeethylenediaminetetraacetate method described by Kaback (8). Membranes were stored in 0.1 M potassium phosphate buffer, pH 6.6, at -70 C and thawed in an ice-water bath when required. Uptake experiments. Assays for amino acid uptake were carried out essentially as described by Kaback (8). Portions of membrane vesicles were incubated for 15 min in a medium containing 50 mM potassium phosphate, pH 6.6, and 10 mM magnesium sulfate. Then the energy source and, 15 s thereafter, the radioactive amino acid were added. The incubation of each sample was terminated by a 200-fold dilution with 0.1 M lithium chloride at room temperature. Each sample was rapidly filtered through a membrane filter (Millipore Corp.; pore size, 0.45 Am; diameter, 24 mm). Each filter was washed once with an equal volume of 0.1 M lithium chloride, dried under an infrared lamp, and placed in a counting vial containing 10 ml of a toluene-based liquid scintillation solution purchased as the concentrate Permafluor from the Packard Instrument Corporation. Samples were counted in a Packard Tri-Carb liquid scintillation counter. Unless otherwise indicated in the text or in the figure and table legends, the assays were carried out at 30 C with lithium D-lactate at a final concentration of 20 mM as the energy source, and the specific activities and final concentrations of the radioactive amino acids employed were as follows: L-[U- 'C ]proline (251 mCi/mmol), 7.5 MM; L-[U-_4C]lysine (312 mCi/mmol), 4 MM. Pretreatment experiments. A frozen membrane preparation was thawed in an ice-water bath and then distributed to a series of chilled tubes. One portion (the untreated control) remained in the ice-water bath throughout the pretreatment procedure. The other portions were diluted sixfold in a pretreatment incubation medium containing final concentrations of 50 mM potassium phosphate (pH 6.6), 10 mM magnesium sulfate, and, where indicated in the table legends, 20 mM lithium D-lactate and 0.25 mM levorphanol. All tubes except the untreated control were transferred to a 30 C waterbath. After 15 min of incubation these portions were diluted 40-fold in ice-cold 0.1 M potassium phosphate buffer at pH 6.6. The membrane vesicles were harvested by centrifugation. Then the initial rate of proline uptake by all portions, including the untreated control, was determined as previously described. Efflux experiments. At the beginning of each efflux experiment membrane vesicles were loaded with ["4C]proline to a steady-state level by 15 min of incubation at 35 C in a medium containing 50 mM potassium phosphate (pH 6.6), 10 mM magnesium sulfate, 20 mM lithium D-lactate, L-[U-_4C]proline (260 mCi/mmol, 7.5 MM), and 0.04 mg of membrane protein, in a final volume of 30 Ml. The amount of
531
["4C proline accumulated by several 30-,ul samples of these vesicles was determined by filtration as previously described. As indicated in the figure legends, various additions were made to the remaining samples several of which were assayed for ["4C]proline accumulation over the next few minutes of incubation. The effect of these additions on net efflux of proline could be calculated by comparing the nanomoles of intravesicular proline in samples before and after these additions. RESULTS Effect of levorphanol and related drugs on the initial rate of transport of the amino acids proline and lysine. Unless otherwise indicated all studies on the initial rate of transport were carried out at 30 C and at pH 6.6. Uptake of proline by membrane vesicles in the absence of levorphanol was studied. Both the initial rate of transport and the total amount of accumulation at the steady state varied considerably from one membrane preparation to another, but in all the preparations the shape of the curve resembled that shown in Fig. 1. Uptake generally remained linear for slightly less than a minute, whereas a steady-state level of accumulation was reached in about 15 min. The effect of levorphanol on the initial rate of amino acid transport was studied by adding various concentrations of levorphanol along with the energy source, i.e., 15 s before the addition of the radioactive amino acid. The data in'Fig. 2 show the inhibition of proline transport by 0.25 mM levorphanol during the first 2 min of incubation. Transport of the basic
0'~~~~~~~~~~~~~~~~~
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532
ANTIMICROB. AGENTS CHEMOTHER.
HOLLAND AND SIMON
since the degree of inhibition by levorphanol is
-
not independent of the external proline
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Time (min) Pia. 2. Effect of 0.25 mM levorphanol on proline uptake by E. coli W 3110 mnembrane vesicles. Each point represents the average of three samples, each containing 0.04 mg of membrane protein in a finhl volume of 60 ul, with (A) and without (0) levor.
phanol.
amino acid lysine is inhibited by levorphanol in a similar manner (data not shown). The results of an experiment in which transport studies were cartied out in the presence of various concentrations of levorphanol are summarized in Table 1. The inhibition of proline transport increases with increasing levorphanol concentration. Pretreatment experiments were carried out to determine whether levorphanol irreversibly alters the ability of membrane vesicles to transport proline. The results of one pretreatment experiment are shown in Table 2. Two other pretreatment experiments produced similar results (data not shown). The rate of transport by drug pretreated vesicles is at least as great as that by control vesicles pretreated in an identical manner. This result does not depend on the presence or absence of an exogenous energy source in the pretreatment incubation medium. However the rate of transport by pretreated vesicles (both drug and control) is less than that obtained by untreated control membranes. Apparently the preincubation and washing procedure results in some loss of transport activity. Kinetic experiments were carried out to determine the type of inhibition produced by levorphanol. Clearly, levorphanol is not a purely noncompetitive inhibitor of proline transport,
concen-
tration. For example, in the kinetics experiment shown in Fig. 3, levorphanol (0.1 mM) inhibits proline transport by 80% at 0.45 AM proline, by 60% at 7.5 gM proline, and by 50% at 22.5 ,M proline. However, the data shown in Fig. 3 do not suggest that levorphanol is a purely co)noetitive inhibitor of proline transport. Furthermore; in other kinetics experiments the veloeity of transport in the presence of 0.25 mM levorphanol is considerably less than the velocity in the absence of levorphanol, even at proline concentrations as high as 100 ,M (35 times the Km for proline transport as determined in these experiments). Webb (21) refers to this type of inhibition as mixed because both the maximum velocity and the Km are modified by the inhibitor. The inhibitor constant (Ki) for levorphanol inhibition of proline transport ig approximately 3 x 10-4 M when calculated by the slope ratio method from the Lineweaver-Burk plot shown in Fig. 3. Levorphanol inhibition of proline transport is independent of the energy source. When succiTABLE 1. Inhibition by levorphanol of the initial raite of [14C]proline uptake by E. coli membrane vesicles Levorphanol concna (M)
Percent 92 83 77 59 42 30 26
1 x10-l 5 x 10-' 2.5 x 10-4 1 x 10-4 2.5 x 10-5 1 x 10-' 1 x 10-6
a Three samples each containing 0.051 mg of menibrane protein in a final volume of 60 ,Ll were employed for each drug concentration. Data are expressed as the percent lihibition of proline transport which was 950 pmol/mg of membrane protein per min in the control.
TABLE 2. The effect of exposure to 0.25 mM levorphanol for i5 min at 30 C on the ability of E. coli membrane vesicles to accumulate proline Additions to pretreatment inculbation medium
None D-Lactateb Levorphanol Levorphanol + D-lactate5
Proline transport after pretreatmenta (pmollmg of protein/min)
83 99 116 104
a The untreated control vesicles accumulated 206 pmol/mg of protein per min. b 20 MM D-lactate final concentration.
LEVORPHANOL AND TRANSPORT IN E. COLI VESICLES
VOL. 7, 1975
35
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FIG. 3. Lineweaver-Burk plot of kinetics of proline uptake by E. coli membrane vesicles in the presence (0) and absence (0) of 0.1 mM levorphanol. The concentrations of [14C]proline used were 0.38, 0.45, 0.55, 0.75, 1.5, 7.5, and 22.5 M1M at the specific activity of 260 mCi per mmol. Samples containing 0.066 mg of membrane protein in a final volume of 60 ul were assayed after 1 min of incubation. Initial rates were calculated in terms of nanomoles of uptake per milligram of membrane protein per minute. The data were fitted by the method of least squares.
nate is substituted for D-lactate in the incubation medium, the inhibition of proline transport remains approximately the same for a given levorphanol concentration. Moreover, levorphanol inhibits transport in a similar manner in the absence of an exogenous energy source (Fig. 4). The effects of temperature and pH on levorphanol inhibition of the initial rate of proline transport were investigated. At each of several drug concentrations in an incubation medium at pH 6.6, the same degree of inhibition of
533
proline transport was observed when experiments were carried out at 30, 37, and 45 C. Similarly in experiments carried out at 30 C, the same degree of inhibition of the rate of proline transport by each of several concentrations of levorphanol was observed in incubation media at pH 6.6, 7.0, and 7.7. Two other drugs of the morphine series, dextrorphan (the enantiomorph of levorphanol) and and levallorphan, inhibit thevesicles. transport proline lysine by membrane As ofshown in Table 3, dextrorphan and levallorphan inhibit proline transport to the same extent as levorphanol at each of several drug concentrations. Effect of levorphanol on steady-state accumulation of proline. Accumulation of proline by membrane vesicles reaches the steady state in about 15 min at 30 C whether or not levorphanol is present in the incubation medium. However, membrane preparations incubated in the presence of 0.25 mM levorphanol accumulate slightly less than half as much proline as control preparations. The effect of temperature on the steady-state accumulation of proline by membrane vesicles - / in the presence of levorphanol was investigated. The amount of proline accumulated at the steady state in the presence of 0.25 mM levorphanol was 47% of control at 30 C, 44% of control at 37 C, and 52% of control at 45 C.
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Time (min) FIG. 4. The effect of 0.25 mM levorphanol on proline transport by E. coli W3110 membrane vesicles
in the absence of an exogenous energy source. Each point represents the average of three samples, each containing 0.14 mg of membrane protein in a final volume of 75 Ml, with (A) and without (0) levorphanol.
534
ANTIMICROB. AGENTS CHEMOTHER.
HOLLAND AND SIMON
TABLE 3. Inhibition of proline uptake in E. coli membrane vesicles by drugs of the morphine series Inhibition of uptakeb
(D
M
)
Levorphanol
Dextrorphan
Levallorphan
44 59 73 96
43 61 78 96
35 51 69 95
(%)
0.05 0.10 0.25 2.00
(%)
(%)
a The lower two drug concentrations were employed in an experiment in which each sample contained 0.025 mg of membrane protein in a final volume of 60 A1 and in which the control samples accumulated an average of 670 pmol of proline per mg of membrane protein. The higher two drug concentrations were employed in an experiment in which each sample contained 0.051 mg of membrane protein in a final volume of 60 Al and in which the control samples accumulated an average of 1.3 nmol of proline per mg of membrane protein. b The data are expressed as percentages of inhibition of uptake after 1 min of incubation.
Steady-state levels of proline accumulation represent a balance between rates of influx and efflux. When levorphanol is added to membrane vesicles which have been preloaded to the steady state in the absence of levorphanol, the intravesicular proline level falls to a new steadystate level. Thus, when 0.25 mM levorphanol (final concentration) is added to membrane vesicles preloaded to the steady state at 35 C, a net efflux of 0.25 nmol of proline per mg of membrane protein per min is observed (Fig. 5). Within 10 min after the addition of levorphanol the intravesicular proline falls from a steadystate level of 2.35 nmol of proline per mg of membrane protein in the absence of levorphanol to a new steady-state level of 1.06 nmol of proline per mg of membrane protein in the presence of 0.25 mM levorphanol. Effect of levorphanol on heati4nduced efflux and on proline exchange. Steady-state levels of proline accumulation exhibit an optimum at 35 C (13). Hence, when membrane vesicles are preloaded with proline to the steady-state at 35 C and then are shifted to 47 C, a net effldx of proline is observed. The results of an experiment in which levorphanol was added to preloaded membrane vesicles at the onset of the temperature change are shown in Fig. 6. In this experiment and two others (data not shown), little net efflux of proline was observed in the control vesicles during the first 15 s at 47 C. Presumably some time was required for the temperature to rise in the tubes.
However, in each experiment the vesicles to which levorphanol was added show considerable net efflux during the first 15 s. At each subsequent time point net efflux was observed in all vesicles. Apart from the initial stimulation of efflux produced by the addition of levorphanol, there appears to be little effect on the rate of heat-induced efflux. When membrane vesicles are preloaded with ["4C]proline to the steady state followed by addition of [12C ]proline, efflux of ["4C ]proline is observed. The addition of various concentrations of levorphanol along with the ["2C]proline has little apparent effect on the rate of this efflux (Fig. 7).
DISCUSSION The data presented in this paper demonstrate that levorphanol is a potent inhibitor of amino acid transport by E. coli membrane vesicles. Inhibition of proline uptake by these vesicles was observed at concentrations as low as 1 ,M levorphanol. Proline uptake was inhibited by more than 50% by 50 MM levorphanol. In earlier
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Time( min) FIG. 5. Stimulation of proline efflux by levorphanol. Membrane vesicles were loaded with [14Cjproline to a steady-state level as described in Materials and Methods. At this time (i.e., zero time in the figure) one set of samples received no additions (0). To the remaining samples was added 5 MI of 50 mM potassium phosphate (pH 6.6) containing 10 mM magnesium sulfate and levorphanol at the following final concentrations: none (0), 0.25 mM (A), and 0.75 mM (A). Incubation was continued at 35 C and samples were assayed at the times indicated. The data are expressed as percentages of control samples assayed at zero time; the uptake of proline in the control samples was 2.35 nmol per mg of membrane protein.
VOL . 7, 1975
LEVORPHANOL AND TRANSPORT IN E. COLI VESICLES
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30 60 90 120 Time (sec) FIG. 6. Effect of levorphanol on heat-induced efflux of proline from E. coli membrane vesicles. Membrane vesicles were preloaded with [14C]proline to a steady-state level as described in Materials and Methods. At this time (i.e., zero time in the figure), all samples were shifted to 47 C with the simultaneous addition of 5 Il of potassium phosphate (pH 6.6) containing 10 mM magnesium sulfate and levorphanol at the following final concentrations: none (0), 0.25 mM (A), and 0.75 mM (A). Incubation was continued at 47 C and samples were assayed at the times indicated. The data are expressed as percentages of control samples assayed at zero time; the uptake of proline in the control samples was 1.54 nmol per mg of membrane protein.
studies involving E. coli intact cells, putrescine uptake was more sensitive to the effect of levorphanol than any other biochemical process studied but still required approximately 0.2 mM levorphanol for effective inhibition (17). Marked effects on growth, RNA synthesis, RNA phage reproduction, amino acid transport, putrescine efflux, and phospholipid metabolism were demonstrated only at levorphanol concentrations of 1 mM or higher (15-19, 23). The results presented here do not support the hypothesis that the primary action of levorphanol is on the cellular level of ATP. Kaback and others (1, 11; W. L. Klein et al., Fed. Proc., 29:341, 1970) h'ave shown that ATP is not required for coupling of energy to the transport of amino acids by E. coli membrane vesicles and yet levorphanol is a potent inhibitor of this transport. Our results indicate that levorphanol produces a readily reversible alteration in the cell membrane and as a consequence inhibits amino acid transport. The findings reported here may be inter-
535
preted in terms of the conceptual model for amino acid transport by membrane vesicles developed by Kaback and Barnes (9, 10) in which the carriers are depicted as electron transfer intermediates which undergo reversible oxidation-reduction. Kaback suggests that electrons which flow from the energy source through one or more flavoproteins reduce a critical disulfide in the carrier molecule resulting in release of an amino acid molecule on the inside of the membrane. The carrier molecule is then oxidized by cytochrome b1 and the electrons flow through the remainder of the cytochrome chain to reduce molecular oxygen. We suggest that levorphanol may inhibit amino acid transport by interrupting the flow of electrons between cytochrome b1 and molecular oxygen. We postulate this possible site of action for levorphanol based upon the following evidence: (i) levorphanol inhibition of uptake is independent of the energy source; hence, it seems unlikely that levorphanol produces its effect by acting directly on the primary dehydrogenase for a particular energy source. (ii) Membrane vesicles incubated in the presence of levor100 tCto- 80 _ \
60
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Time (min) FIG. 7. Effect of levorphanol on proline exchange by E. coli membrane vesicles. Membrane .vesicles were loaded with [14C1proline to a steady-state level as described in Materials and Methods. At this time (i.e., zero time in the figure) [12CJproline at a final concentration of 1.87 mM was added to each sample along with levorphanol at the following final concentrations: none (0), 0.25 mM (A), and 0.75 mM (A). Incubation was continued at 35 C and samples were assayed at the times indicated. The data are expressed as percentages of control samples assayed at zero time; the uptake of proline in the control samples was 2.35 nmol per mg of membrane protein.
536
HOLLAND AND SIMON
phanol accumulate much less proline at the steady state than do control vesicles, suggesting that levorphanol does not act exclusively by interrupting the flow of electrons before cytochrome b. Inhibitors such as oxamate and amytal which act before cytochrome b1 produce marked inhibition of the initial rates of transport but have little effect on accumulation at the steady state (9, 10, 13). On the other hand, anaerobiosis, potassium cyanide, and 2-heptyl-4-hydroxyquinoline-4-oxide, which interrupt the flow of electrons after cytochrome b1, produce profound inhibition of steady-state accumulation (9, 10, 13). (iii) Levorphanol produces rapid efflux of proline from membrane vesicles preloaded to the steady state. Electron transfer inhibitors which act after cytochrome b, cause marked efflux from preloaded membrane vesicles, whereas those inhibitors which act before cytochrome b, produce little or no stimulation of efflux (9, 10, 13). (iv) Levorphanol does not block either temperatureinduced efflux or exchange of external proline with ["4C]proline present in the intravesicular pool. This finding argues against the possibility that levorphanol inactivates the sulfhydryl groups of the carrier at the site of energy coupling. Both uptake and efflux are carrier mediated, and sulfhydryl agents such as pchloromercuribenzoate and N-ethylmaleimide block both temperature-induced efflux and proline exchange (9, 10, 13). The evidence presented here does not rule out the possibility that levorphanol produces its effects by some mechanism not explicable in terms of the Kaback model. For example, the model does not account for the mode of action of 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and azide, each of which inhibits amino acid transport but has no significant effect on the rate of D-lactate oxidation by membrane vesicles (1, 4). Since levorphanol and 2,4-dinitrophenol produce a number of similar inhibitory effects in E. coli intact cells (20), it will be of considerable interest to determine whether levorphanol exhibits a similar lack of inhibition of D-lactate oxidation by membrane vesicles of E. coli. In a number of studies with E. coli, levorphanol and related drugs inhibited growth (15), RNA synthesis (specifically ribosomal RNA synthesis; 19, 20), and the production of infectious RNA phages (16). These drugs also have been shown to inhibit the transport of proline, lysine, and aspartate in Staphylococcus aureus (4) and putrescine, leucine, serine, tryptophan, K+, and Mg2+ in E. coli (3, 17). Effects of levorphanol and related drugs on membrane
ANTIMICROB. AGENTS CHEMOTHER.
phospholipid metabolism and composition in S. aureus (5) and E. coli (23) have been reported. These and other studies have provided evidence that levorphanol alters the properties of bacterial cell membranes. However, the mechanism by which levorphanol produces these effects is not understood. A number of differences between the action of levorphanol and related drugs on intact cells and on membrane vesicles should be pointed out. Levorphanol and its enantiomorph, dextrorphan, are equally potent inhibitors of proline and lysine transport by E. coli membrane vesicles (Table 3), whereas dextrorphan is 20 to 30% less effective than levorphanol as an inhibitor of growth in E. coli (15). Levallorphan, the N-allyl analogue of levorphanol, is a more potent inhibitor than levorphanol of growth and RNA and protein synthesis in E. col'i (6, 15), and of amino acid transport in S. aureus whole cells (4). However, levallorphan is no more effective than levorphanol as an inhibitor of amino acid transport by E. coli membrane vesicles. Levorphanol inhibition of proline transport by E. coli membrane vesicles does not demonstrate the dependence on pH which has been reported for most, if not all, of levorphanol effects in intact cells. In E. coli increasing the pH of the medium increases levorphanol effectiveness as an inhibitor of growth (15), RNA synthesis (18), and transport (17). This dependence on pH is often quite marked. For example, 1 mM levorphanol is required for complete growth inhibition at pH 7 (15). Similarly, 1.3 mM levorphanol, a concentration which markedly stimulates efflux of putrescine from E. coli cells at pH 7.8, produces no detectable efflux from such cells at pH 5.8 (17). In contrast, levorphanol is an equally potent inhibitor of proline transport in E. coli membrane vesicles at pH 6.6 and 7.7. With E. coli decreasing the Mg2+ concentration increases the effects of levorphanol or levallorphan on growth (6), RNA synthesis (E. J. Simon, D. Van Praag, F. L. Aronson, and N. Burton, Fed. Proc., 24:655, 1965), putrescine efflux (17), and arginine uptake (12). This reported dependence on Mg2+ concentration is quite pronounced. The growth of E. coli in a medium containing 0.01 mM Mg2+ is completely inhibited by 0.5 mM levallorphan, whereas the same concentration of levallorphan in the presence of 10 mM Mg2+ produces only a slight inhibition of growth (3). Similarly, 1.35 mM levorphanol markedly stimulates putrescine efflux from E. coli cells in the presence of 0.1 mM Mg2+ but is without effect in the
V VOL. ,LEVORPHANOL AND TRANSPORT IN E. COLI VESICLES 7, 1975
presence of 10 mM Mg2+ (17). In contrast to these studies with intact cells of E. coli, low concentrations of levorphanol produce profound inhibition of proline uptake by E. coli membrane vesicles in the presence of 10 mM Mg2+. The effect of varying the Mg2+ concentration on the potency of levorphanol as an inhibitor of amino acid transport in membrane vesicles was not investigated. In summary, levorphanol is a potent inhibitor of amino acid transport by E. coli membrane vesicles. Transport by these vesicles is more sensitive to the effects of levorphanol than any of the biochemical processes studied in intact cells of E. coli. This paper does not give a detailed mechanism, but the data presented here indicate that levorphanol produces a direct effect on the cytoplasmic membrane and that this effect is not related to ATP levels as suggested by Greene and Magasanik (6). In terms of the Kaback model (9, 10) a possible site of action is suggested. However, more work is needed to determine the mechanism for levorphanol inhibition of transport, ACKNOWLEDGMENTS We thank H. R. Kaback for his helpful suggestions on membrane vesicle preparation. This research was supported by grant DA-0017 from the National Institute of Mental Health. M.J.C.H. holds Public Health Service fellowship no. 4 F02 GM53182-03 from the National Institute of General Medical Sciences. E.J.S. is a Career Scientist of the Health Research Council of the City of New York.
LITERATURE CITED 1. Bames, E. M., Jr., and H. R. Kaback. 1970. ,B-Galactoside transport in bacterial membrane preparations: energy coupling via membrane-bound D-lactic dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 66:1190-1198. 2. Davis, B. D., and E. S. Mingiolo. 1950. Mutents of Escherichia coli requiring methionine or vitamin B,2. J. Bacteriol. 60:17-28. 3. Devynck, M. A., P. L. Boquet, P. Fromageot, and E. J. Simon. 1971. On the mode of action of levallorphan on Escherichia coli: effects on cellular magnesium. Mol.
Pharmacol. 1:605-610. 4. Gale, E. F. 1970. Effects of diacetylmorphine and related morphinans on some biochemical activities of Staphylococcus aureus. Mol. Pharmacol. 6:128-133. 5. Gale, E. F. 1970. Effects of morphine derivatives on lipid metabolism in Staphylococcus aureus. Mol. Pharma-
col. 6:134-145. 6. Greene, R., and B. Magasanik. 1967. The mode of action of levallorphan as an inhibitor of cell growth. Mol. Pharmacol. 3:453-472.
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