Oxidative Phosphorylation in Fractionated - Journal of Bacteriology

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at a concentration of 1 mg/ml appeared to inter-. 'This is the 51st paper in a series dealing with oxidative phosphorylation in fractionated bacterial systems. 1017.
Vol. 108, No. 3

JOURNAL OF BACTERIOLOGY, Dec. 1971, p. 1017-1025

Printed in U.S.A.

Copyright 0 1971 American Society for Microbiology

Oxidative Phosphorylation in Fractionated Bacterial Systems: Effect of Chloramphenicol1 BEN-ZION CAVARI, VIJAY K. KALRA, AND ARNOLD F. BRODIE Department of Biochemistry, University of Southern California School of Medicine, Los Angeles, California 90033

Received for publication 16 July 1971

Chloramphenicol was found to have a direct effect on the respiratory chain of Mycobacterium phlei cells grown in the presence of this drug. Analysis of the respiratory chain components revealed that the presence of chloramphenicol during growth resulted in a partial inhibition in the synthesis of the cytochromes. However, a stimulation in oxidative phosphorylation was observed with the cell-free extract of cells grown in the presence of chloramphenicol. The oxidation of succinate was found to be stimulated 20 to 130%, depending on the particular extract, whereas the oxidation of reduced nicotinamide adenine dinucleotide (NADH) was found to be similar to that of extracts obtained from cells grown in the absence of the drug. Of particular interest was the finding that the cell-free extract of cells grown in the presence of the drug exhibited an increased level of phosphorylation (17 to 100%) when NADH was used as the electron donor. Chloramphenicol appears to affect a component of the respiratory chain between the flavoprotein and cytochrome c. Fractionation of the electron transport particles revealed an increased level of cytochrome b in the fractions which exhibited a stimulation in oxidative phosphorylation. It has been known for some time that chloramphenicol (CAM) inhibits protein synthesis (9), and attempts have been made to see whether CAM has any effect on the energy generation system (24). It was concluded that the antibiotic at the growth-inhibitory concentration does not affect processes of energy generation and that any such effect, when found, must be a secondary event related to the inhibition of protein synthesis caused by CAM. Recently Linnane and his group (15, 28) demonstrated that, in yeast cells and in mammalian tissue culture cells (18), CAM inhibits the synthesis of mitochondrial proteins without affecting the synthesis of ribosomal proteins. These conclusions were based on the observation that the drug had no effect on a petite mutant of yeast and that, in wild type, synthesis of cytochromes a, a3, b, and cl was completely inhibited and synthesis of succinic dehydrogenase was partially inhibited, but cytochrome c synthesis continued. A number of studies (22, 23, 28) have shown an effect of CAM on either the energy-generating system or on the electron transport chain. Godchaux and Herbert (23) reported that CAM at a concentration of 1 mg/ml appeared to inter'This is the 51st paper in a series dealing with oxidative phosphorylation in fractionated bacterial systems.

fere with adenosine triphosphate formation in intact rabbit reticulocytes. Freeman and Haldar (22) claimed that, at 1.9 mg/ml, CAM was a specific inhibitor of reduced nicotinamide adenine dinucleotide (NADH) oxidation by isolated beef heart mitochondria and that the inhibition occurred at the level of NADH dehydrogenase. Hanson and Hodges (25) reported that CAM acts as an uncoupling agent in maize mitochondria. In the present report, an attempt was made to locate the site of action of CAM on the respiratory chains of Mycobacterium phlei. MATERIALS AND METHODS M. phlei (ATCC 354) cells were grown and harvested by the procedure previously described (10). CAM was added to the medium 2 hr after inoculation. The addition of CAM in concentrations of 10 ig/ml and 20 ,g/ml to the growth medium resulted in a slight inhibition of the bacterial growth. Sonically disrupted cells were separated into particulate and supernatant fractions by differential centrifugation in a Spinco model L preparative centrifuge (10). The electron transport particles (ETP) obtained after centrifugation were washed with a solution of 0.15 M KCI containing 0.01 M MgCI2, and adjusted to pH 7.4 with N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid (HEPES)-KOH buffer (0.01 M).

1017

1018

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CAVARI, KALRA, AND BRODIE

Respiration was measured by the conventional manometric technique at 30 C with a Gilson respirometer, or polarographically (16) with a Clark oxygen electrode. With the oxygen electrode, oxygen uptake was calculated from the percentage change in oxygen saturation over a given interval of time, assuming that I ml of water saturated with air contained 237 nmoles of oxygen at 30 C. Inorganic phosphate was determined by the method described by Fiske and SubbaRow (20). The reduction of cytochromes was measured by usidlg a double-beam spectrophotometer (American Instrument Co.) equipped with a vibrating platinum oxygen electrode, or by using a Cary model 14 recording spectrophotometer. Succinic dehydrogenase activity was assayed spectrophotometrically by following the rate of reduction of dichlorophenol indophenol (molar absorbancy at 600 nm, 19.1 mm-' cm-'; reference 5). Succinate-cytochrome c reductase activity was assayed spectrophotometrically by following the rate of reduction of 3(4, 5-dimethyl thiazolyl 2)-2, 5-diphenyl tetrazolium bromide (MTT; molar absorbancy at 565 nm, 15.0 mM-' cm-'; reference 4) or horse heart cytochrome c (molar absorbancy at 550 nm, 20.3 mM-' cm-'; reference 35). Both MTT and horse heart cytochrome c have been shown to accept electrons at the cytochrome c level in M. phlei (1).

The quinone MK9 (II-H) was extracted by the method of Folch et al. (21) and purified by thin-layer chromatography on Silica Gel G plates (250 um in thickness) impregnated with 0.1% rhodamine G. The solvent used was 12% n-butylether in hexane. The quinone was scraped off the plate and eluted from the adsorbent with diethyl ether. The quinone was dissolved in ethanol containing 0.01 volume of I M ammonium acetate buffer (pH 5.0) and reduced by adding sufficient sodium borohydride from a freshly prepared aqueous solution. The concentration of quinone was determined from the difference in optical density at 245 nm of reduced-minus-oxidized quinone (molar absor-

bancy 25.8 mm- I cm- 1). Protein was determined by the method of Lowry al. (31).

et

RESULTS Effect of CAM on oxidative phosphorylation. CAM in concentrations of 10 yg/ml or 20 ,g/ml in the growth medium was found to inhibit protein synthesis of the cells by about 10% and 30%, respectively. The ability of the ETP derived from cells grown with 10 or 20 og of CAM per ml (CAM particles) to carry out oxidative phosphorylation was compared to the ETP derived from cells grown in the absence of the drug. The CAM particles were found to have an increased level of succinoxidase activity (see Table 6) and an increased level of phosphorylation with NADH as the electron donor. About 10 experiments were run; a representative experiment is shown in Table 1. The changes from one experiment to another did not exceed 10%. Although the level of phosphorylation increased with succinate as substrate, the P/O ratios observed with CAM particles were similar to those of the regular ETP. Increasing the concentration of CAM in the growth medium from 10 gg/ml to 20 ,g/ml resulted in a further increase in both oxidation and phosphorylation. However, a concentration of 30 gg of CAM per ml (data not shown) resulted in a significant inhibition of protein synthesis and a decrease in the level of oxidative phosphorylation. CAM had an effect only where protein synthesis could take place (Table 2). Oxidation and phosphorylation of regular ETP with NADH or succinate as electron donors were measured in

TABLE 1. Oxidative phosphorylation with electron transport particles (ETP) obtained from cells grown with chloramphenicol (CAM)a ETP

Normal ................

0

Substrate

(gatoms)

Succinate NADHC

6.1 17.3

Per cent of normal

ApIb ('smoles)

P/

4.6 6.3

0.75 0.38

Per cent of normal

Jg/ml)

.......

Succinate NADH

7.5 18.0

123 104

6.0 8.8

0.80 0.49

106 129

CAM (20 ,sg/ml)

.......

Succinate NADH

8.8 17.2

144 99

7.2 11.1

0.82 0.65

109 171

CAM (10

aThe system consisted of: M. phlei particles (2 mg of protein); tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4), 100 Jumoles; glucose, 20 ,umoles; orthophosphate (pH 7.4), 15 ,moles; MgCl,2 15 ,Amoles; yeast hexokinase, 3 mg; KF, 25 Amoles; adenosine diphosphate, 2.5 umoles; and water to a final volume of 3.0 ml. The reaction was started by the addition of succinate (50 Jmoles) or NADH (25 Jmoles) from the side arm. Rate of oxygen consumption was measured by the conventional manometric technique (Gilson differential respirometer) at 30 C for 20 min. The reaction was stopped by the addition of 1.0 ml of 10% trichloroacetic acid, and a portion was used for phosphate determination as described by Fiske and SubbaRow. PPi, inorganic phosphate. c Reduced nicotinamide adenine dinucleotide.

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ACTION OF CAM ON RESPIRATORY CHAINS OF M. PHLEI

the absence and presence of 20 jig of CAM per ml. As can be seen from Table 2, the presence of CAM had no effect on either oxidation or phosphorylation. Thus the effect of CAM observed with the ETP from CAM-grown cells appears to result in an alteration of the ETP structure of respiratory carriers. The fact that the phosphorylation of the CAM particles was stimulated with NADH as substrate indicated that the oxidation of the NADH was mediated by electrons moving through the main respiratory chain and not through the nonphosphorylative electron transport bypass associated with this substrate (3). Further indication that the oxidation of NADH occurred through the main respiratory chain was the finding that KCN inhibited NADH oxidation of CAM particles to the same extent as that of regular particles (Table 3). Thus the increased level of phosphorylation associated with NADH oxidation with the CAM particles was not the result of an inhibition in the synthesis of the bypass enzymes. Effect of aging on oxidation and phosphorylation. Aging of the particles was found to have a pronounced effect on both oxidation and phosphorylation. The particles were aged by storage at 4 C. The succinoxidase activity of the CAM particles was 41% higher than that of the regular particles after the first day of storage (Table 4). However, after the second day the succinoxidase activity of the CAM particles was only 28% higher and after the fifth day only 20% higher than that of the normal particles. Although the differences were small, they were significant because they were obtained with the same order of magnitude in all four experiments that were carried out. The results in Table 4 represent a mean of these four experiments. The effect of aging on NADH oxidase activity was slight; however, a stimulation in the level of phosphorylation was observed with this substrate, which continued to increase from 17%

after the first day to 44% after the fifth day and finally decreased to 29% after the seventh day. Particles obtained from cells grown in the absence of the drug exhibited no changes or a slow decrease in both oxidation and phosphorylation during the aging process. Effect of heat on the ETP from CAM-grown cells. Brief exposure to heat (50 C for 10 min) of the suspension of ETP (30 mg of protein/ml) in 0.15 M KCl has been shown to reduce oxidative TABLE 2. Effect of chloramphenicol (CAM) on oxidative phosphorylation with Mycobacterium phlei electron transport particlesa Substrate

Succinate Succinate

CAM

added

...

-

...

+

NADHC .... NADH ...

+

(,uatoms)

0

'IP5b (rmoles)

P

12.0 11.6 7.0 7.5

8.0 8.0 4.2 4.4

0.67 0.69 0.60 0.59

-

a Conditions were similar to those described in Table 1, except that 20 ,ug of CAM per ml was added as indicated. b Pi, inorganic phosphate. c Reduced nicotinamide adenine dinucleotide.

TABLE 3. Effect of KCN on respiration with NADH as substratea

(Mgatoms)

0 ETP

Normal .......... CAM (10 ug/ml) CAM (20 ug/ml)

Per cent ihbto

-KCN

+KCN

inhibition

14.5 11.4 10.3

2.9 2.3 1.7

80 80 84

a Conditions were similar to those described in Table 1, except that KCN (3 x 10-3 M) was added as indicated, and only NADH was used as a substrate. Abbreviations: NADH, reduced nicotinamide adenine dinucleotide; ETP, electron transport particles; CAM, chloramphenicol.

TABLE 4. Effect of aging of the chloramphenicol (CAM) particles on the oxidative phosphorylationa NADHb

Succinate Day

2 5 7

0 uptake (uatoms)

0 uptake (gatoms)

P/O

Normal particles

CAM particles

Normal particles

CAM particles

6.9 6.7 6.1 6.1

9.7 8.6 7.3 7.5

0.78 0.80 0.82 0.75

0.76 0.77 0.88 0.80

Normal particles

23.5 22.6 19.2 17.3

P/O

CAM particles

Normal particles

25.7 21.9 17.9

0.36

18.0

0.33 0.39 0.38

CAM particles

0.42 0.44

0.56 0.49

a Conditions were similar to those described in Table 1. The particles were stored in the cold, and oxidative phosphorylation was examined on the days indicated. The CAM particles were prepared from cells grown in the presence of 10 ,ug of CAM per ml. bReduced nicotinamide adenine dinucleotide.

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CAVARI, KALRA, AND BRODIE

phosphorylation (7). Exposure of normal ETP to heat increased succinoxidase activity (up to 100%), whereas the phosphorylation associated with this oxidation increased about 50%. In contrast, with NADH as the electron donor, exposure to heat resulted in a slight inhibition of oxidation, but the level of phosphorylation increased at least threefold. Thus, it was of interest to determine whether the particles from CAM-grown cells, which exhibit increased oxidation of succinate and increased levels of phosphorylation with NADH, could respond to heat treatment in a manner similar to that exhibited by the normal ETP. The effect of heat treatment of normal and CAM particles on oxidation and phosphorylation with succinate or NADH as substrates is shown in Table 5. The results obtained with normal and CAM particles before heating were similar to those shown in Table 1. However, after heating, an increase in the oxidation of succinate was observed but not with NADH as the electron donor (Table 5). These results were observed with the regular and the CAM particles. A slight increase in the P/O ratios was observed after heat treatment with both succinate and NADH as substrates, with the regular or treated particles. Heat treatment of the ETP has been shown to result in conformational change of the ETP (Kalra, Aithal, and Brodie, manuscript in preparation). This effect of heat treatment was not due to the presence of a heat-labile inhibitor or a regulator. The effect of CAM was thought to be due to the inhibition of the synthesis of a natural inhibitor; however, this appears unlikely, since ETP from CAM-grown cells still exhibit increased phosphorylation after heat treatment with succinate as a substrate. Site of action of CAM. To determine the site of action of CAM, the respiratory carriers of the electron transport chain were examined. Cytochromes a and c content was followed with a double-beam spectrophotometer to determine the rates of reduction of the cytochromes as well as the total amount of the cytochromes in the dif-

J. BACTERIOL.

ferent types of particles. Since a stimulation in the level of oxidation was observed with the CAM particles with succinate, this substrate was used to follow the enzymatic reduction of the cytochromes. The time required to reach the transition point from an aerobic to an anaerobic state was shorter for the CAM particles than for the regular particles. Although an effect on oxidative phosphorylation was observed with particles obtained from cells grown on 10 ,ug of CAM per ml, a more pronounced effect was observed with ETP from cells grown in the presence of 20 Ag of CAM per ml. The pattern of reduction of cytochromes c and a was the same in regular particles as in particles derived from cells grown with 10 ,g of CAM per ml (Fig. 1). However, with particles derived from cells grown with 20

CYTOCHROME c (551 - 540)

NAaSAO4. -- --

T

QCt-0.0044

-

SUCCINArE CYTOCHROME a (601i-62

OD-0.00088 -

-

SMIIN'

SUCCINATE

FIG. 1. Reduction of cytochromes c and a in normal electron transport particles and in chloramphenicol (CAM) particles. The system consisted of: tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4), 100 1tmoles; MgC2, 15 Mlmoles; particles (6 mg of protein); succinate, 20 /gmoles; and water to a final volume of 3.0 ml. The reaction was followed spectrophotometrically with an Aminco double-beam spectrophotometer. Solid line, normal particles or 10 ug/ml CAM particles; dashed line; 20 ugg/ml CAM particles.

TABLE 5. Effect of heat on oxidation and phosphorylation of normal and chloramphenicol (CAM) particlesa Before heating

0 (luatoms)

Normal

CAM (10

After heating

Substrate

ETP

gg/ml)

AP,

P/O

0

(gatoms)

______

AP,

P/O

Succinate NADH

3.3 7.8

2.0 1.6

0.61 0.21

6.0 7.2

4.3 1.9

0.72 0.26

Succinate NADH

4.9 7.9

3.3 2.4

0.67 0.30

7.8 8.0

5.6 2.9

0.72 0.36

a Conditions were similar to those described in Table 1. To obtain heat-treated particles, suspensions of 30 mg of protein/ml of normal or CAM particles were incubated at 50 C for 10 min. Abbreviations: ETP, electron transport particles, P1, inorganic phosphate; NADH, reduced nicotinamide adenine dinucleotide.

VOL.

108, 1971

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ACTION OF CAM ON RESPIRATORY CHAINS OF M. PHLEI

per ml, the reduction of cytochromes and a differed from that found in the regular particles. The total amounts of chemically reducible cytochromes c and a were 60% and 50%, respectively, of that found in the normal particles. In normal ETP as well as in CAM particles, all cytochrome c was enzymatically reduced; 94% of cytochrome a in ETP was reduced enzymatically, whereas in the CAM particles only 70% of the total amount of cytochrome a was enzymatically reducible, as based on protein concentration of both types of the particles. The M. phlei system has been shown (8) to contain two different hemochromogens which exhibit absorption between 557 or 558 nm and 562 nm. Of particular interest was the finding that one of these hemochromogens, presumably b type, which has maximum absorption at 433 and 562 nm, was reduced by NADH (Fig. 2), whereas the other component (absorption at 430 and 557 or 558 nm) was reduced when succinate was used as the electron donor. The characteristic profiles of the b-type cytochromes were assayed by using ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TPD) in both the reference and sample absorption cell. Since ascorbate and TPD enter the respiratory chain at the level of cytochrome c (29), both cytochrome c and a are reduced in both absorption cells, thus permitting the reduction of cytochrome b to be followed in the absence of reduction of cytochromes c and a. It was of interest to determine whether CAM inhibits the synthesis of only one

Ag of CAM

c

REGULAR

PARTICLES

type of cytochrome b. In contrast to regular par-

ticles, the particles from CAM-grown cells exhibited only one type of cytochrome b, with maximum absorption at 560 nm, which was irrespective of the electron donor used (Fig. 3). The total amount of cytochrome b (560 nm) in the CAM particles as measured by reduction with sodium dithionite was 80% of that found in the regular particles, assuming that the cytochrome b (560) has the same molar absorbancy (562-563 nm) as the cytochrome b (36). The segment of the respiratory chain on the substrate side of cytochrome b was compared in both types of particles by measuring the succinic dehydrogenase activity. Different dyes were used as electron acceptors. Although the oxidation of succinate by the particles was stimulated by 48 and 70% in the ETP from cells grown on 10 and 20 .g of CAM per ml, respectively, there was a slight decrease in the succinic dehydrogenase activity when dichlorophenol indophenol was used as the electron acceptor (Table 6). This dye accepts the electrons at the flavoprotein level; however, when an electron acceptor was employed which accepts electrons from endogenous cytochrome c, such as MTT or mammalian cytochrome c (1), the CAM particles were found to be more active than the regular particles. In addition, the stimulation observed in succinate cytochrome c reductase activity of the CAM particles was found to occur to the same extent as that observed in the stimulation of succinic oxidase activity. This finding indicates that CAM affects a component on the succinate chain which lies in the segment between the flavoprotein and cytochrome c. Another indication that CAM acts by affecting a component of the respiratory chain between the flavoprotein and cytochrome c on the succinoxidase pathway was obtained by studying the TPD shunt (Kalra, Krishna Murti, and Brodie, CAM

PARTICLES

B

A

SUCCINATE

NADH

~~~~~~~~~~560

0.03 1 C

550

600

650

nm

550

600

-85O

FIG. 2. Difference spectrum of cytochrome b. The system consisted of 100 1umoles of tris(hydroxymethyl) aminomethane-hydrochloride buffer (pH 7.4), 100 nmoles of N, N, N', N'-tetramethyl-p-phenylenediamine, electron transport particles equivalent to 3 mg ofprotein, 10 uimoles of ascorbate, and water to a final volume of 1.0 ml. The reaction was started by the addition of 25 timoles of reduced nicotinamide adenine dinucleotide (A) or 25 Mmoles of succinate (B). The reduction of cytochrome b was followed spectrophotometrically with a Cary model 14 spectrophotometer.

IXX,

V

560

0.02 0.0l

I_

e

550

e

600

6rnm

650

550

600

650

FIG. 3. Difference spectrum of cytochrome b. Conditions were as in Fig. 2, except that chloramphenicol (CAM) particles were used instead of regular normal electron transport particles and either reduced nicotinamide adenine dinucleotide (A) or succinate (B) was used to reduce the cytochrome.

1022

CAVARI, KALRA, AND BRODIE

TABLE 6. Effect of growth with chloramphenicol (CAM) on succinoxidase, succinic cytochrome c reductase, and succinic dehydrogenase activities of Mycobacterium phlei

Elecrontranespr panticles

Electron transport

Succinoxidase activitya

Succinic dehydrogenase and succinic cytochrome c reductase

activity"

Cyto- c DCIP MTT chrome

Normal CAM (10 Ag/ml) CAM (20 Ag/mI)

127 188 216

55 57 47

4.6 5.3 9.8

6.3 8.5 10.6

a Succinoxidase activity was measured polarographically with Clark's oxygen electrode. The system for succinoxidase activity contained particles (4 mg of protein), 100 gmoles of HEPES-KOH buffer (pH 7.4), 15 ,moles of MgCI2, and water to a final volume of 3.0 ml. Results are expressed as nanoatoms of oxygen produced per milligram of protein per minute. b The system for succinic dehydrogenase and succinic cytochrome c reductase activity consisted of: tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4), 100 umoles; MgCl2, 15 ,moles; dichlorophenol indolephenol (DCIP), 3(4, 5-dimethylthiazolyl-2)-2, 5diphenyl tetrazolium bromide (MTT), or horse heart cytochrome c, 200 nmoles; particles, 200 ug of protein with DCIP or MTT and I mg with cytochrome c; KCN (9 ymoles) was added when DCIP or cytochrome c was used as electron acceptor; water to a final volume of 3 ml. The reaction was started by the addition of succinate (50 umoles), and the rate of reduction was measured at 600 nm with DCIP, at 565 nm with MTT, and at 550 nm with cytochrome c.

J. BACTERIOL.

iron was observed. This does not mean that nonheme iron is not functioning. The segment between the flavoprotein and cytochrome c in the NADH chain contains quinone and cytochrome b (2). To determine whether the stimulation in the phosphorylation activity coupled with NADH oxidation was due to a difference in the amount of the quinone in the regular and in CAM particles, the quinone was extracted from the different particle preparation and measured spectrophotometrically (Table 8). The amount of the quinone was not significantly different in the regular and in the CAM particles. Distribution of the oxidative phosphorylation activity with different particulate fractions. The ETP from M. phlei have been shown to be composed of a heterogenous particle population (26), which differs in quinone content, cytochromes, size, composition, and activity (11, 26). It was of interest to see whether CAM affected all types of particles or whether the effect of the drug was primarily on only one type of particle. Normal ETP and particles derived from cells grown with 15 gg of CAM per ml were dispersed in 0.25 M sucrose. Portions (0.5 ml) were layered on top of a 4.5-mI linear sucrose gradient (1.0 to 1.25 M) and centrifuged in an SW39 rotor at 36,000 rev/min for 15 hr in a Spinco model L untracentrifuge. Fifteen fractions of 0.33 ml each were collected. Protein content was determined, TABLE 7. Restoration of the NQNO-blocked succinoxidase activity by TPDa Succinoxidase activityb

unpublished data). When the respiratory chain was blocked by 2 nonyl-8-hydroxyquinoline-Noxide or by irradiation at 360 nm, the addition of TPD was found to restore the oxidation of NADH or succinate by bypassing the block. TPD was shown to accept the electrons from flavoproteins, transferring them to cytochrome c + cl (32). As can be seen in Table 7, the succinoxidase activity was more than two times higher in the CAM particles than in the regular particles. At 3.3 Ag/ml, 2 nonyl-8-hydroxyquinoline-Noxide inhibited the oxidation of the regular and the CAM particles. The addition of TPD restored the oxidation of both the regular and the CAM particles to the same level, so that higher activity was no longer observed in the CAM particles. It thus appears that the site of CAM action in the succinoxidase pathway is located after the flavoproteins and before cytochrome c. This segment of the succinoxidase chain contains an unidentified light-sensitive component, nonheme iron, and cytochrome b (30). In ETP of CAMgrown cells, no detectable amount of nonheme

Electron transport particles

TPD

Normal .......... CAM (20 ug/ml)

175 263

aThe system was the same as in Table 6. NQNO (10 ug per mg of protein) and TPD (300 nmoles) were added as indicated. Abbreviations: NQNO, 2 nonyl-8hydroxyquinoline-N-oxide; TPD, N,N,N',N'-tetramethyl-p-phenylenediamine; CAM, chloramphenicol. Results expressed as nanoatoms of oxygen produced per minute per milligram of protein. TABLE 8. Concentration of quinones in Mycobacterium phlei particles from cells grown in presence and absence of chloramphenicol (CAM) Electron transport particles

Quinones (nmoles/ mg of protein)a

Normal CAM (15 jig/ml) CAM (20 jg/ml)

16.8 18.0 15.2

a Quinones were extracted and the amount was determined as described in Materials and Methods.

VOL. 108, 1971

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ACTION OF CAM ON RESPIRATORY CHAINS OF M. PHLEI

and oxidation and phosphorylation were examined with succinate and NADH as the electron donors. The results are summarized in Table 9. Only the results for fractions 5 to 10 are given in Table 9, since very low activity was found in fractions I to 4 and 11 to 15. The distribution of the oxidative and phosphorylative activities in the regular particles was not the same when NADH or succinate were used as substrates. Maximal oxidative activity for succinate was found in fraction 8, whereas, for NADH, maximal activity was found in fraction 5. Maximal phosphorylative activity was found in fraction 6 when succinate served as an electron donor and in fractions 8 and 9 when NADH was the substrate.

content. The difference spectra (reduced minus

oxidized) of the different particulate fractions were taken, and the amount of cytochrome (nanomoles per milligram of protein) was plotted against the fraction number (Fig. 4). Cytochromes a and c were almost equally distributed throughout fractions I to 9; however, cytochrome b exhibited a peak at fractions 5 to 7 corresponding to the fraction exhibiting the stimulation in oxidative phosphorylation. The distribution of the cytochromes was the same in normal ETP as that described for the CAM particles; however, the amount of cytochromes b, c, and a + as decreased in the particles derived from CAM-grown cells (40, 20, and 50%, respectively, as compared to regular particles). DISCUSSION CAM has been shown to inhibit protein synthesis; relatively high concentrations of this drug are necessary to inhibit protein synthesis in M. phlei. A concentration of CAM from 10 to 20 ,ug/ml resulted in only a slight inhibition of protein synthesis but caused a marked effect on oxidative phosphorylation. At 10 ,ug/ml, CAM does not inhibit the synthesis of cytochromes, whereas 20 ,g/ml was found to decrease the level of cytochrome c, b, and a to 40, 20, and 50%, respectively. It was surprising to find that the cell-free

It is of interest that the stimulation of oxidation and phosphorylation in the CAM particles exhibited slightly different patterns of distribution in the different types of particles. Stimulation in succinoxidase activity in the CAM particles was concentrated in fractions 6 and 7, whereas stimulation in phosphorylation with NADH as substrate was located in fractions 5, 6, and 7. Distribution of cytochromes b, c, and a + as was examined in the different particulate fractions to determine whether there was any correlation between increased activity and cytochrome TABLE 9. Oxidative phosphorylation in fractionated particlesa

CAM particles (% change regular particles) from

Normal particles

Fraction

Substrate 0 (juatoms/ mg of protein)

P,1 (umoles/ mg of protein)

P/0

O (uatoms)

P/0

5

Succinate NADHC

3.1 12.5

1.2 0.4

0.39 0.03

0 + 10

+46 + 100

6

Succinate NADH

2.7 11.6

2.5 1.3

0.93 0.11

+67 +20

+ 12 +91

7

Succinate NADH

4.6 9.8

2.9 2.1

0.63 0.27

+63 +1

+33 +86

8

Succinate NADH

5.8 9.9

3.8 3.8

0.64 0.38

0 +I

+23 0

Succinate NADH

4.0 4.7

2.4 1.9

0.60 0.41

-20 +32

- 13

Succinate NADH

3.1 3.3

1.3 0.9

0.41 0.28

-80 - 10

0 -80

9 10

0

a Normal particles and chloramphenicol (CAM) particles derived from cells grown with 15 jig of CAM per ml were dispersed in 0.25 M sucrose. Samples (0.5 ml) were layered on top of a 4.5-ml linear sucrose gradient (1.0 to 1.25 M) and centrifuged in a Spinco model L preparative ultracentrifuge with an SW39 rotor at 36,000 rev/min for 15 hr. Fifteen fractions of 0.33 ml each were collected. The protein content of each fraction was determined, and the oxidative phosphorylation was measured by using the system described in Table 1. Ppi, inorganic phosphate. c NADH, reduced nicotinamide adenine dinucleotide.

i_

1024

J. BACTERIOL. CAVARI, KALRA, AND BRODIE normal particles. Furthermore, the level of qui-

2

eZ

3o a.

0

A0

d

8

00 u0 6 D

4

Q

w

none was found to be the same in the CAM and

\the regular particles.

/

22

'

CYT.c

\

CYT. b _r\ /

\

zCYT.aa °

cytochrome



_

.________________________________ 2

4

6

8

10

Succinic cytochrome c reductase activity was \ to be stimulated in the CAM particles to found the same extent as succinoxidase activity. This finding indicated that the stimulatory effect occurred before cytochrome c. In addition, when measuring the succinoxidase activity by using the TPD shunt, thus bypassing the cytochrome b, no stimulation was observed. It appears that the site of action of CAM is in the cytochrome b region. Support for this hypothesis was the finding that the of and that bmost was of the cytosynthesis inhibited by CAM chrome b was located in those fractions which .exhibited stimulation in the oxidative phosphorylation. Two types of hemochromogens which appear to be of of the b type have been demonstrated in Mhei. Of paru interestwasthe finding that On petwar Interest was the f

12

FRACTION NO. FIG. 4. Distribution of the cytochromes (Cyt) in

chioramphenicol particle fractions separated by sucrose density gradient. The difference spectrum of each fraction was taken by adding a few grains of sodium di-

thionite to the sample cuvette. The molar absorbancy used to calculate the amount of cytochrome b (560) was based on the assumption that this b-type cytochrome has a molar absorbancy similar to that described for cytochrome b (562) by Freeman and

Haldar (22).

extract from CAM-grown cells exhibited stimulation in succinoxidase and stimulation in phosphorylation with NADH as substrate, an effect that was even more marked in the concentration of CAM that caused inhibition of cytochrome synthesis. Although there was a loss in cytochrome content, it was of interest to find an increase in the level of respiration and coupled activity. This finding suggests that the total content of the terminal cytochromes may not be required for the energy-generating pathway. CAM has also been shown to inhibit the synthesis of cytochromes a, a3, b, and cl in the actively growing liver tissue of rat (6, 19). Because of the differential effects of CAM on the respiratory chains with NADH or succinate as substrates, it was assumed that the site of action of CAM was in the region where the chains are separate, i.e., between the substrate and cytochrome b. In M. phlei, those two separate parts of the chain contain different components: quinone in the NADH chain and a light-sensitive component and non-heme iron in the succinate chain. Examination of the respiratory carriers failed to reveal major differences in the components between substrate and cytochrome b in the ETP from cells grown with or without CAM. The succinic dehydrogenase activity in the CAM particles was found to be the same as that in

only slightly

tTP

NADH, whereas the other was reduced rapidly (8). Since the ETP from CAM-grown cells exhibited an increased level of succinoxidase activity and the

by succinate and slowly by NADH

stimulation appeared to be due to a component between the flavoprotein and cytochrome c, the

effect of CAM on the level of the two b-type cytochromes was examined. In contrast to the regular ETP, the particles from CAM-grown cells contained only one type of cytochrome b (maximal absorption at 560 nm) which differed from either b type from regular particles. The 560-nm b-type cytochrome was reduced by either NADH or succinate and at the same rate. The amount of cytochrome b, assuming the same molar absorbancy, was reduced 20% in the particles from CAM-grown cells. The studies of the nature of cytochrome b fail to explain the observed results with the particles from CAMgrown cells. Another explanation which has been suggested for a role for cytochrome b is that this respiratory carrier serves a dual function: one in electron transport and the other as a structural organizer of the respiratory components (12, 13, 17, 34). The ETP from CAM-grown cells exhibited an increased level of phosphorylation with NADH as the electron donor. This is particularly surprising since the level of phosphorylation with succinate as an electron donor was not increased. CAM may act by preventing the synthesis of a substance which inhibits or regulates the respiratory chain. Furthermore, the particles from CAM-grown cells, like regular particles subjected to heat treatment, do not require the addition of soluble coupling factors for phosphoryla-

VOL. 108, 1971

ACTION OF CAM ON RESPIRATORY CHAINS OF M. PHLEI

tion (7). An inhibitor similar to that suggested above has been suggested by several groups (14, 27, 33). ACKNOWLEDGMENTS The technical assistances of Patricia Brodle and Hiroko Sakamoto is gratefully appreciated. This investigation was supported by Public Health Service grant Al 05637 from the National Institute of Allergy and Infectious Diseases, by grant GB 6257XI from the National Science Foundation, and by a grant from the Hastings Foundation of the University of Southern California School of Medicine. LITERATURE CITED 1. Asano, A., and A. F. Brodie. 1964. Respiratory chains of Mycobacterium ph/li. J. Biol. Chem. 239:4280-4291. 2. Asano, A., and A. F. Brodie. 1965. Phosphorylation coupled to different segments of the respiratory chains of Mycobacterium pheli. J. Biol. Chem. 240:4002-4010. 3. Asano, A., and A. F. Brodie. 1965. The properties of the nonphosphorylating electron transport bypass enzymes of Mycobacterium phlei. Biochem. Biophys. Res. Commun. 19:121-126. 4. Asano, A., T. Kaneshiro, and A. F. Brodie. 1965. Malatevitamin K reductase-A phospholipid requiring enzyme. J. Biol. Chem. 240:895-905. 5. Basford, R. E., and F. M. Huennekens. 1955. Oxidation of thiol groups by 2, 6-dichlorophenol-indophenol. J. Amer. Chem. Soc. 77:3873-3877. 6. Beattie, D. S. 1968. Studies on the biogenesis of mitochondrial protein components in rat liver slices. J. Biol. Chem. 243:4027-4033. 7. Bogin, E., T. Higashi, and A. F. Brodie. 1970. Effects of heat treatment of electron transport particles on bacterial oxidative phosphorylation. Proc. Nat. Acad. Sci. U.S.A. 67:1-6. 8. Bogin, E., T. Higashi, and A. F. Brodie. 1969. Extraparticulate chain interaction between different electron transport particles. Science 165:1364-1367. 9. Brock, T. D. 1961. Chloramphenicol. Bacteriol. Rev. 25:3248. 10. Brodie, A. F., and C. T. Gray. 1956. Phosphorylation coupled to oxidation in bacterial extracts. J. Biol. Chem. 219:853-862. 11. Brodie, A. F., B. Revsin, V. Kalra, P. Phillips, E. Bogin, T. Higashi, C. R. Krishna Murti, B. Cavari, and E. Marquez. 1970. Natural substances formed biologically from mevalonic acid, p. 119-143. In T. W. Goodwin (ed.), Biological function of terpenoid quinones. Academic Press Inc., London. 12. Bruni, A., and E. Racker. 1968. Reconstitution of the succinate-ubiquinone reductase. J. Biol. Chem. 243:962971. 13. Chance, B., and B. Schoener. 1966. High and low energy states of cytochromes. J. Biol. Chem. 241:4567-4573. 14. Chance, B., G. R. Williams, W. F. Holmes, and J. Higgins. 1955. A mechanism for oxidative phosphorylation. J. Biol. Chem. 217:439-451. 15. Clark-Walker, G. D., and A. W. Linnane. 1967. A comparison between cytoplasmic respiratory deficient mutant yeast and chloramphenicol-inhibited wild type cells. J. Cell Biol. 34:1-14. 16. Clark, L. C., Jr., R. Wolf, D. Granger, and Z. Taylor. 1953. Continuous recording of blood oxygen tensions by

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