JOURNAL OF BACTERIOLOGY, Dec. 1971, p. 1017-1025Copyright 0 1971 American Society for Microbiology

Vol. 108, No. 3Printed in U.S.A.

Oxidative Phosphorylation in FractionatedBacterial Systems: Effect of Chloramphenicol1

BEN-ZION CAVARI, VIJAY K. KALRA, AND ARNOLD F. BRODIE

Department of Biochemistry, University ofSouthern California School of Medicine, Los Angeles, California90033

Received for publication 16 July 1971

Chloramphenicol was found to have a direct effect on the respiratory chain ofMycobacterium phlei cells grown in the presence of this drug. Analysis of the res-

piratory chain components revealed that the presence of chloramphenicol duringgrowth resulted in a partial inhibition in the synthesis of the cytochromes. How-ever, a stimulation in oxidative phosphorylation was observed with the cell-freeextract of cells grown in the presence of chloramphenicol. The oxidation of suc-

cinate 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 ofthe drug. Of particular interest was the finding that the cell-free extract of cellsgrown in the presence of the drug exhibited an increased level of phosphorylation(17 to 100%) when NADH was used as the electron donor. Chloramphenicol ap-

pears to affect a component of the respiratory chain between the flavoprotein andcytochrome c. Fractionation of the electron transport particles revealed an in-creased level of cytochrome b in the fractions which exhibited a stimulation in oxi-dative phosphorylation.

It has been known for some time that chlor-amphenicol (CAM) inhibits protein synthesis (9),and attempts have been made to see whetherCAM has any effect on the energy generationsystem (24). It was concluded that the antibioticat the growth-inhibitory concentration does notaffect processes of energy generation and thatany such effect, when found, must be a sec-ondary event related to the inhibition of proteinsynthesis caused by CAM. Recently Linnane andhis group (15, 28) demonstrated that, in yeastcells and in mammalian tissue culture cells (18),CAM inhibits the synthesis of mitochondrialproteins without affecting the synthesis of ribo-somal proteins. These conclusions were based onthe observation that the drug had no effect on apetite mutant of yeast and that, in wild type, syn-thesis of cytochromes a, a3, b, and cl was com-pletely inhibited and synthesis of succinic dehy-drogenase was partially inhibited, but cytochromec synthesis continued.A number of studies (22, 23, 28) have shown

an effect of CAM on either the energy-gener-ating system or on the electron transport chain.Godchaux and Herbert (23) reported that CAMat a concentration of 1 mg/ml appeared to inter-

'This is the 51st paper in a series dealing with oxidativephosphorylation in fractionated bacterial systems.

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fere with adenosine triphosphate formation inintact rabbit reticulocytes. Freeman and Haldar(22) claimed that, at 1.9 mg/ml, CAM was aspecific inhibitor of reduced nicotinamide ade-nine dinucleotide (NADH) oxidation by isolatedbeef heart mitochondria and that the inhibitionoccurred at the level of NADH dehydrogenase.Hanson and Hodges (25) reported that CAMacts as an uncoupling agent in maize mitochon-dria.

In the present report, an attempt was made tolocate the site of action of CAM on the respira-tory chains of Mycobacterium phlei.

MATERIALS AND METHODSM. phlei (ATCC 354) cells were grown and har-

vested 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/mland 20 ,g/ml to the growth medium resulted in a slightinhibition of the bacterial growth. Sonically disruptedcells were separated into particulate and supernatantfractions by differential centrifugation in a Spincomodel L preparative centrifuge (10). The electrontransport particles (ETP) obtained after centrifugationwere washed with a solution of 0.15 M KCI containing0.01 M MgCI2, and adjusted to pH 7.4 with N-2-hy-droxyethylpiperazine-N'-2'-ethanesulfonic acid (HE-PES)-KOH buffer (0.01 M).

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Respiration was measured by the conventional man-ometric technique at 30 C with a Gilson respirometer,or polarographically (16) with a Clark oxygen elec-trode. With the oxygen electrode, oxygen uptake wascalculated from the percentage change in oxygen satu-ration over a given interval of time, assuming that I mlof water saturated with air contained 237 nmoles ofoxygen at 30 C. Inorganic phosphate was determinedby the method described by Fiske and SubbaRow (20).The reduction of cytochromes was measured by usidlg a

double-beam spectrophotometer (American InstrumentCo.) equipped with a vibrating platinum oxygen elec-trode, or by using a Cary model 14 recording spectro-photometer.

Succinic dehydrogenase activity was assayed spectro-photometrically by following the rate of reduction ofdichlorophenol indophenol (molar absorbancy at 600nm, 19.1 mm-' cm-'; reference 5). Succinate-cyto-chrome c reductase activity was assayed spectropho-tometrically by following the rate of reduction of3(4, 5-dimethyl thiazolyl 2)-2, 5-diphenyl tetrazoliumbromide (MTT; molar absorbancy at 565 nm, 15.0mM-' cm-'; reference 4) or horse heart cytochrome c

(molar absorbancy at 550 nm, 20.3 mM-' cm-'; refer-ence 35). Both MTT and horse heart cytochrome c havebeen shown to accept electrons at the cytochrome c levelin M. phlei (1).

The quinone MK9 (II-H) was extracted by themethod of Folch et al. (21) and purified by thin-layerchromatography on Silica Gel G plates (250 um inthickness) impregnated with 0.1% rhodamine G. Thesolvent used was 12% n-butylether in hexane. The qui-none was scraped off the plate and eluted from theadsorbent with diethyl ether. The quinone was dis-solved in ethanol containing 0.01 volume of I M am-monium acetate buffer (pH 5.0) and reduced by addingsufficient sodium borohydride from a freshly preparedaqueous solution. The concentration of quinone was

determined from the difference in optical density at 245nm of reduced-minus-oxidized quinone (molar absor-

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

al. (31).

RESULTSEffect of CAM on oxidative phosphorylation.

CAM in concentrations of 10 yg/ml or 20 ,g/mlin the growth medium was found to inhibit pro-

tein synthesis of the cells by about 10% and 30%,respectively. The ability of the ETP derived fromcells grown with 10 or 20 og of CAM per ml(CAM particles) to carry out oxidative phospho-rylation was compared to the ETP derived fromcells grown in the absence of the drug. The CAMparticles were found to have an increased level ofsuccinoxidase activity (see Table 6) and an in-creased level of phosphorylation with NADH as

the electron donor. About 10 experiments wererun; a representative experiment is shown inTable 1. The changes from one experiment toanother did not exceed 10%. Although the levelof phosphorylation increased with succinate assubstrate, the P/O ratios observed with CAMparticles were similar to those of the regularETP. Increasing the concentration of CAM inthe growth medium from 10 gg/ml to 20 ,g/mlresulted in a further increase in both oxidationand phosphorylation. However, a concentrationof 30 gg of CAM per ml (data not shown) re-sulted in a significant inhibition of protein syn-thesis and a decrease in the level of oxidativephosphorylation.CAM had an effect only where protein syn-

thesis could take place (Table 2). Oxidation andphosphorylation of regular ETP with NADH orsuccinate as electron donors were measured in

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

ETP Substrate 0 Per cent ApIb P/ Per cent(gatoms) of normal ('smoles) of normal

Normal ................ Succinate 6.1 4.6 0.75NADHC 17.3 6.3 0.38

CAM (10 Jg/ml) ....... Succinate 7.5 123 6.0 0.80 106NADH 18.0 104 8.8 0.49 129

CAM (20 ,sg/ml) ....... Succinate 8.8 144 7.2 0.82 109NADH 17.2 99 11.1 0.65 171

aThe system consisted of: M. phlei particles (2 mg of protein); tris(hydroxymethyl)aminomethane-hydro-chloride 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 ofoxygen consumption was measured by the conventional manometric technique (Gilson differential respirometer) at30 C for 20 min. The reaction was stopped by the addition of 1.0 ml of 10% trichloroacetic acid, and a portion wasused for phosphate determination as described by Fiske and SubbaRow.

PPi, inorganic phosphate.c Reduced nicotinamide adenine dinucleotide.

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VOL. 108, 1971 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 ofCAM had no effect on either oxidation or phos-phorylation. Thus the effect of CAM observedwith the ETP from CAM-grown cells appears toresult in an alteration of the ETP structure ofrespiratory carriers.The fact that the phosphorylation of the CAM

particles was stimulated with NADH as sub-strate indicated that the oxidation of the NADHwas mediated by electrons moving through themain respiratory chain and not through the non-phosphorylative electron transport bypass asso-

ciated with this substrate (3). Further indicationthat the oxidation of NADH occurred throughthe main respiratory chain was the finding thatKCN inhibited NADH oxidation of CAM parti-cles to the same extent as that of regular parti-cles (Table 3). Thus the increased level of phos-phorylation associated with NADH oxidationwith the CAM particles was not the result of aninhibition in the synthesis of the bypass enzymes.

Effect of aging on oxidation and phosphoryla-tion. Aging of the particles was found to have a

pronounced effect on both oxidation and phos-phorylation. The particles were aged by storageat 4 C. The succinoxidase activity of the CAMparticles was 41% higher than that of the regularparticles after the first day of storage (Table 4).However, after the second day the succinoxidaseactivity of the CAM particles was only 28%higher and after the fifth day only 20% higherthan that of the normal particles. Although thedifferences were small, they were significant be-cause they were obtained with the same order ofmagnitude in all four experiments that were car-

ried 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 ofphosphorylation was observed with this sub-strate, which continued to increase from 17%

after the first day to 44% after the fifth day andfinally decreased to 29% after the seventh day.Particles obtained from cells grown in the ab-sence of the drug exhibited no changes or a slowdecrease in both oxidation and phosphorylationduring the aging process.

Effect of heat on the ETP from CAM-growncells. Brief exposure to heat (50 C for 10 min) ofthe suspension of ETP (30 mg of protein/ml) in0.15 M KCl has been shown to reduce oxidativeTABLE 2. Effect ofchloramphenicol (CAM) on

oxidative phosphorylation with Mycobacterium phleielectron transport particlesa

Substrate CAM 0 'IP5b Padded (,uatoms) (rmoles)

Succinate ... - 12.0 8.0 0.67Succinate ... + 11.6 8.0 0.69NADHC .... - 7.0 4.2 0.60NADH ... + 7.5 4.4 0.59

a Conditions were similar to those described in Table1, except that 20 ,ug of CAM per ml was added as indi-cated.

b Pi, inorganic phosphate.c Reduced nicotinamide adenine dinucleotide.

TABLE 3. Effect ofKCN on respiration with NADHas substratea

0 (Mgatoms) Per centETP ihbto

-KCN +KCN inhibition

Normal .......... 14.5 2.9 80CAM (10 ug/ml) 11.4 2.3 80CAM (20 ug/ml) 10.3 1.7 84

a Conditions were similar to those described in Table1, except that KCN (3 x 10-3 M) was added as indi-cated, and only NADH was used as a substrate. Ab-breviations: NADH, reduced nicotinamide adeninedinucleotide; ETP, electron transport particles; CAM,chloramphenicol.

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

Succinate NADHb

Day 0 uptake (uatoms) P/O 0 uptake (gatoms) P/O

Normal CAM Normal CAM Normal CAM Normal CAMparticles particles particles particles particles particles particles particles

6.9 9.7 0.78 0.76 23.5 25.7 0.36 0.422 6.7 8.6 0.80 0.77 22.6 21.9 0.33 0.445 6.1 7.3 0.82 0.88 19.2 17.9 0.39 0.567 6.1 7.5 0.75 0.80 17.3 18.0 0.38 0.49

a Conditions were similar to those described in Table 1. The particles were stored in the cold, and oxidativephosphorylation was examined on the days indicated. The CAM particles were prepared from cells grown in thepresence of 10 ,ug of CAM per ml.

bReduced nicotinamide adenine dinucleotide.

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phosphorylation (7). Exposure of normal ETP toheat increased succinoxidase activity (up to100%), whereas the phosphorylation associatedwith this oxidation increased about 50%. In con-trast, with NADH as the electron donor, expo-sure to heat resulted in a slight inhibition of oxi-dation, but the level of phosphorylation increasedat least threefold. Thus, it was of interest to de-termine whether the particles from CAM-growncells, which exhibit increased oxidation of suc-cinate and increased levels of phosphorylationwith NADH, could respond to heat treatment ina manner similar to that exhibited by the normalETP. The effect of heat treatment of normal andCAM particles on oxidation and phosphorylationwith succinate or NADH as substrates is shownin Table 5. The results obtained with normal andCAM particles before heating were similar tothose shown in Table 1. However, after heating,an increase in the oxidation of succinate wasobserved but not with NADH as the electrondonor (Table 5). These results were observedwith the regular and the CAM particles. A slightincrease in the P/O ratios was observed afterheat treatment with both succinate and NADHas substrates, with the regular or treated parti-cles.

Heat treatment of the ETP has been shown toresult in conformational change of the ETP(Kalra, Aithal, and Brodie, manuscript in prepa-ration). This effect of heat treatment was not dueto the presence of a heat-labile inhibitor or a reg-ulator. The effect of CAM was thought to be dueto the inhibition of the synthesis of a natural in-hibitor; however, this appears unlikely, sinceETP from CAM-grown cells still exhibit in-creased phosphorylation after heat treatmentwith succinate as a substrate.

Site of action of CAM. To determine the siteof action of CAM, the respiratory carriers of theelectron transport chain were examined. Cyto-chromes a and c content was followed with adouble-beam spectrophotometer to determine therates of reduction of the cytochromes as well asthe total amount of the cytochromes in the dif-

ferent types of particles. Since a stimulation inthe level of oxidation was observed with theCAM particles with succinate, this substrate wasused to follow the enzymatic reduction of thecytochromes. The time required to reach thetransition point from an aerobic to an anaerobicstate was shorter for the CAM particles than forthe regular particles. Although an effect on oxi-dative phosphorylation was observed with parti-cles obtained from cells grown on 10 ,ug of CAMper ml, a more pronounced effect was observedwith ETP from cells grown in the presence of 20Ag of CAM per ml. The pattern of reduction ofcytochromes c and a was the same in regularparticles as in particles derived from cells grownwith 10 ,g of CAM per ml (Fig. 1). However,with particles derived from cells grown with 20

FIG. 1. Reduction ofcytochromes c and a in normalelectron transport particles and in chloramphenicol(CAM) particles. The system consisted of: tris(hydrox-ymethyl)aminomethane-hydrochloride buffer (pH 7.4),100 1tmoles; MgC2, 15 Mlmoles; particles (6 mg ofpro-tein); succinate, 20 /gmoles; and water to a final volumeof 3.0 ml. The reaction was followed spectrophoto-metrically with an Aminco double-beam spectropho-tometer. Solid line, normal particles or 10 ug/ml CAMparticles; dashed line; 20 ugg/ml CAM particles.

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

Before heating After heatingETP Substrate ______

0(luatoms) AP, P/O 0 (gatoms) AP, P/O

Normal Succinate 3.3 2.0 0.61 6.0 4.3 0.72NADH 7.8 1.6 0.21 7.2 1.9 0.26

CAM (10 gg/ml) Succinate 4.9 3.3 0.67 7.8 5.6 0.72NADH 7.9 2.4 0.30 8.0 2.9 0.36

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

CYTOCHROME c (551 - 540)

T NAaSAO4. -QCt-0.0044 -- --

SUCCINArE

CYTOCHROME a (601i-62

OD-0.00088- -

SMIIN'SUCCINATE

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Ag of CAM per ml, the reduction of cytochromesc and a differed from that found in the regularparticles. The total amounts of chemically reduc-ible cytochromes c and a were 60% and 50%,respectively, of that found in the normal parti-cles. In normal ETP as well as in CAM parti-cles, all cytochrome c was enzymatically re-

duced; 94% of cytochrome a in ETP was reducedenzymatically, whereas in the CAM particlesonly 70% of the total amount of cytochrome a

was enzymatically reducible, as based on proteinconcentration of both types of the particles.The M. phlei system has been shown (8) to

contain two different hemochromogens whichexhibit absorption between 557 or 558 nm and562 nm. Of particular interest was the findingthat one of these hemochromogens, presumablyb type, which has maximum absorption at 433and 562 nm, was reduced by NADH (Fig. 2),whereas the other component (absorption at 430and 557 or 558 nm) was reduced when succinatewas used as the electron donor. The character-istic profiles of the b-type cytochromes were as-

sayed by using ascorbate and N,N,N',N'-tetra-methyl-p-phenylenediamine (TPD) in both thereference and sample absorption cell. Since as-corbate and TPD enter the respiratory chain atthe level of cytochrome c (29), both cytochromec and a are reduced in both absorption cells, thuspermitting the reduction of cytochrome b to befollowed in the absence of reduction of cyto-chromes c and a. It was of interest to determinewhether CAM inhibits the synthesis of only one

REGULAR PARTICLES

550 600 650 nm 550 600 -85O

FIG. 2. Difference spectrum of cytochrome b. Thesystem consisted of 100 1umoles of tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4), 100nmoles of N, N, N', N'-tetramethyl-p-phenylenedi-amine, electron transport particles equivalent to 3 mgofprotein, 10 uimoles of ascorbate, and water to a finalvolume of 1.0 ml. The reaction was started by the addi-tion of 25 timoles of reduced nicotinamide adenine di-nucleotide (A) or 25 Mmoles of succinate (B). The re-

duction of cytochrome b was followed spectropho-tometrically with a Cary model 14 spectrophotometer.

type of cytochrome b. In contrast to regular par-ticles, the particles from CAM-grown cells ex-hibited only one type of cytochrome b, with max-

imum absorption at 560 nm, which was irrespec-tive of the electron donor used (Fig. 3). The totalamount of cytochrome b (560 nm) in the CAMparticles as measured by reduction with sodiumdithionite was 80% of that found in the regularparticles, 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 inboth types of particles by measuring the succinicdehydrogenase activity. Different dyes were usedas electron acceptors. Although the oxidation ofsuccinate by the particles was stimulated by 48and 70% in the ETP from cells grown on 10 and20 .g of CAM per ml, respectively, there was a

slight decrease in the succinic dehydrogenase ac-

tivity when dichlorophenol indophenol was usedas the electron acceptor (Table 6). This dye ac-

cepts the electrons at the flavoprotein level; how-ever, when an electron acceptor was employedwhich accepts electrons from endogenous cyto-chrome c, such as MTT or mammalian cyto-chrome c (1), the CAM particles were found tobe more active than the regular particles. In ad-dition, the stimulation observed in succinate cy-tochrome c reductase activity of the CAM parti-cles was found to occur to the same extent as

that observed in the stimulation of succinic oxi-dase activity. This finding indicates that CAMaffects a component on the succinate chain whichlies in the segment between the flavoprotein andcytochrome c.Another indication that CAM acts by affecting

a component of the respiratory chain betweenthe flavoprotein and cytochrome c on the succin-oxidase pathway was obtained by studying theTPD shunt (Kalra, Krishna Murti, and Brodie,

CAM PARTICLES

0.03

C

0.02

0.0l

550 600 6506rnm 550 600 650

FIG. 3. Difference spectrum of cytochrome b. Con-ditions were as in Fig. 2, except that chloramphenicol(CAM) particles were used instead of regular normalelectron transport particles and either reduced nicotin-amide adenine dinucleotide (A) or succinate (B) was

used to reduce the cytochrome.

B ASUCCINATE NADH

1 ~~~~~~~~~~560560IXX, V

I_ e e

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TABLE 6. Effect ofgrowth with chloramphenicol(CAM) on succinoxidase, succinic cytochrome creductase, and succinic dehydrogenase activities of

Mycobacterium phlei

Succinic dehydrogenaseand succinic cyto-

Electron transport Succin- chrome c reductaseElecrontranespr oxidase activity"panticles activitya

DCIP MTT Cyto-chrome c

Normal 127 55 4.6 6.3CAM (10 Ag/ml) 188 57 5.3 8.5CAM (20 Ag/mI) 216 47 9.8 10.6

a Succinoxidase activity was measured polarograph-ically with Clark's oxygen electrode. The system forsuccinoxidase activity contained particles (4 mg of pro-tein), 100 gmoles of HEPES-KOH buffer (pH 7.4), 15,moles of MgCI2, and water to a final volume of 3.0ml. Results are expressed as nanoatoms of oxygen pro-duced per milligram of protein per minute.

b The system for succinic dehydrogenase and succiniccytochrome c reductase activity consisted of: tris(hy-droxymethyl)aminomethane-hydrochloride buffer (pH7.4), 100 umoles; MgCl2, 15 ,moles; dichlorophenolindolephenol (DCIP), 3(4, 5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT), or horse heartcytochrome c, 200 nmoles; particles, 200 ug of proteinwith DCIP or MTT and I mg with cytochrome c; KCN(9 ymoles) was added when DCIP or cytochrome c wasused as electron acceptor; water to a final volume of 3ml. The reaction was started by the addition of suc-cinate (50 umoles), and the rate of reduction wasmeasured at 600 nm with DCIP, at 565 nm with MTT,and at 550 nm with cytochrome c.

unpublished data). When the respiratory chainwas blocked by 2 nonyl-8-hydroxyquinoline-N-oxide or by irradiation at 360 nm, the addition ofTPD was found to restore the oxidation ofNADH or succinate by bypassing the block.TPD was shown to accept the electrons from fla-voproteins, transferring them to cytochrome c +cl (32). As can be seen in Table 7, the succinoxi-dase activity was more than two times higher inthe CAM particles than in the regular particles.At 3.3 Ag/ml, 2 nonyl-8-hydroxyquinoline-N-oxide inhibited the oxidation of the regular andthe CAM particles. The addition of TPD re-stored the oxidation of both the regular and theCAM particles to the same level, so that higheractivity was no longer observed in the CAM par-ticles.

It thus appears that the site of CAM action inthe succinoxidase pathway is located after theflavoproteins and before cytochrome c. Thissegment of the succinoxidase chain contains anunidentified light-sensitive component, nonhemeiron, and cytochrome b (30). In ETP of CAM-grown cells, no detectable amount of nonheme

iron was observed. This does not mean that non-heme iron is not functioning. The segment be-tween the flavoprotein and cytochrome c in theNADH chain contains quinone and cytochromeb (2). To determine whether the stimulation inthe phosphorylation activity coupled withNADH oxidation was due to a difference in theamount of the quinone in the regular and inCAM particles, the quinone was extracted fromthe different particle preparation and measuredspectrophotometrically (Table 8). The amount ofthe quinone was not significantly different in theregular and in the CAM particles.

Distribution of the oxidative phosphorylationactivity with different particulate fractions. TheETP from M. phlei have been shown to be com-posed of a heterogenous particle population (26),which differs in quinone content, cytochromes,size, composition, and activity (11, 26). It was ofinterest to see whether CAM affected all types ofparticles 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 dispersedin 0.25 M sucrose. Portions (0.5 ml) were layeredon top of a 4.5-mI linear sucrose gradient (1.0 to1.25 M) and centrifuged in an SW39 rotor at36,000 rev/min for 15 hr in a Spinco model Luntracentrifuge. Fifteen fractions of 0.33 ml eachwere collected. Protein content was determined,

TABLE 7. Restoration of the NQNO-blockedsuccinoxidase activity by TPDa

Succinoxidase activitybElectron transport

particles

Normal ..........

CAM (20 ug/ml)

TPD

175263

aThe system was the same as in Table 6. NQNO(10 ug per mg of protein) and TPD (300 nmoles) wereadded as indicated. Abbreviations: NQNO, 2 nonyl-8-hydroxyquinoline-N-oxide; TPD, N,N,N',N'-tetra-methyl-p-phenylenediamine; CAM, chloramphenicol.

Results expressed as nanoatoms of oxygen pro-duced per minute per milligram of protein.

TABLE 8. Concentration ofquinones inMycobacterium phlei particles from cells grown inpresence and absence of chloramphenicol (CAM)

Electron transport Quinones (nmoles/particles mg of protein)a

Normal 16.8CAM (15 jig/ml) 18.0CAM (20 jg/ml) 15.2

a Quinones were extracted and the amount was de-termined as described in Materials and Methods.

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and oxidation and phosphorylation were exam-ined with succinate and NADH as the electrondonors. The results are summarized in Table 9.Only the results for fractions 5 to 10 are given inTable 9, since very low activity was found infractions I to 4 and 11 to 15. The distribution ofthe oxidative and phosphorylative activities inthe regular particles was not the same whenNADH or succinate were used as substrates.Maximal oxidative activity for succinate was

found in fraction 8, whereas, for NADH, max-

imal activity was found in fraction 5. Maximalphosphorylative activity was found in fraction 6when succinate served as an electron donor andin fractions 8 and 9 when NADH was the sub-strate.

It is of interest that the stimulation of oxida-tion and phosphorylation in the CAM particlesexhibited slightly different patterns of distribu-tion in the different types of particles. Stimula-tion in succinoxidase activity in the CAM parti-cles was concentrated in fractions 6 and 7,whereas stimulation in phosphorylation withNADH as substrate was located in fractions 5, 6,and 7.

Distribution of cytochromes b, c, and a + aswas examined in the different particulate frac-tions to determine whether there was any corre-

lation between increased activity and cytochrome

content. The difference spectra (reduced minusoxidized) of the different particulate fractionswere taken, and the amount of cytochrome (nan-omoles per milligram of protein) was plottedagainst the fraction number (Fig. 4). Cyto-chromes a and c were almost equally distributedthroughout fractions I to 9; however, cyto-chrome b exhibited a peak at fractions 5 to 7corresponding to the fraction exhibiting the stim-ulation in oxidative phosphorylation. The distri-bution of the cytochromes was the same innormal ETP as that described for the CAM par-ticles; however, the amount of cytochromes b, c,and a + as decreased in the particles derivedfrom CAM-grown cells (40, 20, and 50%, respec-tively, as compared to regular particles).

DISCUSSIONCAM has been shown to inhibit protein syn-

thesis; relatively high concentrations of this drugare 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 pro-tein synthesis but caused a marked effect on oxi-dative phosphorylation. At 10 ,ug/ml, CAM doesnot inhibit the synthesis of cytochromes, whereas20 ,g/ml was found to decrease the level of cyto-chrome c, b, and a to 40, 20, and 50%, respec-tively. It was surprising to find that the cell-free

TABLE 9. Oxidative phosphorylation in fractionated particlesa

Normal particles CAM particles (% change fromregular particles)Fraction Substrate

0 (juatoms/ P,1 (umoles/ P/0 O (uatoms) P/0mg of protein) mg of protein)

5 Succinate 3.1 1.2 0.39 0 +46NADHC 12.5 0.4 0.03 + 10 + 100

6 Succinate 2.7 2.5 0.93 +67 + 12NADH 11.6 1.3 0.11 +20 +91

7 Succinate 4.6 2.9 0.63 +63 +33NADH 9.8 2.1 0.27 +1 +86

8 Succinate 5.8 3.8 0.64 0 +23NADH 9.9 3.8 0.38 + I 0

9 Succinate 4.0 2.4 0.60 -20 - 13NADH 4.7 1.9 0.41 +32 0

10 Succinate 3.1 1.3 0.41 -80 0NADH 3.3 0.9 0.28 - 10 -80

a Normal particles and chloramphenicol (CAM) particles derived from cells grown with 15 jig of CAM per mlwere dispersed in 0.25 M sucrose. Samples (0.5 ml) were layered on top of a 4.5-ml linear sucrose gradient (1.0 to1.25 M) and centrifuged in a Spinco model L preparative ultracentrifuge with an SW39 rotor at 36,000 rev/min for 15hr. Fifteen fractions of 0.33 ml each were collected. The protein content of each fraction was determined, and theoxidative phosphorylation was measured by using the system described in Table 1.

Ppi, inorganic phosphate.c NADH, reduced nicotinamide adenine dinucleotide.

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2 normal particles. Furthermore, the level of qui-none was found to be the same in the CAM and

A0 / \the regular particles.e0Z Succinic cytochrome c reductase activity wasi_ d \found to be stimulated in the CAM particles to8 the same extent as succinoxidase activity. This

3oa. finding indicated that the stimulatory effect oc-00 CYT.c \ curred before cytochrome c. In addition, whenu0 6 measuring the succinoxidase activity by using the

TPD shunt, thus bypassing the cytochrome b, noCYT. b stimulation was observed. It appears that the siteD 4 _r\ / \ of action of CAM is in the cytochrome b region.w Q zCYT.a Support for this hypothesis was the finding that

2 a the synthesis of cytochrome b was only slightly2 ' ° _ ~° inhibited by CAM and that most of the cyto-chrome b was located in those fractions which

.________________________________ .exhibited stimulation in the oxidative phosphoryl-2 4 6 8 10 12 ation.

FRACTION NO. Two types of hemochromogens which appearto be of the b type have been demonstrated in

FIG. 4. Distribution of the cytochromes (Cyt) in tTP of Mhei. Of paru interestwasthechioramphenicol particle fractions separated by sucrose finding that On petwar Interest was the

density gradient. The difference spectrum of each frac- ftion was taken by adding a few grains of sodium di- NADH, whereas the other was reduced rapidlythionite to the sample cuvette. The molar absorbancy by succinate and slowly by NADH (8). Since theused to calculate the amount of cytochrome b (560) ETP from CAM-grown cells exhibited an in-was based on the assumption that this b-type cyto- creased level of succinoxidase activity and thechrome has a molar absorbancy similar to that de- stimulation appeared to be due to a componentscribed for cytochrome b (562) by Freeman and between the flavoprotein and cytochrome c, theHaldar (22). effect of CAM on the level of the two b-typeextract from CAM-grown cells exhibited stimu- cytochromes was examined. In contrast to thelation in succinoxidase and stimulation in phos- regular ETP, the particles from CAM-grownphorylation with NADH as substrate, an effect cells contained only one type of cytochrome bthat was even more marked in the concentration (maximal absorption at 560 nm) which differedof CAM that caused inhibition of cytochrome from either b type from regular particles. Thesynthesis. Although there was a loss in cyto- 560-nm b-type cytochrome was reduced by eitherchrome content, it was of interest to find an in- NADH or succinate and at the same rate. Thecrease in the level of respiration and coupled ac- amount of cytochrome b, assuming the sametivity. This finding suggests that the total content molar absorbancy, was reduced 20% in the parti-of the terminal cytochromes may not be required cles from CAM-grown cells. The studies of thefor the energy-generating pathway. CAM has nature of cytochrome b fail to explain the ob-also been shown to inhibit the synthesis of cyto- served results with the particles from CAM-chromes a, a3, b, and cl in the actively growing grown cells. Another explanation which has beenliver tissue of rat (6, 19). suggested for a role for cytochrome b is that this

Because of the differential effects of CAM on respiratory carrier serves a dual function: one inthe respiratory chains with NADH or succinate electron transport and the other as a structuralas substrates, it was assumed that the site of ac- organizer of the respiratory components (12, 13,tion of CAM was in the region where the chains 17, 34).are separate, i.e., between the substrate and cyto- The ETP from CAM-grown cells exhibited anchrome b. In M. phlei, those two separate parts increased level of phosphorylation with NADHof the chain contain different components: qui- as the electron donor. This is particularly sur-none in the NADH chain and a light-sensitive prising since the level of phosphorylation withcomponent and non-heme iron in the succinate succinate as an electron donor was not increased.chain. Examination of the respiratory carriers CAM may act by preventing the synthesis of afailed to reveal major differences in the compo- substance which inhibits or regulates the respira-nents between substrate and cytochrome b in the tory chain. Furthermore, the particles fromETP from cells grown with or without CAM. CAM-grown cells, like regular particles sub-The succinic dehydrogenase activity in the CAM jected to heat treatment, do not require the addi-particles was found to be the same as that in tion of soluble coupling factors for phosphoryla-

1024 CAVARI, KALRA, AND BRODIE J. BACTERIOL.

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VOL. 108, 1971 ACTION OF CAM ON RESPIRATORY CHAINS OF M. PHLEI

tion (7). An inhibitor similar to that suggestedabove has been suggested by several groups (14,27, 33).

ACKNOWLEDGMENTS

The technical assistances of Patricia Brodle and HirokoSakamoto is gratefully appreciated.

This investigation was supported by Public Health Servicegrant Al 05637 from the National Institute of Allergy andInfectious Diseases, by grant GB 6257XI from the NationalScience Foundation, and by a grant from the Hastings Founda-tion of the University of Southern California School of Medi-cine.

LITERATURE CITED

1. Asano, A., and A. F. Brodie. 1964. Respiratory chains ofMycobacterium ph/li. J. Biol. Chem. 239:4280-4291.

2. Asano, A., and A. F. Brodie. 1965. Phosphorylation cou-

pled to different segments of the respiratory chains ofMycobacterium pheli. J. Biol. Chem. 240:4002-4010.

3. Asano, A., and A. F. Brodie. 1965. The properties of thenonphosphorylating electron transport bypass enzymes

of Mycobacterium phlei. Biochem. Biophys. Res.Commun. 19:121-126.

4. Asano, A., T. Kaneshiro, and A. F. Brodie. 1965. Malate-vitamin 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 mito-chondrial protein components in rat liver slices. J. Biol.Chem. 243:4027-4033.

7. Bogin, E., T. Higashi, and A. F. Brodie. 1970. Effects ofheat treatment of electron transport particles on bac-terial oxidative phosphorylation. Proc. Nat. Acad. Sci.U.S.A. 67:1-6.

8. Bogin, E., T. Higashi, and A. F. Brodie. 1969. Extraparti-culate chain interaction between different electron trans-port particles. Science 165:1364-1367.

9. Brock, T. D. 1961. Chloramphenicol. Bacteriol. Rev. 25:32-48.

10. Brodie, A. F., and C. T. Gray. 1956. Phosphorylation cou-pled 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 biologicallyfrom mevalonic acid, p. 119-143. In T. W. Goodwin(ed.), Biological function of terpenoid quinones. Aca-demic Press Inc., London.

12. Bruni, A., and E. Racker. 1968. Reconstitution of the suc-

cinate-ubiquinone reductase. J. Biol. Chem. 243:962-971.

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. Hig-

gins. 1955. A mechanism for oxidative phosphorylation.J. Biol. Chem. 217:439-451.

15. Clark-Walker, G. D., and A. W. Linnane. 1967. A com-parison between cytoplasmic respiratory deficient mutantyeast 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

polarography. J. Appl. Physiol. 6:189-193.17. Dawson, A. P., G. F. Cox, and M. J. Selwyn. 1968. An

effect of malate of the redox state of a cytochrome bcomponent in mitochondria from various source.Biochem. Biophys. Res. Commun. 32:579-587.

18. Firkin, F. C., and A. W. Linnane. 1968. Differential effectsof chloramphenicol on the growth and respiration ofmammalian cells. Biochem. Biophys. Res. Commun. 32:398-402.

19. Firkin, F. C., and A. W. Linnane. 1969. The effect ofchloramphenicol on regenerating rat liver. Exp. Cell.Res. 55:68-76.

20. Fiske, C. H., and Y. SubbaRow. 1925. The colorimetricdetermination of phosphorus. J. Biol. Chem. 66:375-400.

21. Folch, J., 1. Ascoli, M. Lees, J. A. Meath, and F. N. Le-Baron. 1951. Preparation of lipid extracts from braintissue. J. Biol. Chem. 191:833-841.

22. Freeman, K. B., and D. Haldar. 1967. The inhibition ofNADH oxidation in mammalian mitochondria by chlor-amphenicol. Biochem. Biophys. Res. Commun. 28:8-12.

23. Godchaux, W., and E. Herbert. 1967. The effect of chlor-amphenicol in intact erythroid cells. J. Mol. Biol. 21:537-553.

24. Hahn, F. E. 1967. Chloramphenicol, p. 308-330. In D.Gottlieb and P. D. Shaw (ed.). Spring-Verlag, NewYork.

25. Hanson, J. B., and T. K. Hodges. 1963. Uncoupling actionof chloramphenicol as a basis for the inhibition of ionaccumulation. Nature (London) 200:1009.

26. Higashi, T., E. Bogin, and A. F. Brodie. 1969. Separationof a factor indispensable for coupled phosphorylationfrom the particulate fraction of Mycobacterium phlei. J.Biol. Chem. 244:500-502.

27. Horstman, L. L., and E. Racker. 1970. Interaction be-tween mitochondrial adenosine triphosphatase inhibitorand mitochondrial adenosine triphosphatase. J. Biol.Chem. 245:1336-1344.

28. Huang, M., D. R. Biggs, G. D. Clark-Walker, and A. W.Linnane. 1966. Chloramphenicol inhibition of the forma-tion of particulate mitochondrial enzymes of saccharo-myces cerevisiae. Biochim. Biophys. Acta 114:434-436.

29. Jacobs, E. D., and D. R. Sanadi. 1960. Phosphorylationcoupled to electron transport mediated by high potentialelectron carriers. Biochim. Biophys. Acta 38:12-34.

30. Kurup, C. K. R., and A. F. Brodie. 1967. Studies on theinvolvement of metal in Mycobacterium phlei. J. Biol.Chem. 242:197-203.

31. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

32. Mustafa, M. G., M. L. Cowger, R. F. Labbe, and T. E.King. 1968. General nature of "Wurster's Blue Shunts"in the respiratory chain. J. Biol. Chem. 243:1908-1918.

33. Pullman, M. E., and G. C. Monroy. 1963. A naturallyoccurring inhibitor of mitochondrial adenosine triphos-phatase. J. Biol. Chem. 238:3762-3769.

34. Tyler, D. D., R. W. Estabrook, and D. R. Sanadi. 1965.Electron and energy requirements for cytochrome b re-

duction during the oxidation of tetramethyl-p-phenylenediamine. Biochem. Biophys. Res. Commun. 18:264-269.

35. Van Gelder, B. F., and E. C. Slater. 1962. The extinctioncoefficient of cytochrome c. Biochim. Biophys. Acta 58:593-595.

36. Zaugg, W. S., and J. S. Rieske. 1962. The quantitative es-

timation of cytochrome b in submitochondrial particlesfrom beef heart. Biochem. Biophys. Res. Commum. 9:213-217.

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