Na+-dependent amino acid transport in isolated membrane vesicles of a marine pseudomonad energized by electron donors

Vol. 47, No. 4, 1972

BIOCHEMICAL

Na+-DEPENDENT

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AMINO ACID TRANSPORT IN ISOLATED MEMBRANE VESICLES

OF A MARINE PSEUDOMONAD ENERGIZED BY ELECTRON DONORS G. Dennis

Sprott

and Robert

A. MacLeod

Department of Microbiology Macdonald Campus of McGill University Montreal 870,Quebec,Canada

Received

April

17, 1972

SUMMARY: Transport of L-alanine and AIB into isolated membrane vesicles of marine pseudomonad B-16 (ATCC 19855) was stimulated by NADH and ascorbate-TPD. Other electron donors tested and ATP had no stimulatory effect. The u take of both amino acids required Na+ specifically. Neither Li+, K+ nor RbP could replace Na+ for transport. Using L-alanine as the test amino acid, it was found that both endogenous transport and NADH stimulated transport was inhibited by anaerobiosis, HOQNO and CN-. Transport in the presence of ascorbate-TPD was inhibited by anaerobiosis and by CN- but not by HOQNO. PEA induced the loss of L-alanine from membrane vesicles which had been allowed to accumulate the amino acid, suggesting that L-alanine had been concentrated inside the vesicles against a gradient. Thus, the Na+-dependent transport process is an active one and is coupled to the oxidation of electron donors. The site of energy coupling appears to lie below the quinone in the respiratory chain. INTRODUCTION Most

bacteria

In the case

(1). this for

marine

requirement

of a marine for

Na+ for

Na+ to transport

be required

Bacillus and for

for

the

pseudomonad growth

metabolites transport

Zicheniformis

typhimuriwri marine

the

transport

Na+ specifically

require

into

(MacLeod, of thiomethyl

shown to reflect

the cells.

of glutamate Thurman

growth

and metabolism

and Photobacterium fischeri

(2)

has been

for

into

Na+ has also

been

Escherichia coZi

and Rogers,

unpublished

S-D-galactopyrancsLde

into

(3)

a requirement (4)

shown

to

and

observations)

SnlmoneZla

(5).

The mechanism pseudomonad

of Na+-dependent transport of AIB into B-16 has been examined in some detail.

to decrease the Km for does for Na+-dependent

intact cells of Na+ has been shown

AIB transport into the marine pseudomonad (6), as it solute transport into animal cells (7), suggesting

Abbreviations: AIB, a-aminoisobutyric.acid; TPD, N,N,N',N'-tetramethyl-pHOQNO, 2-heptyl-4-hydroxyquinoline-N-oxide; phenylenediamine dihydrochloride; PEA, phenethylalcohol; PMS, phenazine methosulfate. 838 Copyright@1972,by

Academic Press, Inc.

BIOCHEMICAL

Vol. 47, No. 4, 1972

that

Na+ increases

compound

being

the affinity

observations

membrane

have led

facilitated

a gradient

intracellular

requires

coupling

is

Na+ is

the

the penetration

system.

required

while

These

for

the

of energy

in animal

believed

shown that

(11,

the

transport

to the

oxidation

on electron

the

accumulation

and the presence

have been

developed

pseudomonad

B-16 which

dependent

transport

the

(7).

of

acids,

sugars

electron

ATP dependent

and other

donors

free

membrane

vesicles

of cell

wall

depends

procedures of marine

material

of this

can be

which

Since membranes

vesicles

have

metabolites

in a system

of cytoplasmic

are completely

and co-workers

membrane

phosphorylation.

the isolation

of energy

Kaback

bacterial

oxidative

by isolated

cell

isolated

of various but not

for

the mechanism through

in the

12) using of amino

transport

cells

to be mediated

of Na+ and K+ gradients

and others

coupled

for

to permit

uncoupled

the membrane

the expenditure

transport

generally

maintenance 10)

that

or carrier

shown

K+ (8).

In Na+-dependent

(9,

conclusion

of AIB across

RESEARCH COMMUNICATIONS

protein been

by AIB in an energy

to the

diffusion

against

of a binding Na+ has also

transported.

of the cytoplasmic

AND BIOPHYSICAL

(13),

organism

Na+-

has been

examined. MATERIALS Cells

were

described

previously

pressure

cell

treated

(13).

DNA'ase

in a solution HCl.

in a complex

operating

with

referred

0.2 M; KCl, with

grown

0.01

medium

Protoplasts

at 15,000 (50 rig/ml),

were

- 16,000 RNA'ase

0.05

within at 75,000

sedimented

washed

suspension

previously intact

microscopy viable those

was adjusted

calibrated cells

curve.

(13),

0.05

preparations by phase

by Kaback

of serological

(9).

per ml with

Tris.

were

suspension

complete-salts

Tris

and 0.1 ml of the suspension tubes. The electron donor,

judged

free

of a of

and electron numbers

a modification was diluted solution,

The

the aid

insufficient

The membrane

839

and the

contrast

to contain

to pH 8.5

x g was discarded.

complete-salts

cells to account for membrane transport activity. The procedures used in the transport studies were described

of NaCl,

M adjusted

at 4,080

were

(150 pg/ml)

to 10 mg/ml with

as determined count

of a French

fragments

35 minutes

with

turbidimetrically

and shown by plate

aid

and comprised

base,

x g for

twice

The membrane

and protoplasts

dry wt. of membranes otherwise indicated, series

were

the

and lysozyme

15 minutes

was centrifuged

by methods

The membrane

Tris

The supernatant final

psi.

phase with

(50 ug/ml)

membranes

which

broken

M; and Trizma

sedimentable

METHODS

to mid log

to as complete-salts

M; MgSOb,

Material

and

of

of to 5 mg

unless

was added

to each

of a

dissolved

in complete-salts

Vol. 47, No. 4, 1972

Tris in

BIOCHEMICAL

solution,

was added

complete-salts

Tris

was 0.17 the

ml.

After

reaction

The suspension

liquid

diphenyloxazole/l wt. tions

performed

other

spectrometer

using

solution were

labeled,

14C-AIB

(8.6

M final

concentration

is

at 25'C, solution.

overlaid

with

Tris

a

solution.

Isocap/300 fluid

reported

represent

mixture

Tris

5.0 ml scintillation activity

dissolved

shaker

Chicago

(5 g 2,5-

as cpm/mg dry

the average

of determina-

Amersham

from an ascorbic

base.

Where necessary,

Trizma

base.

Searle,

Corp.)

was used

in were

of all

x 10T6 M and

Nuclear)

at 5.5

x 1Ou5

mixture.

of residual digested

solution

(156 mCi/m mole,

at 1.8

New England

the reaction

the level

acid

pH adjustments

14C-L-alanine

carboxyl-labeled,

Na+ in membrane

with

preparations

HN03 and Hc101, (14)

prepared

and analyzed

photometrically. RESULTS Compounds

systems

(9,

shown

10, 11,

and AIB transport increased ascorbate-TPD is

12) were

for

of interest

of both

the

uptake

that

as electron

tested

isolated

for

donors

their

membrane

1, show that

the uptake

DISCUSSION

and

to be active

into

Table

The results,

It

filter

in a Nuclear

used was prepared

made with

samples

Na+,

on a rotary

4.0 ml of complete-salts

recorded

Trizma

mCi/m mole,

To determine without

of the reaction

u Millipore

Transport

results

acid

in duplicate.

solutions

flame

with

to pH 8.5 with

uniformly

a 0.8 counted

All

The ascorbate adjusted

times

14C-amino

0.4 ml of complete-salts

and then

toluene).

of membranes.

specified

and washed dried

scintillation

volume

with

using

by the

The final for

was diluted

was filtered were

immediately

solution.

Whatman 1/3 prefilter, The filters

followed

incubation

mixture

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

capacity

vesicles

two of these,

amino

ascorbate-PMS

L-alanine

pseudomonad

B-16.

NADH and ascorbate-TPD,

somewhat

which

bacterial

to stimulate

of marine

NADH was equally

acids.

of AIB but

in other

less

is active

as effective

active

for

as

alanine.

as an artificial

elec-

tron donor in the other systems tested (11, 12) was inhibitory or ineffective here. As can be seen, this was due to inhibition produced by PMS. ATP was also into

somewhat

inhibitory

A highly

specific

requirement

membrane

vesicles

isolated

and absence could

replace

respond than activity

of an added less

was used

of the AIB

level for

tested. Na+ for

the

transport

could

be demonstrated both donor, Figure 1. Neither The uptake of AIB could system.

electron

Na+ in this

to Na+ but

alanine

at the

dramatically. in an effort

(see Materials

Since

a higher

inthepresence Li+, K+ nor also

for

the

and Methods),

the higher

lower

Rb+

be shown

concentration

to compensate

840

of L-alanine

to

of AIB specific

non-Na+-dependent

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL

TABLE Effect

RESEARCH COMMUNICATIONS

1

of potential electron donors and ATP on the uptake of L-alanine AIB by isolated membrane vesicles of marine pseudomonad B-16 L-alanine Compound

and

AIB

tested Uptake*

Ratio+

Ratio

Uptake cpmlmg

cpmlmg None

5,558

1.00

1,732

1.00

NADH

9,554

1.72

5,496

3.17

NADPH

5,798

1.04

2,045

1.18

Succinate

5,956

1.07

1,301

0.75

D(-)-Lactate

3,983

0.72

1,051

0.61

Ascorbate-TPD

15,100

2.72

5,667

3.27

Ascorbate-PMS

2,593

0.47

2,321

1.34

PMS

1,408

0.25

Ascorbate

4,836

0.87

ATP

4,241

0.76

D,L-a-Glycerophosphate

3,981

0.72

819

0.47

1,279

0.74

932

0.54

1,218

0.70

Compounds tested were at 20 mM with the following exceptions: NADH, 25 mM; NADPH, 25 mM; TPD, 150 nM; PMS, 100 pM. The compound tested was added just prior to the 14C-amino acid. *Uptake by membrane vesicle preparation after 5 minutes incubation. SRatio of the uptake in the presence, to uptake in the absence, of an added electron donor. uptake

of AIB observed

membranes.

The level

The stimulation

is

probably

due to non-specific

of residual

Na+ in the

of L-alanine

transport

in other the

bacterial

membrane

acid

transport

electron

donor

Kaback the site between

D-lactic

E. cozi

(9,

appear

to 02 as the

and co-workers

of energy

vesicle

would

systems

dehydrogenase

for

so far

depressed donor.

examined

lactose

evidence and amino

(9,

the transport Thus,

as

10, 11,

12),

of electrons

and cytochrome

bl,

indicating acid

inhibited NADH, but

it

on transport

in the presence

841

that

transport

from

in E. cozi is

localized

in the respiratory

15).

had no effect

required

by a flow

In the E. coli system, the quinone lactose uptake (9). In the case of marine endogenous L-alanine transport and transport

inhibited

was 1.1 mM.

acceptor.

have presented

coupling

Np markedly electron

to be energized terminal

of AIB to the

mixture

by ascorbate-TPD

presence of 02, Figure 2. Replacing air with both in the presence and absence ofthcartificial amino

binding

incubation

inhibitor,

chain

of

HOQNO,

pseudomonad B-16, in the presence of ascorbate-TPD,

HOQNO of

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

“6 t= P ia 8 $12 4 t =a % ii

Y ‘3 t0 2 ;’ 3 !? ?’

24

Y :o

CON1

NaCl

LiCl

0 KCI

RbCl

CONT

N&l

LICI

KCI

RbCl

FIG. 1. Requirement for Na+ for the uptake of L-alanine and AIB by isolated membrane vesicles of marine pseudomonad B-16. Left figure, L-alanine; right figure, AIB. Shaded bars, no added electron donor (ENDOG); hatched bars, ascorbate-TPD (A-TPD); control (CONT), no added salt. Membranes were prepared by substituting LiCl for NaCl in the complete-salts Tris solution during the final stages of isolation. The membranes were then collected by centrifugation and resuspended to 10 mg/ml in Mg-K-Tris solution (MgSOb, 0.05 M; KCl, 0.01 M; Tris-HCl, 0.05 M; pH 8.5). Aliquots of this suspension were1 diluted to 5 mg/ml with Mg-K-Tris solution containing either no added salt (CONT) or sufficient NaCl, LiCl, KC1 or RbCl to give 0.2 M in the final incubation mixture. For the transport studies the procedures described in Materials and Methods were used with the exceptions of adding the electron donor and the 14C-alanine in Mg-K-Tris solution and of washing the membranes on the filter with complete-salts Tris solution containing LiCl in place of NaCl. Ascorbate, TPD and 14C-alanine concentrations as in Table 1. Incubation time, 5 min.

1

FIG. 2. Effect of aerobic and anaerobic conditions on L-alanine uptake by isolated membrane vesicles of marine pseudomonad B-16 in the presence and absence of ascorbate-TPD. The membrane suspensions contained in serological tubes were either left open to the atmosphere or sealed with serum caps and flushed with N2 for 5 min. In the case of the sealed tubes, subsequent additions to the membrane suspension were made by injection through the cap. Curve 1, air, no added electron donor; ascorbate-TPD; Curve 2, air, Curves 3, 4, N2, with or without ascorbate-TPD. 842

BIOCHEMICAL

Vol. 47, No. 4, 1972

Table

2.

Transport

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the presence

of both

electron

donors,

as well

as

was inhibited markedly by cyanide. These findings endogenous transport, would suggest that in the case of marine pseudomonad B-16, the site of energy coupling

for

transport

respiratory

of L-alanine Further

chain.

irradiating the

inhibits

quinone

transport

Zicheniformis

6346

(MacLeod,

pseudomonad

and transport

to ascorbate-TPD

fact

that

electron

not

this

into

uptake

transport

endogenous i'e have not

Thurman

donor

and Rogers,

2 that

is known

chain

(16)

phZei

unpublished

by membrane

of the marine

transport

coupled

vesicles

be substantially

is

a relatively

of the marine reduced

Figure

in the membranes

identifiedthisindigenous

high

capable

pseudomonad.

by inhibition

2, suggests electron

endogenous

that

of energizing donor,

but

is

Uptake*

None

10,677

NADH

None

12,807

Ascorbate-TPD

None

46,607

None

HOQNO

NADH

HOQNO

3,363

Ascorbate-TPD

HOQNO

43,622

3,009

None

CN-

NADH

CN-

2,435

Ascorbate-TPD

CN-

2,214

2,034

HOQNO and KCN were present at 2.0 x 10S5 M and 1.0 x 10S3 M, respectively. The inhibitors were added 15 minutes prior to the addition of exogenous donor and 14C-L-alanine. Concentrations of added electron donor as in Table 1. "as in Table 1. 843

an

transport. since

2

Inhibitor added

The of

there

cpmlmg None

and B.

observations).

of inhibitors added in the presence and absence of electron donors on the capacity of isolated membrane vesicles of marine pseudomonad B-16 to take up L-alanine

Electron donor added

to

and

(11)

vesicles

of NADH, but not

there

TABLE Effect

procedure

transport

by

shown).

membrane

could

This

and 02 in the

was obtained

of Mycobacterium

transport

or anaerobiosis,

electron yet

vesicles

the quinone

conclusion

electron

in the presence

can be seen in Table of L-alanine

this

360 nm light.

endogenous

(data

between

for

of the

in membrane

inhibited

It

with

component

Irradiation

uptake

support

the membranes

destroy

lies

HOQNO is

Vol. 47, No. 4, 1972

able

to inhibit

natural

BIOCHEMICAL

endogenous

electron

a level

donor

preceding

Evidence active

the

that

transport

leakage late time. quickly

the

of PEA, but

the membrane

resuspended

with per

using

evident

that

a gradient.

per

of this

L-alanine results

membrane

into

appear

that

the

the respiratory

membrane

not

chain

at

was being

vesicles

Figure

3.

of PEA they

were

into active

pseudomonad

24

water

(data

extraction.

the membrane

vesicles

30

of not

vesicles It

B-16

exposed

to be capable

as L-alanine.

transportof

with

was

PEA treatment

by hot

transported

reaction

followed

in membrane

remained

in a

to the

found

the

to accumu-

When the membranes

before

be recovered

12 TIME

added

radioactivity

Na+-dependent

6

organism allowed

vesicles

absence,

activity

of marine

0

were

is an

to increase

in its

prevailing

could

known

of this

vesicles

of the radioactivity

recovered

vesicles

in the membrane

vesicles,

cent

PEA is

PEA was then

the absence

show that

PEA.

Membrane

to levels

14C-L-alanine

cent

against

These

in

again

Ninety-eight

would

by the membrane

cytoplasmic (17).

of radioactivity

preloaded

AIB into

was obtained

manner

up L-alanine

Ninety

of L-alanine

and the level

shown).

electrons

to the maximum level.

from

it

quinone.

In the presence

to PEA were

activity,

introducing

through

reversible

lost

taking

is

process

14C-L-alanine

mixture

transport

the uptake

of solutes

completely

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

L-alanine

can be coupled

is

thus

and to the

36

- i%

FIG. 3. PEA induced loss of "C-L-alanine from isolated membrane vesicles. Vesicles were allowed to accumulate L-alanine for 10 min in the absence of exogenous electron donors. At zero time either complete-salts Tris solution, The final curve 1, or PEA in complete-salts Tris solution, curve 2, was added. PEA concentration was 0.25%.

844

BIOCHEMICAL

Vol. 47, No. 4, 1972

oxidation

of electron and inhibitors

transport,

oxidative

in this

process

further

evidence

and eucaryotic

In consideration

donors.

the donors

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

used,

and the

fact

phosphorylationand

as was previously that cells

of the that

ATP are supposed

the mechanisms

(8).

effects

ATP failed

of

to stimulate

probably

not

These

findings

of Na+-dependent

on transport

transport

directly

involved

provide in procaryotic

may be different.

ACKNOWLEDGMENTS This Canada. McGill

work G.D.S.

was supported is

the holder

by a grant

from

of a McConnell

the National

Research

Council

pre-doctoral

fellowship

from

of

University.

REFERENCES 1. MacLeod, R.A., Bacterial. Rev., 2, 9 (1965). 2. Drapeau, G.R., and MacLeod, R.A., Biochem. Biophys. Res. Commun. 12, 111 (1963). and MacLeod, R.A., J. Bacterial. 92, 63 3. Drapeau, G.R., Matula, T.I., (1966). I., J. Bacterial. 100, 329 (1969). 4. Frank, L., and Hopkins, 5. Stock, .I., and Roseman, S., Biochem. Biophys. Res. Commun. 46, 132 (1971). 6. Wong, P.T.S., Thompson, J., and MacLeod, R.A., J. Biol. Chem. 244, 1016 (1969). 7. Schultz, S.G., and Curran, P.F., Physiol. Rev. so, 637 (1970). 8. Thompson, J., and MacLeod, R.A., J. Biol. Chem., 246, 4066 (1971). 9. Kaback, H.R., and Barnes, E.M., Jr., J. Biol. Chem., 246, 5523 (1971). Chem. 247, 298 (1972). 10. Short, S.A., White, D.C., and Kaback, H.R., J. Biol. 11. Hirato, H., Asano, A., and Brodie, A.F., Biochem. Biophys. Res. Commun. 44, 368 (1971). 12. Konings, W.N., and Freese, E., FEBS Letters, I&, 65 (1971). 13. Martin, E.L., and MacLeod, R.A., J. Bacterial. 105, 1160 (1971). 14. Sanui, H., and Pace, N., J. Gen. Phys., 42, 1325 (1959). 15. Barnes, E.M., and Kaback, H.R., J. Biol.-Them. 246, 5518 (1971). 16. Kashket, E.R., and Brodie, A.F., J. Biol. Chem., 238, 2564 (1963). 17. Thompson, J., and DeVoe, I.W., Can. J. Microbial., in press.

845