Stimulation of proline transport by cupric ion in membrane vesicles from mycobacterium phlei

Stimulation of proline transport by cupric ion in membrane vesicles from mycobacterium phlei

BIOCHEMICAL Vol. 69, No. 2, 1976 AND BIOPHYSICAL RESEARCH COMMUNICATIONS STIMULATIONOF PROLINETRANSPORT BY CUPRICION IN MEMBRANE VESICLESFROMMYCOB...

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BIOCHEMICAL

Vol. 69, No. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

STIMULATIONOF PROLINETRANSPORT BY CUPRICION IN MEMBRANE VESICLESFROMMYCOBACTERIUM PHLEI S.A. Yankofsky* and Arnold F. Brodie Department University of School Los Angeles, Received

January

of Biochemistry Southern California of Medicine California 90033

26,1976

Membranevesicles of M. phtei actively take up proline in the absence of exogenously provided electron donors. Evidence is provided that endogenous transport is coupled to the oxidation-reduction of lowpotential electron carriers, but does not involve the cytochromes. The endogenous transport was found to be enhanced under either aerobic or anaerobic conditions by the addition of certain artificial electron acceptors such as Cu". SUkU44RY:

Like other bacterial

INTRODUCTION:

transport

systems, the uptake of

amino acid by membranevesicles of Mycobacteriwn phlei requires substrate oxidation,

does not require high energy phosphate bonds and is inhibited

by anaerobic conditions proline

requires,

specific

less effective electron proline

proline

In the

M.

phlei system, the uptake of

to substrate oxidation,

Na+ ion (5), and

(6), but does not require a proton gradient

that substrates such as generated NADHor succinate are

Thus, one might conclude that active

into membranevesicles of

M.

transport

of

phlei requires the input of an

substrate and involves the participation

of the respiratory

This communication demonstrates that the active transport of 2+ in the M. phlei system can be mediated by Cu under both aerobic

and anaerobic conditions. *On leave from Department of Microbiology, Ramat Aviv, Tel-Aviv, Israel Copyright All rights

(7).

for proline uptake than electrons derived from artificial

donors.

oxidizable chain.

in addition

phospholipids

It is of interest

(l-4).

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

455

University

of Tel-Aviv,

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

M4TERIALSANDMETHODS: The growth conditions and the preparation of the electron transport particles (ETP) from M. phlei (ATCC354) have been previously described (8). The uptake of proline, glutamine and glutamic acid was measured as previously described (3,s) except that NaCl was omitted from the assay system when glutamine and glutamic acid were used as the solute. The rate of oxidation was measured polarographically at The identification of radioactive 30' with a Clark oxygen electrode. materials accumulated by membranevesicles was carried out as described by Hirata et. aZ. (2). RESULTSAND DISCUSSION: The relationship total

ml/mg protein

(9).

Thus, incubation of membranevesicles with 25 nmoles/ml

should, in the absence of energy transduction,

uptake of 42.5 pmoles of amino acid per mg protein. conditions,

volume and

in M. phlei membranevesicles has been shown to be 1.7 X 10-3

protein

of proline

between vesicular

uptake of proline

does not occur.

result

in an

Under anaerobic

In contrast,

aerated membrane

vesicles in the absence of added substrate accumulated someproline.

This

endogenousuptake may range from 40 to 80 pmoles of proline/mg protein. Under these conditions,

the uptake of proline

corresponding values obtained with an electron tetramethyl

phenylene-diamine.

appear to establish a two-fold

is ten times less than the donor such as ascorbate-

However, the endogenous levels of transport concentration

gradient.

Endogenousproline uptake is coupled to endogenousrespiration, suggesting that inhibitors the transport

process.

o-phenanthroline

of endogenousrespiration

should also inhibit

It was observed that amytal, atebrin and

abolish both endogenousrespiration

and endogenoustransport

(Table I).

However, inhibitors completely inhibit transport. transport carriers

of the cytochromes such as NHQNO or sodium aside the endogenousrespiration,

but not endogenousproline

The results suggest that the energy for the endogenous process is derived from electron (flavoprotein

Abbreviations:

flow through low potential

or nonhemeiron).

NHQNO,2-n-nonyl-hydroxy-quinoline-N-oxide.

456

Vol. 69, No. 2, 1976

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Effect

of Various Respiratory

Inhibitors

on the EndogenousProline Transport

Inhibitor Inhibition

Proline Uptake

Concentration

pmoles/mg protein

Percent Inhibition

Expt. I ----

88

----

5mM

41

100*

1omM

42

100

5mM

41

100

NHQNO

25ug/ml

85

6.5

NaN3

1omM

78

1.2

KCN

5mM

79

13.1

None Atebrin Sodium amytal o-Phenanthroline

14 Incubation mixtures contained, per ml, 25nmoles C-L-proline, (10,000 CPM/nmole), 50umoles HEPESbuffer, pH7.5, lOumoles MgC12, 20umoles NaCl and 1.86 mg of ETP protein. The reaction was carried out wrth vigorous stirring at 30' for ten minutes and then terminated by 20-fold dilution into 0.05M potassium phosphate buffer, pH7.5. *An uptake of 40-45 pmoles of proline/mg protein is observed in the absence of energy transduction and may be due to diffusion and nonspecific binding of the amino acid.

Endogenousproline transport carriers

transport

pathway involving by oxygen.

appears to be coupled to an electron

direct

reoxidation

of relatively

low potential

Thus, it should be possible to drive active

transport

of proline under anaerobic conditions by simply replacing oxygen with an artificial potential.

electron acceptor system of appropriate A suitable electron

oxidation-reduction

acceptor for this purpose was found to be Cu2+ .

Incubation of membranevesicles with 0.15 mMCuC12 (or CuS04) for just one minute prior

to proline

addition resulted in a four to five

457

fold

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BIOCHEMICAL

2

4

6

8

IO0

T

Fig. 1.

Effect

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2

I

4

6

8

IO

E (min)

M

of Cu2+ on aerobic proline

transport

The mixture contained in a total volume of 2 ml: 1.8 mg ETP protein, 50 umoles HEPES,pH 7.5 buffer, 40umoles NaCl, 20 umoles MgC12, and 50 umoles of 14C-L-proline (10,000 CPM/nmole). Where pertinent, CuCl was present at 150 nmoles/ml, potassium succinate (pH 7.5) at 25 umoles/m1 , or NADH(12.5 umoles/ml). (A) Without added substrate; (B) With succinate; (C) With NADH. Incubation was at 30' and 0.3 ml samples were taken at the indicated time points. Symbols are: -a-o- without CuC12, -o-o- with CuC12. stimulation

of the steady state level of proline uptake (Fig. I).

It

should,

however, be noted that variation preincubation of proline

of the CuC12input or prolongation of the 2+ period reduced the stimulatory effect of Cu . The uptake

mediated by Cu2+ (150-200 PM), is 40 to 50 percent less than

that observed with succinate and NADHas electron donors.

The addition of

Cu2+ ion in the presence of energy donors such as succinate and NADH reduced the steady state level of uptake of proline (Table II).

(Fig. 1B & C), as well 2+ Proline uptake mediated by Cu also

occurred under anaerobic conditions.

Except for Fe3+, other fourth period

as the rate of oxidation

transition tion,

metals were ineffective.

However, even at its optimal concentra2+ (0.6mM), Fe3+ is only one-tenth as effective as Cu .

That electron

flow through low-potential carriers is required for 2+ enhancement of endogenoustransport by Cu is indicated by the fact that amytal, atebrin and o-phenanthroline However, irradiation the uptake of proline.

inhibit

this process (Table III).

of membranevesicles at 360nmlight It is pertinent

458

does not affect

to mention that irradiation

of

Vol. 69, No. 2,1976

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TABLE II Effect

of Cu2+ on ETP Respiratory

Capacity

Oxygen Consumption natoms/mg/min

Additions

ETP

Relative Rates

10.8

1.00

7.0

0.70

54.2

5.02

8.1

0.75

ETP + NADH

67.8

6.28

ETP + NADH+ CuC12

21.1

1.95

ETP + CuC12 ETP + Succinate ETP + Succinate + CuC12

Reaction mixture compositions are identical to Fig. 1.

membranevesicles of M. phZei with light naphthoquinone and results

to those given in the legend

at 360nminactivates

in an inhibition

of oxidation

the natural

via the cytochrome (10)

Except for cyanide, inhibitors of cytochrome function do not significantly 2+ 2+ inhibit the Cu promoted proline uptake. Inhibition of Cu -promoted 2+ transport by cyanide is due to the removal of Cu from the system as a consequence of its The reduction

chemical interaction

with cyanide (11).

of cytochromes b,c and a+a3 in the presence of CuC12

did not occur in the absence of added substrate.

Although reduction of 2+ the cytochromes occurred on addition of substrate and Cu , the rate of reduction was decreased.

2+ Cu also had no affect

on the capacity of

M. phZei cytochromes to undergo chemical reduction by dithionite. above results (e.g.,

indicate

flavoprotein

the participation

and nonhemeiron)

of only low potential

The carriers

in the cupric-promoted proline

process.

459

uptake

Vol. 69, No. 2, 1976

Effect

BIOCHEMICAL

of Inhibitors

TABLE III 2+ on Cu Mediated Proline Transport Under Aerobic Conditions

+cu+2

Inhibitors

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-CU+2

(A-B)

Percent of Inhibition

pmoles/mg protein Expt.

I

None

310

55

255

Amytal

141

7

134

48

Atebrin

62

0

62

76

o-Phenanthroline

16

0

16

94

NHQNO

270

51

219

14

NaN3

303

46

257

0

KCN

54

47

7

97

290

31

259

0

Expt. II 360nmlight

The concentrations of inhibitors and ETP protein were as in Table I, the concentration of CuC12was 150 nmoles/ml.

The role of Cu2+ may be to serve as an efficient

bridge between a part

of the respiratory chain and the transport process. It is pertinent to 2+ mention that Cu has been shown to stimulate glucose transport in fat cells

(12,13) and to play an important role in copper-containing oxygenases (14). 2+ Although the concentrations of Cu required to stimulate transport are nonphysiological

and membranevesicles preparations

systems, the cupric-mediated

transport

appear to be perturbed

process may serve as a model system

for studying the events and the nature of the protein(s) mechanismof active

transport.

460

involved

in the

Vol. 69, No. 2, 1976

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

This work was supported by grants from the National Institutes of Health, US Public Health Service (AI 05637), the National Science Foundation (GB 32351X), and the Hastings Foundation of the University of Southern California School of Medicine. The authors wish to acknowledge the technical assistance of Mrs. Marlene Cartter.

ACKIVOWLEDGMENTS:

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Kaback, H.R., Biochem. Biophys. Acta, 265, 367, 1972. Hirata, H., Asano, A. and Brodie, A.F., Biochem. Biophys. Res. Commun. 44, 368, 1971. Prasad, R., Kalra, V.K. and Brodie, A.F., Biochem. Biophys. Res. Commun.63, 50, 1975. Berger, G.A., Proc. Natl. Acad. Sci. U.S.A., 2, 1514, 1973. Hirata, H., Kosmakos, F.C. and Brodie, A.F., J. Biol. Chem., 249, 6965, 1974. Prasad, R., Kalra, V.K. and Brodie, A.F., J. Biol. Chem., 250, 3699-3703 (1975) Hinds, T.R. and Brodie, A.F., Proc. Natl. Acad. Sci. U.S.A., 7& 1202, 1973. Brodie, A. F., J. Biol. Chem. 243, 398, 1959. Hirata, H. and Brodie, A.F., Biochem. Biophys. Res. Commun.,47, 633,

10. 11. 12. 13. 14.

1972.

Asano, A. and Brodie, A.F., J. Biol. Chem. 9, Yonetani, T., Biochem.Biophys. Res. Commun.,3, Czech, M.P. and Fain, J.N., J. Biol. Chem. 247, Czech, M.P., Lawrence, J.C. (Jr.) and Lynn, W.S., 1001, 1974. Gunsulas, I.C., Pederson, T.C. and Sligar, S.G., 44,

377,

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