Eur. J. Biochem. 23 (1971) 528-532

Release of Respiratory Control in Particles from Micrococcus denitrijcans by Ion-Translocating Antibiotics

Philip JOHN and W. Allan HAMILTON Biochemistry Department, University of Aberdeen

(Received April 3O/July 28, 1971)

A study was made of the ability of ion-translocating antibiotics to uncouple oxidative phosphorylation in particles prepared from Micrococcus denit r i f icuns. These particles possess respiratory control. Uncoupling was observed as an approximately 3-fold stimulation of the basal rate of respiration.

Addition of valinomycin uncoupled in the presence of NH,+ or a combination of K+ and nigericin. Addition of a high concentration of gramicidin (10 pg/mg protein) uncoupled in the absence of added cations or other antibiotics. Addition of a low concentration of gramicidin (0.1 pg/mg protein) uncoupled only in the presence of NH,+ or a combination of Na+ and monensin.

It is concluded that the orientation of the particle membrane of Micrococcus denitrificam is opposite to that of the intact cell membrane.

Extensive investigations have been made of the effects of ion-translocating antibiotics of the vali- nomycin and nigericin groups on energy conserva- tion in mitochondria [l -31, chloroplasts [4,5], sub- mitochondria1 particles [6 -81, subchloroplast par- ticles [9,10] and chromatophores of photosynthetic bacteria [ll-141. The effects of the antibiotics on these energy-transducing membrane systems have been interpreted in terms of the ability of the anti- biotics to increase the permeability of artificial and natural membranes to specific cations [2,15 -171. Although antibiotics have been shown to facilitate ion transport across the cell membrane of chemo- synthetic bacteria [18-201 little is known of the effect of these antibiotics on bacterial oxidative phosphorylation.

The present paper describes the effect of vali- nomycin, gramicidin, nigericin and monensin on respiratory control in phosphorylating particles pre- pared from Micrococcus denitrificans [21,22]. A slight modification of the previously published [21] isola- tion procedure is also described. This modification resulted in a significant decrease in the basal rate of respiration observed with the particles. Studies of the respiratory-chain system of M . denit r i f icans [23 -251, respira,tion-driven proton translocation by whole cells [26], and the proton conductance of the intact cell membrane [27] suggest that the mechanism

Unusual Abbreviation. CCCP, carbonyl cyanide m-chloro- phenylhydrazone.

Enzymes. Alcohol : NAD oxidoreductase or alcohol de- hydrogenase (EC 1.1.1.1); mucopeptide N-acetylmuramyl- hydrolase or lysozyme (EC 3.2.1.17).

of energy conservation in M . denit r i f icuns is essen- tially similar to that of mitochondria.

MATERIALS AND METHODS

M . denit r i f icans NClB 8944 was grown, and the particles were isolated, as described previously [21] except that in the present study 10mM ATP was added to the 100mM Tris-acetate in which the lysozyme-treated cells were initially suspended. This suspension was subsequently diluted 10-fold with water in order to osmotically lyse the cells, and therefore the concentration of ATP decreased to 1 mM during lysis. The particles could be stored a t 4 "C for up to one week without significantly affecting the properties described in the present paper.

Oxygen consumption was measured with a Clark oxygen electrode; ADP/O ratios were calculated by the procedure of Chance and Williams [28]; and protein was determined by the biuret method, all as described previously [22].

The uncouplers and antibiotics were added as small volumes (1 -4 pl) of acetone solutions. Pre- liminary experiments revealed that up to 20p1 of acetone could be added without significantly affecting the activities studied.

NAD+ and ATP were obtained from Sigma Chemical Co. (London, England) ; yeast alcohol dehydrogenase, egg-white lysozyme and ADP from Boehringer Mannheim GmbH (Mannheim, Germany) ; carbonyl cyanide m-chlorophenylhydrazone (CCCP), valinomycin and gramicidin from Calbiochem Ltd. (London, England). Nigericin (Antibiotic X-464,

Vo1.23, N0.3, 1971 P. JOHN and W. A. HAMILTON 529

see [I]) from Hoffman La Roche Inc. (New Jersey, U.S.A.), monensin and dianemycin from Eli Lilly and Co. (Indianapolis, U.S.A.) were generous gifts.

All other chemicals were of reagent grade.

Table 2. Effect of CCCP, 2,4-dinitrophenol, and gramicidin on the rate of respiration

Conditions were as for Table 1, except that CCCP, 2,4-di- nitrophenol and gramicidin were added to the basic reaction medium as indicated; and 1 mg of particle protein was

present

RESULTS

Effect on Respiratory- Control Ratios of the Particles of Adding A T P

to the Lysis Medium From Table 1 it can be seen that inclusion of ATP

in the medium in which the lysozyme-treated cells were osmotically broken resulted in the particles possessing a lower basal rate of respiration, without affecting in a consistent way their state 3, state 4, or CCCP- stimulated rates of respiration. Thus par- ticles prepared in this way had higher CCCP-induced respiratory-control ratios than particles prepared in the absence of ATP, although the ADP-induced respiratory-control ratios and the ADPI0 ratios did not differ between the two types of particle. Because of their higher CCCP-induced respiratory-control ratios, particles isolated from cells lysed in the pres- ence of ATP were used in the present investigation.

Table 1 also shows that, with either type of particle, higher respiratory rates were observed on addition of 10 pM CCCP than in the presence of ADP and Pi. No simple explanation is available of the relatively slow ADP-induced state 3 rate observed with these particles.

Effect of Uncouplers on the Respiratory Rate In Table 2 the relative effectiveness of CCCP,

2,4-dinitrophenol and gramicidin as uncoupling

Table 1. Rates of respiration and ADPIO ratios of particla prepared from cells lysed in the presence ( A ) and absence (B)

of ATP The basic reaction medium contained, in a total volume of 3 mi, 50 mM Tris-acetate at pH 7.3, 5 mM magnesium ace- tate, 15 pl ethanol, 12 units dialysed alcohol dehydrogenase, and 0.1 ml of particle suspension containing 1.0-1.6 mg protein. The reaction was started by the addition of 0.2 mM NAD+. The temperature was 30 "C. Sodium phosphate (Pi) (3.3 mM), ADP (0.2 mM) and CCCP (10 pM) were added as

indicated

Respiratory- control ratio Respiratory rate

ADP/O Expt no. + +Pi + ADP +CCCP State 3

Control cccp

Respiratory- control ratio Respiratory rate

natoms 0 x min-' x mg protein-'

1 A 83 232 189 109 2.8 1.7 1.3 B 109 226 177 103 2.1 1.7 1.3

2 A 63 180 143 83 2.9 1.7 1.3 B 92 177 140 80 1.9 1.8 1.3

3 A 63 160 140 80 2.5 1.8 1.3 B 126 197 140 77 1.6 1.8 1.4

Rate of Respiratory-control ratio

Addition Oxygen 0, uptake, with additions 0, uptake, no additions uptake

natoms x min-' x mg protein-'

- None 72 CCCP 1p.M 134 1.9 CCCP l0p.M 197 2.7 CCCP 30pM 186 2.6 2,4-Dinitrophenol

0.1 mM 80 1.1 2,4-Dinitrophenol

1 mM 112 1.6

Gramicidin 1 pg 109 1.5 Gramicidin 10 p.g 217 3.0

Gramicidin 0.1 pg 72 1.0

Table 3. Effect of valinonzycin on the rate of respiration in the presence of nigericin, Kf and NH4f

Conditions were as for Table 1, except that valinomycin (1 pg), nigericin (1 pg), potassium acetate and ammonium acetate were added to the basic reaction medium as indi-

cated; and 1 mg of particle protein was present

Respiratory-control ratio 0, uptake, with additions 0, uptake, no additions

Rate of oxygen uptake Additions

Control + Valino- mvcin

Control +Valino- mvcin

natoms x min-'x mg protein-'

- - None 83 77 K+ (10 mM) 92 92 1.1 1.1 Nigericin 83 80 1 .o 1.0 K+ ( 10 mM) + nigericin 92 240 1.1 2.9 NH,+ (10 mM) 103 149 1.2 1.8 NH4+ (100 mM) 132 234 1.6 2.8

agents may be compared. It can be seen that the optimum concentration of CCCP was about 10 pM (cf. [23]) that 2,4-dinitrophenol was a relatively poor uncoupler (cf. [21,23]), and also that under these conditions gramicidin did not uncouple at a concen- tration of 0.1 pglmg protein, although it did markedly uncouple at a concentration of 10 pglmg protein.

Effect of Cation-Translocating Antibiotics on the Respiratory Rate

Addition of either valinomycin or nigericin alone had no significant effect on the rate of respiration, either in the presence or in the absence of K+ (Table 3). However, addition of both valinomycin and nigericin resulted in a K+-dependent increase

35 Eur. J. Biochem., Vo1.23

530

300

- - c .- $ 200

F n

7 .- E

Release of Respiratory Control in Microcoww denitrificans

-

-

Eur. J. Biochem.

+ Valinomycin t Nigericin

f 5 I /

I I 5 10

0 ' 0

[Potassium acetate] (mM)

Fig. 1. Effect of potassium-acetate concentration on the rate of respiration in the presence of valinomycin ( 0 ) and both va- linmycin and nigericin ( A ) . Conditions were as for Table 1, except that valinomycin (1 pg) and nigericin (1 pg) were added to the basic reaction medium which contained the indicated concentrations of potassium acetate; and 1 mg

of particle protein was present

in the respiratory rate (Table 3, Fig.l). Increasing concentrations of NH,+ resulted in some increase in the respiratory rate, but this effect was greatly enhanced by the presence of valinomycin (Table 3, Fig.2).

The pattern of uncoupling observed with vali- nomycin, nigericin and K+ was repeated when gramicidin, rnonensin and Na+ were used ; gramidicin being employed a t a concentration (0.1 pg/mg pro- tein) which when added alone did not uncouple (Tables 2 and 4). Thus, addition of either gramicidin or monensin alone had no significant effect on the respiratory rate, either in the presence or in the absence of Na+ (Table 4). However, addition of both gramicidin and monensin resulted in a Na+-dependent increase in the respiratory rate (Table 4, Fig.3). Furthermore, gramicidin, like valinomycin, stimulat- ed the respiratory rate in the presence of NH,+ (Table 4).

The effects with nigericin and monensin noted above, have also been demonstrated with dianemycin

Comparison of the data shown in Tables 1, 2, 3 and 4 reveals that the respiratory-control ratios

(4 pg).

300

- - c .- 2 200 g F - 'c .- E

E u)

0

c a, -x

a

L - L

z l 100 5 m s 0 0 m c

c

i9

50 100 0

0 [Ammonium acetate] (mM)

Fig.2. Effect of ammonium-acetate concentration on the rate of respiration in the presence ( A ) and absence (0 ) of valirw- mycin. Conditions were as for Table 1, except that valino- mycin (2 pg) was added to the basic reaction medium which contained the indicated concentrations of ammonium ace-

tate; and 1 mg of particle protein was present

+Gramkidin 200 - ._ m

c Q

c

z 8 L

m c n

0- 0 5 10

[Sodium acetate] (mM)

Fig.3. Effect of sodium-acetate concentration on the rate of respiration in the presence of gramicidin (0 ) and both grami- cidin and monensin ( A ) . Conditions were as for Table 1, except that gramicidin (0.1 pg) and monensin (3 pg) were added to the basic reaction medium which contained the indicated concentrations of sodium acetate; and 1 mg of

particle protein was present

Vo1.23, No.3, 1971 P. JOHN and W. A. HAMILTON 531

Table 4. Ejfect of gramicidin on the rate of respiration. in. the. presence of monensin, Na+ and NH,+

Conditions were as for Table 1, except that gramicidin (0.1 pg), monensin (3 pg), sodium acetate and ammonium acetate were added to the basic reaction medium as indicat-

ed; and 1 mg of particle protein was present Respiratory-control ratio 0; uptake,withadditions

Additions 0; uptake, no additions Rate of oxygen uptake

Control + Grami- cidin Control + Grami.

cidiu

natoms x m i x ' x mg protein-'

- - None 80 80

Monensin 80 80 1.0 1.0 Na+ (10 mM) + monensin 94 192 1.2 2.4

Naf(10mM) 94 94 1.2 1.2

NH4+ (10 mM) 84 229 1.1 2.9 NH4+ (100 mM) 123 269 1.5 3.4

observed in the presence of the appropriate combina- tions of antibiotics and cations (Tables 3 and 4) were similar to the respiratory control ratios observed on addition of CCCP (10 pM) or gramicidin (10 pg/mg protein).

DISCUSSION

Mitochondria may be uncoupled by valinomycin in the presence of K+ [29-311, whereas submito- chondrial particles [6,7,14], chromatophores of Rhodospirillurn rubrum [l 1,141 and subchloroplast particles [9,10,14] are uncoupled by the addition of valinomycin only in the presence of either NH4+ or a combination of K+ and nigericin. This difference has been attributed [14,32] to the opposite polarities ofthe coupling membranes. The direction of the oxido- reduction-driven proton translocation is outwards in the intact mitochondrion, and in whole cells of M. denitrificuns [26], but inwards in the submito- chondrial particle, bacterial chromatophore and sub- chloroplast particle.

In the present work it has been demonstrated that particles prepared from M. denitrificuns are similarly only uncoupled by valinomycin in the presence of either NH,+ or a combination of K+ and nigericin.

Analogous results were obtained with gramicidin. The antibiotic is known to confer on membranes a limited permeability to protons and a high perme- abilitiy to NH,+ and Na+ [IS]. At a concentration of 10 pg/mg protein gramicidin appears to translocate protons across the membrane of the M. denitrificuns particles a t a sufficiently high rate to induce uncoupl- ing in the absence of any additional combinations of antibiotics and cations. At the lower concentration of 0.1 pg/mg protein however, although gramicidin cannot translocate protons a t a rate sufficient to induce detectable uncoupling when added alone, it is capable of translocating NH,+ and Na+ a t sufficient- 36.

ly high rates to induce uncoupling in the presence of either NH,+ or a combination of Na+ and monensin.

We conclude therefore that the mode of action of these antibiotics in the M. denitrificans particles is similar to their mode of action in other membrane systems, and that uncoupling is brought about by the dissipation of the pH gradient and membrane po- tential generated by an inwardly directed oxido- reduction-driven translocation of protons.

This inferred polarity of the membrane of the M. denitrificuns particles has been supported by some preliminary observations. NADH-dependent re- spiration by the particles caused a CCCP-inhibited, valinomycin-enhanced reversible increase in the pH of a lightly buffered KC1 medium. Under similar conditions, respiration of endogenous substrates by the whole cells caused a CCCP-inhibited, valino- mycin-dependent reversible decrease in the pH of the medium.

The authors wish to thank the Medical Research Council for financial support.

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P. John's present address : Department of Physiology-Anatomy University of California, Berkeley, California 94720, U.S.A.

W. A. Hamilton Department of Biochemistry, University of Aberdeen Aberdeen, AB9- 1 AS, -Great".Britain