THE JOURNAL OF BIOLOWXL CREXISTRY Vol.247, No. 8,Issue of April 25, pp. 2408-2418, 1972

Printed in U.S.A.

Amino Acid Transport in Membrane Vesicles of

Bacillus subtilis

(Received for publication, September 23, 1971)

WILHELMUS N. KONINGS" AND ERNST FREESE

From the Laboratory of Molecular Biology, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Public Health Service, United States Department of Health, Education and Welfare, Bethesda, Maryland 20014

SUMMARY

Membrane vesicles of BdciZlus subfilis actively transport amino acids in the presence of electron donors. The effec- tiveness of each physiological electron donor depends on the presence of the necessary membrane proteins that allow sub- strate oxidation and couple it to amino acid transport. Some of these proteins are present in all membranes while others (e.g. oxidizing glycerol phosphate) appear only in certain growth media (containing glycerol). Physiological electron donors are NADH, NADPH, L-a-glycerol phosphate, L-lac- tate, and succinate. NADH stimulates amino acid uptake at the highest initial rate whereas glycerol phosphate is the most effectrve electron donor since it energizes the transport of the largest number of amino acid molecules per nmole of sub- strate oxidized per min.

Reduced phenazine methosulfate (PMS) is a very efficient nonphysiological electron donor which energizes amino acid transport at a high initial rate.

At the membrane concentrations usually employed (1 to 5 mg of membrane protein per ml), NADH, NADPH, and re- duced PMS consume oxygen so rapidly that special oxygena- tion is required to maintain the uptake of amino acids at a high rate. This uptake stops when the energy source has been oxidized; the accumulated amino acid then leaks out at a rate of 8% per min.

With reduced (by ascorbate) PMS as electron donor, the K, and V,,, values of the uptake of 18 amino acids were determined. The Km values range from 10e5 to 10e6 M whereas the V,,, values vary widely for the different amino acids. Competition experiments have shown the presence of at least nine different amino acid transport carriers in the vesicles.

The energy for active transport appears to be produced in the cytochrome-linked electron transport chain: all electron donors eifective in amino acid transport cause the reduction of the membrane-bound cytochromes. The electron trans- port inhibitors antimycin, Z-heptyl-4-hydroxyquinoline- N- oxide, cyanide, and azide also inhibit amino acid transport, for all energy sources. Rotenone and oligomycin inhibit only the lactate-energized transport. Under conditions

* Present address, Laboratory of Microbiology, Biological Center, University, Groningen, The Netherlands.

under which amino acids are transported no net ATP synthe- sis has been observed.

The isolation of membrane vesicles from bacteria has made it possible to examine the energy requirement of substrate trans- port independent from the general metabolism of the cells (1). Membrane vesicles from gram-negative as well as gram-positive bacteria perform concentrative uptake of glucose and fructose by the phosphoenolpyruvate transferase system (2). Recently Kaback and Mimer (3) reported that membrane vesicles from Escherichiu coli concentrate amino acids in the presence of either D-hi&k or, less effectively, L-lactate, succinate, and NADH. n-Lactate also energizes the uptake of fl-galactosides (4), man- ganese (5), and potassium (in the presence of valinomycin) (6) in E. coli vesicles.

In a brief publication we have recently reported that mem- brane vesicles of Bacillus subtilis take up L-serine in the pres- ence of n-lactate or, if sufficient oxygenation is provided, in the presence of NADH, NADPH, or reduced phenazine metho- sulfate (7). The present paper determines rate constants and establishes by competition results that the transport carriers of most amino acids differ from one another. It concludes that the amino acid uptake is coupled to the cytochrome-linked electron transport chain but apparently not to ATP production. Vesi- cles isolated from cells grown in a glycerol-containing medium exhibit oxygen consumption and excellent amino acid uptake in the presence of L-or-glycerol phosphate. Part of this work has been presented in an abstract (8).

METHODS

Cell Growth-B. subtilis strain 60015, requiring indole (or tryptophan) and methionine, was grown with forced aeration in 14 L cultures at 37” in the following two media. Casein hydroly- sate medium contained per liter 14 g of K~HPOI, 6 g of KHsPO+ 0.25 g of MgS04 x 7 H20, 2 g of (NH&S04, 1 g of ammonium citrate, 25 mg of L-tryptophan, 1 g of vitamin-free casein hy- drolysate (Nutritional Biochemicals, Cleveland, Ohio), and 10 g of n-glucose. Nutrient sporulation medium contained per liter 8 g of nutrient broth, 0.7 mM CaC12, 0.05 mM MnC12, 1 mM MgC12,

2408

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Issue of April 25, 1972 W. N. Konings and E. Freese 2409

1 X 1OV M FeC13, 100 rnM potassium phosphate, pH 6.5, 25 pg per ml of L-tryptophan, and 10 I.cg per ml of L-methionine.

Measurements of Oxygen Consumption-Oxygen consumption was measured with the Osygraph (Gilson Medical Electronics, Middleton, Wisconsin, model KM) in a 2.0-m] temperature- controlled reaction vessel at 25”. The reaction mixture con- tained 0.05 M potassium phosphate, pH 6.6, and the oxidizable compound at a concentration of 20 mM. The reaction was ini- tiated by the injection of 20 ~1 of the membrane preparation (final concentration 0.06 mg per ml by means of a spring-loaded Hamilton syringe, model CR700). Oxygen consumption was also followed in the presence of PMS’ by injecting it into the complete reaction mixture at a final concentration of 100 or 200

PM. Transport &Vu&es-The cells were harvested during exponen-

tial growth at an absorbance at 600 nm (A6& of 1.5 to 2.0. Cells washed in 0.1 M potassium phosphate, pH 7.3, were sus- pended (80 ml per g wet weight) in this buffer plus 0.5 M sucrose and incubated at 33” in the presence of 250 pg per ml of lysozyme. The incubation was continued for 15 min after at least 95% of the cells were spheroplasts as seen in the phase contrast micro- scope (total time about 45 mm). The spheroplasts were centri- fuged at 0”, lysed by at least 300.fold dilution, and purified by washing with low and high speed centrifugation according to Kaback (9). The final membranes were suspended in 0.1 M

potassium phosphate, pH 6.6, at 6 to 8 mg of protein per ml, distributed in 2-ml aliquots into thin walled plastic tubes, and stored in liquid nitrogen. All experiments were performed within 2 hours after thawing, the rapid utilization of the mem- branes seeming particularly important for energization by lac- tate.

The incubation mixture for transport studies contained final concentrations of 0.05 M potassium phosphate, pH 6.6 (or when specifically st.ated 0.05 M Tris-Cl, pH 6.6), 10 mM MgS04, and U-r4C-amino acids and electron donors at the concentrations stated (final volume 100 ~1 in tubes with 10 to 11 mm internal diameter). Vesicles, inhibitors, U14C-amino acid, and electron donors (usually at 20 mM final concentration) were added in this sequence by means of repeating dispensers attached to syringes (Hamilton Co., Whittier, Cal.). The timing of the experiment started with the addition of the electron donor. In the experi- ments using special oxygenation the tube contents were rapidly stirred by 7 mm long Teflon-coated magnetic stirring bars; tubes were placed in a temperature-controlled water bath which rested on top of a magnetic stirrer (Cole Parmer Co., Chicago). Fol- lowing addition of the electron donor, water-saturated oxygen was blown over the mixture. The reaction was terminated by the addition of 2 ml of 0.1 M LiCl and filtered (Millipore filter HAWP, pore size 0.45 /1, 25 mm diameter). The filters were washed with 2 ml of 0.1 M LiCl, dried on planchets for 10 min at 105”, and counted in a gas flow counter. For all experimental points the value obtained for zero time incubation (LiCl added before amino acid) was subtracted.

Thin Layer Chromatography of Transported Amino Acids- After filtering the vesicle preparation and washing it with 2 ml of 0.1 Y LiCl, the radioactive compounds were extracted from the Millipore filter in 2 ml of Hz0 by heating at 70” for 60 min

1 The abbreviations used are: NSMP, nutrient sporulation me- dium; PMS, phenazine methosulfate; HO&NO, 2.heptyl-4.hy- droxyquinoline-N-oxide; p-CMB, p-chloromercuribenzoate.

In the case of asparagine and glutamine, the membranes were treated with 0.1 ml of chloroform, 0.2 ml of methanol, and then 2 ml of water at room temperature. In all cases, the extract was freeze-dried and the residue dissolved in 20 ~1 of HQO. Five to ten microliters were spotted on thin layer plates (Silica Gel H) and chromatographed with ether-formic acid-Hz0 (7 :2 : 1) ac- cording to Myers and Huang (10). The plates were dried and radioautographs were made on no-screen x-ray film with exposure for 5 days. The spots were identified by using as references the original U-leC-amino acids (1 to 2 ~1 spotted).

Estimation of Vesicle Volume-(a) To 1 ml of membranes (7.23 mg of protein per ml) obtained from NSMP-grown cells 50 ~1 of [curboxyl-r4C]dextran (mol wt = 60,000 to 90,000,50 PCi per ml) were added. After centrifugation (30 min, 12,000 x g) the total water volume in the pellet was determined from the differ- ence of the wet weight and the dry weight (dried 3 hours at 110”). The interstitial volume between the vesicles was determined by dissolving the pellet in 0.5 ml of 10% sodium dodecyl sulfate and counting the radioactivity (of the remaining dextran) in the liquid scintillation counter. The total water minus the inter- stitial volume gave the volume inside the vesicles. (b) The in- ternal vesicle volume was also estimated by incubating the mem- branes (NSMP-grown, 0.86 mg of protein per ml) with ascorbate (20 mM) and PlMS (100 PM) in the presence of a r4C-labeled amino acid for 50 min with special oxygenation. At this time all ascorbate had been oxidized and the transported amino acid had leaked out again, so that the concentration of amino acid inside and outside the vesicles should be the same. By performing this experiment at six different amino acid concentrations a straight line was obtained by plotting the counts inside the vesicles (counts per min after incubation minus the counts per min at the same amino acid concentration but without incubation) against the amino acid concentration. From the amount of amino acid inside the vesicles their volume could be calculated.

Electron Microscopy-A membrane vesicle preparation (7 mg of protein per ml) from NSMP-grown cells was fixed with 2.5% glutaraldehyde, stained with uranyl acetate and osmium tetrox- ide, embedded in Epon according to the method of Kellenberger et al. (II), and examined in thin sections in the electron micro- scope.

Assay for Phosphorylated Intermediates-After incubation of a reaction mixture containing 32Pi and B. subtilis or E. coli mem- brane vesicles, the presence of 32Pi-labeled nucleotides was deter- mined by thin layer chromatography on polyethylenimine as described by Cashel et al. (12).

32P-Labeled Membranes-32P-Labeled membrane vesicles of E. coli K-12 were prepared according to Kaback (9) from cells (late exponential phase) grown in a medium described by Kaempfer and Magasanik (13) which was supplemented with 0.2% casamino acids and contained 0.4 mM 32Pi (lo5 cpm per nmole). The membranes were suspended in 0.1 M Tris-Cl, pH 6.6.

Materials-All 14C-amino acids (uniformly labeled) were obtained from New England Nuclear (Boston, Massachusetts) at the highest specific activity. They were stored at -10” at concentrations of 10m4 to lo+ M. L-Phenylalanine and L-trypto- phan (obtained in 1 N HCI) were adjusted to pH 6.6 with NaOH.

L-[U-liC]Cysteine was prepared by adding dithiothreitol (final concentration I mM) to a SOhtiOn of 7.5 X IOV5 M CySthe jUSt

before use. Nonradioactive L-cysteine was prepared, also just before use, by neutralization of a cysteine-HCI solution.

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2410 Amino Acid Transport in Membrane Vesicles oj B. subtilis Vol. 247, x0. s

TABLE I I I I I I I

Effect of different energy sources on L-serine uptake Membrane vesicles isolated from Bacillus subtilis grown on

casein hydrolysate medium were preincubated for 15 min at 33” in the reaction mixture containing 50 mM potassium phosphate, pH 6.6, and 10 mM MgS04. The energy source was added and immediately followed by n-[‘“Clserine.

I L- PROLINE

COllC.3- I I tration Uptake

Without a-lditional oxygen supply: membrane protein concentration, 2.85 mg per ml; L-[r%]serine concentration, 7.8 X 10” M; incuba- tion for 20 min.

None .................. L-Lactate. ............ n-Lactate. ............. Succinate ............. Malate ................ Pyruvate. ............. D, n-fi-Hydroxybutyrate Acetate ................ Glyoxylate ............ Glyceraldehyde ........ Fumarate ............. Oxaloacetate .......... Glucose ............... ATP .................. ADP .................. GTP .................. GDP. Phosphoenolpyruvate. NADH. ............... NAD .................. NADPH .............. NADP ................ F,4D ..................

...... ...... 15.1

...... ...... 20 83.5

. 20 36.8

...... ...... 20 32.6

...... ...... 20 17.9

...... ...... 20 13.7

...... ...... 20 23.1 20 14.3

...... ...... 20 14.4

...... ...... 20 16.2

...... ...... 20 20.6

...... ...... 20 16.1 . 20 21.0

...... ...... 10 18.0

...... ...... 10 18.8

...... ...... 10 20.2

...... ...... 10 13.7 . . . . . . . 10 14.8

...... ...... 10 28.3

...... ...... 10 17.3

...... ...... 10 25.2

...... ...... 10 16.3 . . 20 15.4

pmo1es/ntg membrane

protein

With azlditional oxygen supply: membrane protein concentration, 3 mg per ml; L-[14C]serine concentration, 15.6 X 10% M; incuba- tion for 5 min.

None..................................... L-Lactate.......................... NADH. . . . NADPH . . . Ascorbate @O m&r) f PMS (100 MM) Succinate (20 m&r) + PMS (200 PM).

L-Malate...........................

20 . . 20 . . . . . 20

. . . . . .

. . . . . 20

23.4 238.3 631.6 637.6

1641.3 615.7

14.2

n-Asparagine and L-glutamine were passed as neutral solutions over a tiny (pencil) column of Dowex 1, chloride form, and washed out with the same volume of water. We learned this method of removing the deaminated compounds (pyro-form and free acid) from Dr. S. B. Prusiner, National Institutes of Health.

The electron donors and inhibitors all were obtained from commercial sources.

RESULTS

NAIJH produced (at 20”) a significant uptake for 1 to 2 min, which was often followed by a loss and a later leveling off or a slow increase at the spontaneous rate found without addition of an energy source. The limited uptake observed in initial membrane preparations without added energy source (Figs. 1 and 2) could consistently be reduced by diluting the protoplasts

Amino Acid Uptake in Presence of Physiological Energy Sources Membrane vesicles were isolated from B. subtilis cells harvested

2 H. Glossmann, W. N. Konings, and E. Freese, unpublished experiments.

DEGREES CENTIGRADE

FIG. 1. Temperature dependence of L-proline (4.6 X 1O-6 M) uptake by membrane vesicles (5.5 mg of protein per ml) of casein hydrolysate-grown cells of Bacillus subtilis in the presence of dif- ferent energy sources (10 mM). Samples taken after 5-min in- cubation.

during exponential growth in casein hydrolysate medium. When their uptake of n-[U-14C]serine was measured after 20-min incu- bation at 33” in the presence of different potential energy sources but without any special oxygenation, n-lactate was most effective (see Table I). A small stimulation of uptake above the spon- taneous level was also observed for nn-fl-hydroxybutyrate, n-lactate, succinate, NADH, and NADPH. Similar results were observed for several other amino acids (such as proline and glycine) ; the temperature optimum was, for all amino acids tested, 33” for L-lactate and 20” for NADH as can be seen for n-proline in Fig. 1. The effects of n-lactate and succinate were too small to exhibit clear temperature optima. The extent of amino acid incorporation, observed after 5-min incubation, in- creased with the concentration of L-lactate and NADH up to 20 mM-a concentration used in all further experiments.

Fig. 2 shows that n-lactate (at 33”) sustained n-proline uptake for 10 min or longer. The rate and final level of this accumula- tion decreased greatly when the membrane preparation was kept, after thawing from the liquid nitrogen temperature, for several hours in an ice bath before it was used. This deterio- ration of transport probably resulted from proteolysis because the acrylamide pattern (in sodium dodecyl sulfate) of proteins changed significantly when the membrane preparation was kept in the cold for several hours2 L-Lactate also stimulated the up- take of all other L-amino acids investigated except phenylala- nine, tryptophan, and methionine. Cysteine and cystine were not tested.

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Issue of April 25, 1972 W. N. Konings ancl E. Freese 2411

1

-1

1

1 80

MINUTES

FIG. 2. L-Proline (4.6 X 1OV M) uptake into membrane vesicles (5.5 mg of protein per ml) of casein hydrolysate-grown cells. Energy sources: Li-L-lactate at 33” (0) and NADH at 20” (A), used at concentrations of 20 mM without special oxygenation. No energy sources (None) at 33” (A) and at 20” (0).

at least 300-fold for the lysis procedure. As previously shown (7), the membranes of casein hydrolysate-grown cells have high NADH (and NADPH) oxidase activities (1042 nmoles per (min x mg of membrane protein)) whereas the L-lactate de- hydrogenase activity is much lower (6 nmoles per (min X mg of membrane protein)). In the presence of NADH and a mem- brane protein concentration of 3 mg per ml, 90% of the dissolved oxygen would therefore be used up in 4.3 sec. To avoid the osygen limitation, which is significant even in the small incuba- tion mixture of 100 ~1, wet oxygen was supplied and the mixture was rapidly stirred to obtain maximal gas exchange. Under these conditions, a much higher incorporation of serine was observed in the presence of NADH and NADPH (Table I). Further- more, the temperature optimum observed for the amino acid uptake with NADH (after 5 min) was 33”, the same as that for L-lactate stimulation.

The kinetics of the NADH stimulation of L-serine uptake with additional oxygen supply is shown in Fig. 3. At a mem- brane protein concentration of 3 mg per ml the uptake continued for 6 to 7 min, at which time all NADH must have been con- sumed, as one can calculate from the rate of oxygen consumption. The accumulated L-serine subsequently leaked out at a rate of 8% per min. When the membrane protein concentration was halved to 1.5 mg per ml, serine uptake continued for 12 to 13 min, because NADH lasted twice as long. The oxygen effect in the presence of NADH has also been observed for all other amino acids which were taken up at the same relative rates as those reported later for ascorbate plus PMS. No uptake could be found for phenylalanine and tryptophan. The amino acid uptake energized by L-lactate, n-lactate, or succinate was not increased by special oxygenation (Table I), in agreement with the low rates of oxygen consumption in the presence of these compounds (see Table II).

Reduced Phenazine Methosulfate as Electron Donor-Table I shows that succinate stimulated serine uptake only slightly. This limitation was caused neither by the exhaustion of oxygen (oxy-

1000

250

0 ,

I I I IO 20 30

MIN.

FIG. 3. L-Serine (1.6 X 10m5 M) uptake into membrane vesicles of casein hydrolysate-grown cells in the presence of 20 mM NADH at 25”. 0 and 0 = 3 mg of membrane protein per ml. A and A = 1.5 mg of membrane protein per ml. 0 and A = with special oxygen supply. l and A = without special oxygen supply.

TABLE II

Oxygen consumption and L-se&e uptake by membrane vesicles in presence of diferent energy sources

The rate of oxygen consumption was measured at 25” in 0.05 M potassium phosphate, pH 6.6 (see “Methods”). The rate of

L-serine uptake was measured in the usual reaction mixture con- taining 1.5 mg of membrane protein per ml and 31.2 X 10e6 M L-[l%]serine. A = membranes of cells grown in casein hydroly-

sate medium. B and C = membranes of cells grown in NSMP.

Energy source

L-Lactate (20 mM) _. . . . . D-Lactate (20 mM). Succinate (20 mM). .

Succinate (20 mM) + PMS (200 FM). .

NADH (20 mM) . NADPH (20 mM). Ascorbate (20 mM) + PMS

(100 PM). . . . .

L-a-Glycerol phosphate (20 rnM).......................

mygen consumptior

* I B

6.0 <0.5 5.3 <0.5

1.7 16.8 10 0.59

216 331.7 80 0.241 1042 314.9 160 0.507 1042 188.9 40 0.211

8ola

<0.5

8328 710 1.736”

27.9 225 8.06

L-Serine uptake

rransport efficiency

ratio

C/B

a With ascorbate (20 mM) + PMS (100 PM) but without mem- branes it was 423 nmoles per min.

* For the calculation of C/B only the enzyme-catalyzed portion of B (832 - 423 = 409) was used.

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I effect was increased by special oxygenation since 90% of the oxygen was used up in 21 see under these conditions. Apparently,

L-Lactate the reduced PMS could not only react directly with oxygen but it could also feed electrons into the electron transport chain and thereby stimulate amino acid transport. PMS could also be reduced chemically by the addition of ascorbate (20 mM), the optimum concentration of PMS being 100 PM, at 25”. The uptake of amino acids after 5-min incubation in the presence of ascorbate plus PMS was about a factor 3 higher than that ob- served in the presence of NADH. Even the effect of NADH

Succinate could be potentiated by the addition of 100 pM PMS which can be chemically reduced by NADH (Fig. 5). Reduced PMS apparently was particularly effective in supplying electrons to

00-d

that part of the electron transport chain that was involved in amino acid transport. (In the presence of 1.5 mg per ml of mem-

MIN. brane protein and 100 pM PMS, NADH was oxidized in 5 min

FIG. 4. L-Serine (1.6 X 10m5 M) uptake into membrane vesicles instead of in 12 min.) PMS did not increase the amino acid

(3 mg of protein per ml) of casein hydrolysate-grown cells in the uptake or oxygen consumption stimulated by L- or n-lactate. presence of 10 mM Li-L(+)lactate (A), 20 mM Nap-succinate (O), and 10 mM Li-L(+)lactate after 5-min incubation with 20 mM Naz-

Properties of Vesicles Obtained from Cells Grown in Nutrient

succinate (0). The reaction mixtures of 800 ~1 stood in tubes in Sporulation iMedium-In addition to the amino acids present in

a 33” water bath. Aliquots (100 pl) were withdrawn at different casein hydrolysate medium, nutrient sporulation medium con-

times and assayed. tains other components, such as 0.47 mM glycerol, but no glucose. The enzyme activities of membranes isolated from NSMP-

2500, I grown cells differed significantly from those of casein hydrolysate- grown cells (Table II). The NADH and NADPH oxidation activities were much lower, whereas the succinate oxidation ac- tivity (measured without PMS) was increased indicating a stronger coupling of the succinic dehydrogenase (measured with PMS) to the electron transport chain. Whereas no L-cy-glycerol phosphate oxidation (or dehydrogenase) activity was observed in extracts or membranes of casein hydrolysate-grown cells, the membranes of NSMP-grown cells had a significant activity.

These results were correlated with those for serine uptake in the presence of the different energy sources (Fig. 6 and Table II). The rate of uptake in the presence of NADH or NADPH was lower than that observed with casein hydrolysate vesicles and a steady state level was reached because these energy sources were not used up during 30 min incubation. L-a-Glycerol phosphate stimulated serine uptake in vesicles of NSMP-grown (but not casein hydrolysate-grown) cells at the same rate as NADH and

2412 Amino Acicl Transport in Membrane Vesicles of B. subtilis Vol. 247, ISo. 8

FIG. 5. L-Serine (1.6 X 10e6 M) uptake at 25” into membrane reached $ of its steady state level, although the activity of

vesicles (1.5 mg per ml of protein) of casein hydrolysate-grown glycerol phosphate oxidation was only & of NADH oxidation. cells in the presence of 20 mM NADH plus 100 PM PMS (0) or 20 Surprising was the finding that succinate did not stimulate mM NADH (0). Reaction mixtures (100 ~1) were incubated with special oxygenation.

serine uptake much more in vesicles of NSMP than in those of casein hydrolysate-grown cells, although the succinate oxidation activity was 10 times higher (Table II).

genation did not help) nor by the accumulation of an inhibitor The highest rate of uptake was again observed in the presence or block in the electron transport chain, because L-lactate could of ascorbate plus PMS; the accumulated serine leaked out when still stimulate serine uptake when it was added 5 min after ascorbate had been oxidized (Fig. 6). succinate (Fig. 4). The low stimulation was apparently due to Table II also shows the ratio of the rates of serine uptake and a loose coupling of succinic dehydrogenase to the electron trans- oxygen consumption. The highest ratio, i.e. the most efficient port chain, because the succinic dehydrogenase activity itself, uptake per molecule oxidized, was obtained for L-oc-glycerol measured in the presence of PMS, was high whereas the succinic phosphate. oxidation activity was low (see Table II). Attempts to increase Volume and Concentrating Power of Closed Membrane Vesicles- the L-serine uptake by the addition of various electron coupling A membrane vesicle preparation of NSMP-grown cells was pre- compounds, such as menaquinones, naphthoquinones, ubiqui- pared by Dr. E. B. Freese for electron microscopy and examined nones, or tetramethyl-p-phenylenediamine at concentrations of in thin sections. Fig. 7 shows an example of the closed vesicles 5 to 250 pM, were unsuccessful. However, the addition of typically seen. PMS (optimal concentration of 200 PM) greatly stimulated the The volume of the membrane suspension occupied by closed amino acid transport in the presence of succinate (see Table I) vesicles was determined with [carbozyl-14C]dextran, which prob- as we have already shown in an earlier paper (7). Again this ably is not, able to enter the vesicles (see “Methods”). The

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vesicle volume thus calculated was 3.7 ~1 per mg of membrane protein. Since not all vesicles are necessarily able to transport amino acids and since the preparation contains some nonmem- branous impurities, this volume represents an upper limit for the transporting vesicles. Even with this measure the concentration power of the vesicles would be remarkable. For instance, in the presence of ascorbate (20 mM) plus PMS (100 PM) as energy source (and with an initial amino acid concentration in the medium equal to the K, value) the concentration inside the vesicles (1 mg of protein per ml) would be, after 1-min incubation, for L-serine 10 times and for L-alanine 50 times higher than the outside concentration.

The internal vesicle volume into which amino acids were actually transported could be assessed by determining the

Ascorbate-PMS

0 NADH

1,600

cerol - P

NADPH

Succinate - PMS

\ ,, L - Lactate A

0 -- Nane I I 1- 1 I

-0 IO 20 30 MIN

FIG. 6. L-Serine (3.1 X 10e5 M) uptake at 25” into membrane vesicles (1.47 mg of protein per ml) of NSMP-grown cells. The concentration of all electron donors was 20 mM and of PMS in the nresence of either ascorbate or succinate was 100 PM.

FIG. 7. Electron micrographs of NSMP-grown membrane vesi- cles. Bar = 1 pm.

amount of label remaining in the vesicles a long t,ime after the energy had been used up. At this time inside and outside con- centrations should be the same. The volume thus calculated, as an average of different amino acid concentrations, was 1.78 ~1 per mg of membrane protein. This value is by a factor 2 lower than the weight value determined above, which might indicate that not all vesicles could transport amino acids and retain them throughout the filtering process needed to measure the uptake.

Determination of Rate Constants-The conditions producing the highest uptake, i.e. ascorbate plus PMS as energy source, were used to determine the K, and V,,, values for the different amino acids in NSMP vesicles. Particular care was needed for L-asparagine and L-glmamine, both of which are unstable and decompose into the pyro-compounds and then into the free deaminated compounds aspartate and glutamate. The amides were therefore purified just before use (see “Methods”).

The results in Table III show the kinetic data for 18 of the natural L-amino acids. No uptake was observed for tryptophan and that for cystine and histidine was so low that no data for Km or Vmax could be obtained. The K, values ranged from 2 x

lo+ to 4 x 10e5 M. The K, values were quite similar for dif- ferent vesicle preparations (varying by about +15 9$ around the values given here). But the V,,,, values varied much more since they depended on variations in the vesicle isolation and pre-

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2414 Amino Acid Transport in Membrane Vesicles of B. subtilis Vol. 247, No. 8

TABLE III

Kinetic constants of amino acid transport into vesicles of B. subtilis

Amino acid uptake was measured in membrane vesicles (0.86

mg of protein per ml) of NSMP-grown Bacillus subtilis at 25”. Immediately after addition of 20 rnM ascorbate and 100 PM PMS, the i4C-amino acid was added and the mixture incubated for 1 min

with special oxygenation. Each amino acid was assayed at eight different concentrations, differing by up to a factor of 10. Each value was corrected for the control value obtained when the incu- bation mixture was first diluted with 2 ml of LiCl before the addi-

tion of the ‘K-amino acid and then immediately filtered. Cys- t,eine, asparagine, and glutamine were prepared immediately before the assay as described under “Methods.” The values in brackets are accurate within a factor of 2 to 3, owing e.g. to low transport (threonine, phenylalanine, methionine) or high binding to the membranes (cysteine), or possible inaccuracy due to the

presence of deaminated asparagine or glutamine.

Amino acid --

Glycine Alanine

M

9 x 10-S

9 x 10-c

nmoles/(min x mg protein)

1.0 3.9

Valine 8 X 1O-5 0.7 Leucine 5 x 10-e 0.2 Isoleucine 9 x 10-s 0.2

Serine 4 x 10-S 3.0 Threonine (10-S) 0.2

Asparagine (3 x 10-S) U-2) Glutamine. (6 X 1O--6) (1.6)

Aspartate Glutamate

2 x 10-s .5 x 10-S

1.5

9.0

Lysine 1.7 x 10-b 0.1

Arginine 7 x 10-c 1.1

Phenylalanine Tyrosine

(10-S)

2 x 10-6 0.1

0.15

Cysteine. (3 x 10-G) 1.8 Methionine.......... (lo-h-10--“) 0.3

Proline. 3.8 x 10-6 1.0

sumahly the extent of proteolysis; the Vmav values given here are the optimal values of particularly active vesicle preparations.

However, the ratio of V mRX values, determined in one vesicle

preparation for different amino acids, remained the same in other

preparations.

To determine whether the transported amino acid retained its structure or was metabolized into another compound the com- pounds were extracted from the membrane vesicles, concentrated, and then chromatographed on thin layer. Excepting asparagine and glutamine, the only radioactive spot visible on the thin layer chromatogram migrated to the same place as the original amino

acid. The amino acids were therefore transported in their original form.

For both asparagine and glutamine the freshly purified com-

pounds contained only a trace ( <lye) of deaminated material (migrating faster on the thin layer plate). The material isolated from the membrane vesicles, however, consisted mostly (80 to 90%) of the faster migrating material. At the concentration of the K, value, 2 to 3% of the asparagine and about 10% of the glutamine were taken up per min by the membrane vesicles. Since this was more than the contamination present in the prepa- ration, the transported amino acids apparently were deaminated either during or after the transport. However, the K, or Vmax

values might have been affected by the contaminant and are therefore placed in brackets in Table III.

Competition of Transport by Di#erent Amino Acids-In order to see whether different amino acids were transported by different carrier proteins, the inhibition of transport of a given amino acid by all other amino acids was determined. The inhibiting (unlabeled) amino acids were used at concentrations of 1 mM, which were at least 30 times higher than the concentrations of the radioactive amino acids whose transport was measured. The results in Table IV show that only a few amino acids signifi- cantly inhibited the uptake of a given amino acid. In most cases the inhibition was mutual, indicating that the two amino acids were transported by the same carrier. Such common groups were: glycine and alanine; valine, leucine, and isoleucine; serine and threonine; asparagine and glutamine (which also had an affinity to the carriers of serine and of aspartate and glutamate) ; aspartate and glutamate; lysine (possibly with histidine for which no accurate data could be obtained); arginine; phenylalanine and tyrosine; cysteine and methionine; and proline.

Cysteine reduced the transport of almost all amino acids except the acidic ones. Presumably, this effect is due to the interaction of the sulfhydryl group with some membrane com-

ponent needed for active transport because 1 mrvr p-CMB also inhibited the initial rate of serine uptake by 70% (see Table V). The uptake of cysteine itself was significantly inhibit.ed by all other amino acids.

In several cases one amino acid partially inhibited the uptake of another but not vice versa. For instance, aspartate and glu- tamate inhibited the uptake of several amino acids which did not inhibit the uptake of aspartate or glutamate; arginine inhibited the uptake of lysine but not vice versa. This might indicate the affinity of one amino acid to (one of) the carrier proteins of another amino acid.

Involvement of Electron Transport System-To verify the in- volvement of the electron transport chain in amino acid trans- port the reduction of cytochromes by the various energy sources and the effect of electron transport inhibitors on amino acid uptake were measured.

23. subtilis produces cytochromes a, b, c, and cl (14). In mem- branes of NSMP-grown cells, the difference spectrum (in Fig. 8) of dithionite-reduced versus oxidized membranes showed bands which corresponded to these cytochromes as follows: bands at about 603 and 442 nm for cytochrome a and bands in the range of 428, 528, and 560 nm which could comprise cytochromes b, c, and cl. (Under the spectroscope at liquid nitrogen tem- perature the band at 560 nm split into two bands indicating the presence of cytochrome b and at least one type of cytochrome c.) When the membranes were exposed to NADH or ascorbate plus PMS the difference spectra against oxidized membranes showed that all bands could be reduced to some extent (Fig. 8). However, the comparison with dithionite reduction revealed that only the band at 603 nm characterizing cytochrome a was

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Issue of April 25, 1972 W. N. Konings and E. Free-se 2415

TABLE IV Percentage of inhibition of transport of amino acids

The amino acid uptake was measured in membrane vesicles (0.8G mg per ml) of NSMP-grown 13. subtilis at 25”. 1 rnM cold amino acid, 20 mM ascorbate, 100 PM PMS, and the ‘G-amino acid were added in rapid succession and the mixture was incubated for 1 min (after the ‘*C-amino acid addition) with special oxygenation. The percentage of inhibition was calculated with respect to the amino acid uptake obtained in the absence of unlabeled amino acid. Cysteine, asparagine, and glutamine were prepared immediately before the assay as described under “Methods.”

The concentrations of the 14C-amino acids were: glycine, 1.25 X 1O-5 M; alanine, 1.4B X 10e6 M; valine, 8.05 X 1O-6 M; leucine, 7.87 X 1Om6 M; isoleucine, 7.87 X lo-” M; serine, 3.12 X 10m5 M; threonine, 1.19 X 10m5 M; asparagine, G.10 X 1O-6 M; glutamine, G.32 X 10-O M; aspartate, 9.10 X 10-e M; glutamate, 9.74 X low6 M; lysine, 7.44 X lo-” M; arginine, 7.9 X low6 M; phenylalanine, 4.33 X 1O-6 M; t,yrosine, 4.39 X 10e6 M; cysteine, 7.5 X 10-e ‘M; methionine, 9.06 X 1O-6 M; proline, 4.6 X lo-” M. -

W-Amino acids transported

VIET PRO

0 13

10 5

11

16 21

12 18

7 18

39

18

43

26 30

32 -

58

54 99

26

45

21

19 20

20

28 0

40

40

39 47

26

19

11

13

11 0

50 12

9

7 99

\RG PHE TYR CSH

29

21

13 0

16 20

40

5

i- GO 40

7 14

12 23 5 10

41 43

45

28 11

0

10 29 6 30

12

17

22

14

0

15

10

0

17

100 100

88

38 1

66

11

Gl

63

26 36

60 58

1B

15 -

98 -

15 15

18

24

82

96 95

23 14

4

2

66 90 35 88 40 68

0 56

Inhibitors

GLY

-

ALA VAL LEU ILE

- 1 rnM

GLY

ALA 95 81 17 13 0 0 0 27 92 93 40 12 31 38 8 41

VAL 0 11 95 72 90 LEU 19 11 89 89 85 ILI? 16 17 100 85 80

SER 19 15 41 20 37 THR 8 0 43 41 35

ASP-NH, 16 10 39 17 25 GLU-NH, 16 15 24 40 30

ASP 25 17 46 50 23 GLU 32 26 38 35 40

LYS 0 33 11 17

HIS 6 13 17 8

ARG 3 5 0 16

PHE TYR TRY

16 30 34 35 0 7 20 2

19 4 6 16

CSH cs-SC MET

3 19 60 50 2 11 0 17

8 16 28 24

PRO

0

1

9

13 1

15

20

15 0

0 0 11 0 19

SER I’HR

37

9 14

91 71

70 51

40 47

19

22

33

15 10 14

28 0

17

12

12

6 13

97 78 89 30

9 14

8 40

9 42

15 23

13 24

0

12

15 12

37 18 11

0

ANS- 2 >LU- NH2

20

10

9

9 0

41 23

ASP

5

31

0

0 20

0 0

44 53

-

70 84

-

5

1

0

0

15 0

0

0 1

21

GLU LYS (

!

i

-

i

0

30 0

1

10

7

9 19

0 0

24 40

4

1G

0

1

12

99 80

71

94

0

0

0 11

70 19

39

34

71

57

33

4

17

10 2

0

35 9

25

6

90 98

8 100

71

81

24 7 0

33 17

27

6 -

can also enter electrons into the cytochrome-linked electron transport chain and reduce all cytochromes, with the exception of some 6- and c-type cytochromes which may be bypassed, partially or completely, by a given electron donor.

The usual inhibitors of the electron transport system also inhibited the oxygen consumption in the presence of NADH, catalyzed by a membrane preparation (see Table V). With the exception of HOQNO the inhibitors were effective only at much higher concentrations than for mitochondria, a phenomenon that is well known for bacterial electron transport (15). Never- theless, antimycin and cyanide apparently inhibit the same area of the electron transport chain as in mitochondria because they inhibit the reduction of the same cytochromes (14). Since the inhibition is not complete, even at high concentrations (0.5 m&l antimycin and 10 mM cyanide) of these inhibitors, the NADH

completely reduced whereas the bands comprising cytochromes b and c were only partially reduced (about 70% for NADH and 33 y. for ascorbate plus PMS). Since cytochrome b precedes cytochrome c in the electron transport chain it probably was cytochrome b which, owing to a bypass, was not completely reduced by these energy sources, in agreement with results by Miki et al. (14). Also the flavoproteins, characterized by the trough in the range of 460 to 510 nm, showed incomplete re- duction by either energy source (Fig. 7). In the presence of 20 mM of either L-lactate, succinate, or L-a-glycerol phosphate, all cytochrome bands were partially reduced, but quantitative comparisons were not feasible because the anaerobic state could not be reached owing to the low oxidase activities with these substrates. The above results indicate that all energy sources capable of stimulating amino acid uptake in membrane vesicles

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2416 Amino Acid Transport in Membrane Vesicles of B. subtilis Vol. 247, No. 8

TABLE V

Inhibition of NADH oxidation and L-[14C]serine uptake

n-Serine uptake was measured at 33” for 20 mM n-lactate, and at 25” with special oxygenation for 20 mM NADH or 20 mM suc- cinat,e plus 200 ,UM PMS. The inhibition in the case of n-lactate,

NADH, and succinate plus PMS was studied in casein hydrolysate membranes (3 mg of membrane protein per ml). Since ethanol inhibited the uptake (2% ethanol by about 10% and 57, ethanol

by 4Oyo) all ethanol-soluble compounds were dissolved at the high- est possible concentration, usually giving a final concentration of 1% ethanol. All values are stated with respect to controls having

the same ethanol concentration. The samples were incubated for 5 min. The inhibition in the case of ascorbate 20 mM plus 100 PM PMS was studied in NSMP membranes (1.44 mg of membrane

protein per ml) at 25”. The samples were incubated for 1 min. T

Inhibitors Concentration

KCN

KCN KCN . . Azide. . . Antimycin. . HOQNO . . Dinitrophenol. Rotenone. . . Amytal.. p-CMB . Valinomycin.. Oligomycin. . Anaerobic. . .

miw

2

10 20 10

0.5 0.02 0.5

0.25 20

1

4 x 10-S 0.1

NADH xidation

31 65

85 73

0

I

I

Serine uptake energy

in presence of

64 33 48

97 78 100 69 60 82 66 82 75 79 70 97

79 17 15

51 0 89

0

96

85

14 73

60

oxidation system may partially bypass the sites inhibited by these compounds as has been concluded by Miki et al. (14).

The above inhibitors of the cytochrome-linked electron trans- port system also inhibited serine uptake energized by L-lactate, NADH, and succinate or ascorbate in combination with PMS (Table V). (However, rotenone and oligomycin inhibited sig- nificantly only the uptake in the presence of L-lactate, indicating that it specifically reacted with the lactate branch entering the electron transport chain.) 2,4-Dinitrophenol, which uncouples oxidative phosphorylation in mitochondria (16), also inhibited serine uptake. The need for oxygen in amino acid uptake was demonstrated by the fact that replacement of oxygen with argon reduced serine uptake by 85 and 80% in the presence of NADH or L-a-glycerol phosphate, respectively (in vesicles of NSMP- grown cells).

Inwolvement of ATP-In collaboration with Dr. M. Cashel (of the National Institutes of Health) the following attempts were made to see if phbsphorylation is involved in amino acid trans- port.

1. Vesicles of cells grown in casein hydrolysate medium were washed three times with 0.1 M Tris-Cl, pH 6.6, each time being thoroughly resuspended. The vesicles (3 mg of protein) were then incubated (in 0.05 M Tris-Cl, pH 6.6, plus 10 mM MgS04) in the presence of electron donors (L-lactate (20 mM), NADH (20 mM), or ascorbate (20 mM) plus PMS (100 PM)), serine (8 x 1OF M), and cold nucleotides (ADP, ATP, or ATP, GTP, UTP,

CTP (0.2 mM)) singly or in combinations, for 1 to 15 min. The vesicles accumulated serine at about the same rate as in 0.05 M

potassium phosphate, pH 6.6. They also transported “Pi (e.g. at concentrations of 5, 50, and 100 PM the uptake of 32Pi after 15.min incubation with 20 mM L-lactate was respectively 50, 530, and 1000 pmoles per mg of membrane protein). But, the amount of 32P-labeled nucleotides (ATP, GTP, UTP, or CTP) found after 15 min was less than 2 pmoles per mg of membrane protein.

2. To test the possibility that the phosphate donor for the phosphorylation of nucleotides was the membrane-bound phos- phate, membrane vesicles were prepared from E. coli K-12 grown in Tris-buffered minimal medium supplemented with 0.2% casamino acids and 32P (total Pi = 0.4 mrvr). The resulting 32P-labeled membrane vesicles were tested for the capacity to perform phosphorylation of nucleotides as described above. The amount of 32P-labeled nucleotides was less than 10 pmoles per mg of protein. No changes in this amount were observed in the presence of n-lactate, NADH, ascorbate plus PMS, casamino acids plus n-lactate or proline plus n-lactate.

These negative observations and the lack of amino acid up- take by ATP or GTP argue against the involvement of a phos- phorylation process for amino acid transport.

DISCUSSION

In membrane vesicles of B. subtilis, the transport of almost all natural L-amino acids can be activated by several electron donors, which can enter the electron transport chain either via dehydro- genases (L-a-glycerol phosphate, L-lactate, NADH, or NADPH) or directly (reduced PMS). The only exception was L-trypto- phan for which no energized transport was observed, in spite of the fact that the B. subtilis strain required L-tryptophan for growth and must therefore be able to take it up into intact cells.

The energy of amino acid transport is derived from some com- ponent in the electron transport system that passes electrons from the physiological substrates via flavoproteins and the cyto- chromes b, cl, c, and a to oxygen. The usual inhibitors of the cytochrome chain (azide, cyanide, HO&NO, and antimycin) inhibit the amino acid transport energized by all of the electron donors used. 2,4-Dinitrophenol, which uncouples oxidative phosphorylation in mitochondria (16), inhibits amino acid up- take in B. subtilis vesicles without affecting NADH oxidation. It might uncouple the amino acid transport carrier from the electron transport chain or alter the permeability properties of the membrane. Rotenone and oligomycin specifically inhibit the amino acid transport stimulated by L-lactate for unknown reasons. The above compounds also inhibit lactose transport in vesicles of E. coli with n-lactate as electron donor (4).

Different electron donors energize amino acid transport with very different efficiencies. A measure for this “efficiency” can be obtained by comparing the maximum rate of amino acid uptake with that of oxygen consumption in the presence of different electron donors (Table II). The ratio of these rates (measured in membranes of NSMP-grown cells) is highest for L-ar-glycerol phosphate, followed by L-lactate (determined in other experiments as 5 to 7) and by ascorbate plus PMS, and it is low for succinate, NADH, or NADPH. L-a-Glycerol phos- phate, which is 40 times more efficient than the least efficient compound (NADPH), can therefore donate a large fraction of the electrons it liberates to those electron transport complexes on the membrane to which one of the different transport carriers

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Issue of April 25, 1972 W. N. Konings and E. Freese 2417

- Na, S, 0,

-- Ascorbate t PMS

-.- NADH

I --- Na, S2 Oe / Ascorbate t PMS

A=0.2

400 450 500 550

“Ill

I’IG. 8. Anaerobic reduction of membrane respiratory compo- nents by ascorbate-PMS, NADH, and dithionite. The difference spectra shown were obtained at room temperature (23”) with an Aminco-Chance split beam spectrophotometer. The oxidized/ oxidized base-line was adjusted for aerobic membrane suspensions (3.0 ml) containing 50 mM potassium phosphate (pH 6.6), 10 rnM MgS04, and 5 mg of membrane protein per ml in both sample and

is coupled. In contrast, most electrons liberated by NADH (or NADPH) apparently bypass the coupling site of transport carriers either spatially or because they enter the electron transport chain downstream from the carrier coupling site.

While reduced PMS can be oxidized by free oxygen, it can also pass electrons into different sites of the electron transport system. At least one of these sites must be at or before the coupling site for the amino acid carriers, because reduced PMS energizes amino acid transport. Since this transport is inhibited by cyanide (which inhibits cytochrome oxidase) and by HOQNO or autimycin (both of which inhibit electron flow into cyto- chrome cr), the carrier site apparently is coupled to the electron trausport system before cytochrome cl. Reduced PMS can even reduce some membrane flavoprotein, as indicated by the absorption trough at 465 nm. Rut the small (14%) inhibition of uptake by 20 mM amytal, which reacts rather specifically with flavoproteins (17), indicates that at most a small portion of reduced PMS energizes amino acid transport by reducing flavo- protein. If NADH (NADPH) or succinate would enter electrons only into cytochrome 5, the coupling site would have to precede cytochrome 5; these inefficient electron donors might then energize amino acid transport by an inefficient reverse electron flow. However, we do not know whether the menaquinone of B. suhtilis accepts electrons preceding or following cytochrome b, and there are indications (14) that NADH can pass electrons directly into cytochrome c, bypassing cytochrome b. We can therefore only conclude that the amino acid carriers are coupled to the electron transport system somewhere after a flavoprotein

reference beams. The substrate was then added to the suspension in the sample beam and the spectrum scanned until no further ab- sorbance changes were observed indicating an anaerobic steady state. After this state had been reached (in about 1 min), the spectra were recorded. The concentrations of the electron donors were 5 mM NADH, 20 mM ascorbate plus 30 FM PMS, and 0.2 mg per ml of dithionite.

(e.g. of glycerol phosphate dehydrogenase) and before either cytochrome b or menaquinone, but in any case preceding cyto- chrome cl.

These data are consistent with the proposal of Barnes and Ka- back (18) for E. coli (which has no c-type cytochromes) that the lactose carrier is located in the electron path between an FAD- linked dehydrogenase and cytochrome b. In contrast to E. coli, the carrier of B. subtilis could not be linked to n-lactate dehydro- genase because n-lactate stimulates amino acid transport ex- tremely weakly.

Although succinic dehydrogenase is present in membranes at a high activity, it apparently is only weakly coupled to the cytochrome system (low succinic oxidation activity) in NSMP- grown cells and not measurably coupled in casein hydrolysate- grown cells. (This also agrees with the finding that B. subtilis cannot grow on succinate as sole carbon source.) The gap in the electron transport from succinate can be bridged by PMS, which can then also energize amino acid transport.

When membrane vesicles are prepared from cells grown in different media, they show different dehydrogenase and oxidation activities as well as different amino acid transport efficiencies with different electron donors. For example membranes of cells grown in casein hydrolysate medium which contains no measur- able glycerol show hardly any oxidation or amino acid transport activity with glycerol phosphate; in contrast, membranes of cells grown in NSMP which contains 0.47 mM glycerol show significant oxidation and amino acid transport activities with glycerol phosphate. Growth in glycerol induces the FAD-linked

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Amino Acid Transport in Membrane Vesicles of B. subtilis Vol. 247, No. 8

glycerol phosphate dehydrogenase in B. subtilis (19). This en- zyme is also inducible in E. coli (20) and membrane vesicles containing it can be energized by L-oc-glycerol phosphate to transport amino acids and B-galactosides (18, 21, 22). This media effect has to be kept in mind when the efficiency of elec- tron donors of different organisms are compared.

Apart from the media effect, different organisms differ in their capacity to produce different dehydrogenases which serve as entry ports to the electron transport chain and energize the amino acid transport system. This is already clear from the different response of E. coli (3) and B. subtilis to L- uersus D-lac- tate or to succinate. It also follows from a finding by Short et al, (23) in Staphylococcus aureus that L-cx-glycerol phosphate seems to be the only physiological electron donor. Our finding of the nonenzymic entry of electrons into the amino acid trans- port system by reduced PMS has made it possible to reveal amino acid transport in vesicles of microorganisms for which no physiological electron donor has so far been found (24).

The K, (and V,,,) values measured for different amino acids in B. subtilis vesicles have to be regarded as preliminary because they were determined within a limited concentration range. It is possible that for some amino acids more than one carrier exists and therefore more than one K, value will be found in more elaborate experiments. The same limitation applies to the competition studies using different amino acids. These studies show that distinct carriers exist for different groups of amino acids and they indicate which amino acids use a common carrier. But they do not exclude the possibility of additional carriers that are not common for the other amino acids of the same group. Such two transport systems with different specificity have been observed for lysine in intact cells (25).

The liquid volume of the membrane vesicles (2 to 4 ~1 per mg of protein) represents only a small percentage of total liquid in the above transport experiments (e.g. 0.3 to 0.6% in Fig. 6). Nevertheless, a large fraction of the added amino acid is taken up in the presence of an efficient energy source. For example in the experiment in Fig. 6, with ascorbate plus PMS as energy source, 8% of the serine was found inside the vesicles after 5-min incubation. Since the m em b rane protein concentration was 1.5 mg per ml, this corresponds to a concentration factor of 13 to 26 above the external concentration. This concentrating power is still smaller than that of metabolizing whole cells (which was about 40 times higher for the uptake of the nonmetabolizable ar-aminoisobutyric acid3). This difference may be due to losses in the membrane isolation procedure and to proteolytic degrada- tion. It is therefore likely that the V,,, values of intact cell membranes would be higher than the values determined here.

The uptake of amino acids starts immediately after the addi- tion of the electron donor to the vesicle preparation. This would not seem to leave time for much of the electron donor to enter the vesicles (in the case of phosphoenolopyruvate transferase experi- ments the vesicles have to be preincubated with the energy donor to ensure a maximal rate of sugar transport), unless one would assume that the vesicles were open without energy source and would close after addition of the energy donor. I f that were the case one would expect the vesicles to open again after the

3 C. Sheu, W. N. Konings, and E. Freese, manuscript in prepa- ration.

energy donor has been used up. This possibility is unlikely because we have ascertained that the vesicles can take up YLol- methylglucoside in the presence of (preloaded) phosphoenol- pyruvate, at the time at which [3H]glycine, which has been taken up in the presence of ascorbate and PMS, starts to leak out be- cause all ascorbate has been oxidized. It therefore appears likely that the electron donor can provide electrons from without the vesicle. This contention will have to be thoroughly exam- ined. Our results also indicate no involvement of a phosphoryl- ated intermediate in the transport of amino acids, but we cannot rule out the possibility of a rapid turnover of such a compound.

Acknowledgments-One of the authors (W. N. K.) is indebted to Dr. H. R. Kaback for his generous advice during this investi- gation. He also would like to thank the National Institute of Neurological Diseases and Stroke for a visiting fellowship and

the Niels Stensen Stichting for additional support.

1. 2.

lLEFERFNCI~‘S i ,

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U. S. A. 66, 1008-1015 BAHNES, E. M., ANU KAI~ACIC, H. I:.. (1970) Proc. Nal. Acad.

Sci. U. S. A. 66, 1190-1198 BHATTACHARYYA, P. (1970) J. Racleriol. 104, 1307-1311 BHATTACHARYYA. P.. EPSTI:IPV‘. W.. ANU SILVER. S. (19711 Proc.

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Sci. Works. Fat. Sci. Osaka Univ. 16. 33-58 SMITH, L. (1961)in I. C. GUNSALUS AND R. Y. STANIER (Edi-

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Wilhelmus N. Konings and Ernst FreeseBacillus subtilisAmino Acid Transport in Membrane Vesicles of

1972, 247:2408-2418.J. Biol. Chem. 

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