Observations on the inhibitor sensitivity of the choline oxidation system

ARCHIVES

OF

BIOCHEYIRTRY

Observations

AXD

BIOPHYSICS

115, 373-384 (1966)

on the Inhibitor

Sensitivity

Oxidation D. D. TYLER, Department

of Biophysics

System’

J. GONZE,

and Physical Pennsylvania,

of the Choline

AND

R. W. ESTABROOK’

Biochemistry, Johnson Research, Foundation, Philadelphia, Pennuylvania

University

of

Received May 6, lQG6 The influence of respiratory chain inhibitors on the choline oxidation system of rat liver mitochondria and submitochondrial particles has been investigated. In both t,ypes of enzyme preparations choline oxidation was inhibited by amytal, antimycin A, and cyanide, but was not affected by rotenone, which specifically inhibited the DPNH oxidation system, or by thenoyltrifluoroacetone, which was inhibitory only to succinate oxidation. Freshly prepared mitochondria suspended in 50 mM potassium phosphate buffer catalyzed an oxidation of endogenous substrate at about 30% of the rate at which choline was oxidized under the same conditions, but at a rate representing only a few percentage of the activity of t’he succinate oxidation system. It is suggested that the previously reported apparent inhibitor-resistant oxidation of choline is due to a concurrent inhibitor-resistant oxidation of endogenous substrate. Spectroscopic studies of the cytochromes (reduced minus oxidized difference spectra) of liver mitochondria reduced by choline or by succinate revealed that t)he same amounts of respiratory pigments were reduced by either substrate. It is tolleluded that in liver mitochondria, the respiratory chain pathways available for the oxidation of DPSH, choline, and succinate are common between cytochrome b and oxygen. A marked difference between the kinetics of inhibition of the respiratory chain by nmytal and by rotenonc is described.

Previous studies have revealed that, in liver and kidney tissue, the flavoprot’ein choline dehydrogenase is functionally linked to t’he cyt80chromc system (l-3). This conclusion is supporkd by the observation that the activit’y of the choline oxidation system of isolated liver mitochondria is suppressed by n number of respiratory chain inhibitors, including amytd, antimycin A, quinoline osidq3 azidc, and cyanide (1, 5). Kimura et 1 This work was snpported ill pari. by 1‘.S. Public Health Service grant GM 12202-01. * Recipient of a U.S. Public IIealth Service liesearch Career J)evelopment Award (GM-K34111). 3 Abbrevint ions used : quinolillp oxide, 2-w hept~l-l-h\-droxyqllirloliue S-oxide; TTB, 4,1,1, Trifluoro-l-(2-~l~ie~r~-l)-l,3-b~~taurdio~~r or then-

al. (3) have suggested t,hat cytochrome b component’s of the choline and t#he sue&ate oxidat8ion systems of liver mitochondria are not, the same and are not directly interlinked. Their c*onclusions were based upon t,he finding that’, when rat liver mitochondria were incubated in phosphate buffer containing exogenous cytochrome c, an inhibitorinsensitive oxidation of choline was observed, apparently due to the presence of a11 aut’ooxidizable component of the choline oxidation system at the level of c*yt,ochrome b. The present’ paper describes a rc-examinao~ltriflnoroacetolle; mCl-CCP, carbonyl cyanide ,,l-chlorophellq-1-h~draz~)l~e; BOTI, p-hydroxybuI yrat e; PUS, phellaxine methosrdfate; TR A, t riethanolamine hydrochloride.

373

373

TYLER,

GONZE,

tion of bhe inhibitor sensitivity of the choline oxidation system and a spectroscopic study of the behavior of cytochrome b during electron t,ransfer from choline to oxygen. Experimental data on the effects of two relachain inhibitors, tively new respiratory rotenone (6, 7), and TTB (8) are included. This work supplements a study of t,he components of the choline oxidation system and the site of action of amytal, which has been reported previously from t,his laboratory (9). A preliminary account, of the results has been presented (10). MATERIALS

AND

METHODS

Preparalive procedures. Rat liver mitochondria were isolated in 0.25 M sucrose by the method of Schneider (11). Submitochondrial pa,rticles were

AND

ESTABROOK

prepared from rat liver mitochondria 1,~ t11tl method of Chatlee et nl. (12) and ihrir fraction, designated “high speed precipit at (x,” w:~s rlsed a$ the stock enzyme suspension. Wnlerin2.s. Chemicals were obtained from the following so~~rces: Rotenone from K and K Laboratories, Inc., New York; sodium amytal from Eli Lilly and Company; ttntimycin A from 1he Kyowa Fermentation Illdustry Compatjy Ltd., Tokyo, Japan; TTB from Eastman Organic Chemicals; mCl-CCP was a gift from Dr. 1’. Hcytlcr of the E. I. d11 Pont de Nemorws and Compatly, Inc., Wilmingtolr, ljelaware; alld DPNH and cytochrome c (Type III) from the Sigma Chemical Company. Aliqlwts of 1 M solutions of potassium succinate, sodirlm (I)L)-P-hydrox?‘t)llt3.r:Ite, and choline chloride were added as indicated. Notcnone, TTB, and antimycin A were added in the form of small volumes (O.Ol-O.OZ ml) of conccntrated ethanolic solutions. A stock solution of 1 rn.v mCl-CCP was prepared by dissolving crystals of the compolmd in dilllte potassirlm hydroxide. Sodium amytal was freshly. prepared as :I 0.4 M solution in distilled water. dssay procedures. Oxygen uptake was measured polarographically by the method of Chappell (13) with a Clark oxygen electrode. The nllmbers beside the oxygen electrode traces presented in the figures denote the rate of oxygen tiptake, expressed as microliters of oxygen rltilized per milligram birwet protein per horlr. Two types of buffer were used for the enzyme assays. One buffer contained sucrose (0.25 M), triethanolamine-TIC1 (0.015 M, pH 7.41, and potassium phosphate (0.005 N, pH 7.J), and is referred to in the figure legends as “sucrose-TI:&phosphate buffer.” The second bllft’er contained potassium phosphate (0.05 M, pH 7.4). Kinetic measlwements of cytochrome oxidation and reduction were made in an Aminco-Chance dual wavelength spectrophot,ometrr (American Instrument Company, Maryland). Lon-temperature difference spectra were obtained by the method of Chance (14). Protein was estimated by the bilwet, method (15) with bovine serum albumin as standard.

RESULTS of rotenone and amytal on FIG. 1. Influence mitochondrial oxidations. The 2%ml reaction medium contained sucrose-TRA-phosphate buffer and eit,her 10 rnM sodium (uL)-P-hydroxybutyrate, in experiment,s A and B, or 7 nw choline chloride, in experiment C. Further additions of mitochondria (0.1 ml, containing 6.3 mg protein) and other reagents were as indicated. Temperature, 22”.

Influence of rotenone and amytal. The oxygen electrode traces presented in Fig. 1 illustrate three separate experiments in which equal samples of a stock suspension of rat liver mitochondria were diluted in a reartion medium containing either P-hydroxybutyrate (Fig. IA and 1B) or choline (Fig.

INHIBITION

OF CHOLINE

l(1). After a brief incubation of the mitoc*hondria, the uwoupling agent mCl-CCP (16) was added to ensure optimal conditions for the oxidation of added suhst)rntes during the subsecluent course of the cxpcriment~s. In the first two experiments, the iqid oxidation of p-hydroxybutyrate was inhibited strongly by the addition of either rotcnone (I’&. 1.4) or amytal (Fig. IH). The further addition of choline restored oxyg:‘en uptake in tjhc rote none-treated mitochondria but not in the :m~yt,al-treated mitochondriu. In the Iat,ter experiment oxygen uptake was restored by t,he addition of succinnte. In the third cxperiment (Fig. lC), the choline oxidation rat,e was found to he largely unuffectcd by rotcnone hut was inhibited st.rongly by t.he furt,hcr addition of nmytal. Control experiments, in which 110 added substrate was present, showed t.hat the rate of oxidation of endogenous substrate in the uncouplertrentcd mit.ochondria was too low (&o* = 6) to have any significant influence on the results. Experiments of the type illustrated in Fig. 1 therefore suggestfed that choline oxidation was largely unaffected in liver mitochondria t.reated with sufficient rot,enonc to inhibit DPN-linked substrate oxidat’ion almost c*ompletcly. Similar results were obt airlwl when 2 ,4dinit,rophenol (30 PM) was used as the uncoupling agent instead of mCl-WI’, and also when rat. kidney mitochondria were used as the enzyme prcparation. The experiments described above did not rule out the possibility that choline was ~ctivst~ing a rotenone-insensit,ive oxidation of endogenous sub&rate. This interpretnt,ion was shown to be incorrect by t)he results of cxpcriment,s with submitochondrial liver particles. The part’icles were found to (*OILtain enzyme systems for l.he oxidation of DPNH, choline, and suocinate (cf. 21), but did not appear to cont#ain endogenous substrates. Experiments performed with these particles were similar to those shown in Fig. 1, cxwpt that DPNH was used as substrat,e instead of P-hydroxybutyrat,c. So n&XCCP was added to the reaction mixture, since the rate of oxidat,ions catalyzed by the partjicales was not affected by uncoupling agents. The oxygc11 elettrodo traws prcwnted irl I’ig. 2

OSIDASE

373

FIG. 2. Influence of roteuone and nm~inl on oxidations catalyzed 1)~ sktbmitochorldrial particles. The 3-ml reaction medillm contained sncrose-TR.~-phosphate buffer, and srtbmit,ochondrixl particles (2.8 mg protein). Frwther additions W-WC made 21s indicated. Tempernture, 22”.

show that a differential effect of rot’enone and amytal on choline oxidation, similar to that demonst.rated wit,h mitlochondrin (Fig. I), could also be demonstrated with t,he submitochondri:tl particle preparat,ion. The changes in oxygen upt,ake observed in t8hc experiments of Fig. 2~2 and 2B were correlated with vhangcs in the oxidation-reduction state of the endogenous cyt,ochromcs of the particles, by repeating the experiments in a dual wavelcngt,h spectrophotomcter set at, 551+X0 1nl.L.This wave1engt.h pair largely measures extinction changes due to the oxidation and reduction of endogenous rytochromes c and cl . A low conwntration of azide was added init,ially t’o inweasc the magnitude of the steady-A&e extinction changes occurring during the course of the

TYLER,

GONZE, AND ESTABROOK

100

OPNH

80

Fro. 3. Influence of rolenone and amytal on the oxidation and redllclion of endogenorls CJYOchrome c + cl dnring the oxidation of added suh-

strates by s~~t)mitocholldri~l pitrticles. .k cnvette containing 3 ml of sl~crose-TRA-phosphate brlffer and submitochondrial particles (3.2 mg protein) was placed ill n dlla1 w:ivefength spectrophotometer, and the absorbance diff’erence at, 551 rnp mintIs 540 rn@was determined. An upward deflection of the tracings indicates a redllc(ion of cytochrome c + c1 . Further

additions

to the reaction

IL Amytol

Concentration, mM

FIG. 1. Illflrle~tce of amy(al cotIwtttr:tlion the rate of oxidation of L)PNII atrd choline

011 1)~

sltbmitochondrial particles. The %ml rr:tctioll medium cant ainsd sl~crose-TI~A-phosph:lteb~~flkr, aud sut,lnitocholldriaf particles (2.8 mg potcin). The efcct of variotls concentrations of amytal on the initial rate of oxygen uptake observed aftcz the addition of eit,her DPNH (0.5 mu) or choline (7 mu) was determined. Temperattlw, 22”.

mixture \vrcreas indicated. TemperakIm, 22”. experiment (cf. 19). As illustrated in Fig. 3, the addition of DPNH caused an abrupt upward deflect,ion of the spectrophotometric trace, indicating that a reduction of cytochrome c + cl had occurred. The addition of either rotenonc (upper tracing) or amyt.aI (lower tracing) caused a reoxidation of cytochrome. At’ t)his stage the addition of choline brought about a rapid reduction of cytochrome in t,he rotenone-treated system, whereas no detectable changes in cytochrome reduction occurred in t)he arnytal-treated sample. Finally, the addition of succinate to restore oxygen upt,ake in the amytal-treated particles caused a shift to a new steady state of cytochrome reduction which persisted until t,he suspension became anaerobic, when a further upward deflection occurred, indicating full reduction of t,he cytochrornes. These obscrvntions correlated satisfactorily with t,he results of previous experiments

(Figs. SA and X3) in which it was found t,hat choline restored oxygen uptalic in tbc DPNH and rotenone-treated part icles but, not, in the DI’KH and nmytnl-treated systern. Titration \vith amytnl of t)he DI’SH aud choline oxidasc activities of t,he subrnitochondrial particle preparation revealed a clifference in hhc inhibit’or sensitivity of t-he two systems (Fig. 4). With DPNH oxidasc, 0.17 rn~ nmytal inhibited the oxygen upt,alcc by 50 %, whereas 0.W ml\1 amytal was required to produce the same degree of inhibition fol the (Lholine oxidation system Witch liver mitochondria, the corresponding concc~ntrations of amytal required for :iO”I; inhibition of oxygen uptake were 0.2 rnRI for P-hydroxybutyrate oxidasc and 0.7 no for (*holine oxidasc, when the titratiorls were perfornletl in the presence of malonate, as reconm~cndccl by Chance and Hollunger (20). These values of amytal con(*entration requirccl to inhibit

0 DPNH . Succinote

t-------.-..JJ

oi

, 0

m Choline

p-.,

I

0.05

,y?-+ 0.10

0.15

0.20

pg Antimycin FIG. 5. Iufiuence of antimgcin A concent.ration on the oxidation of added slthstrates 1;)~ a fixed wncel~tration of snhmitochondrial particles. The 3-ml reaction mixture contained sucrosc-‘!Xhphosphate buffer, and submitochondrial particles (2.1 mg protein). Various concentrations of antimy& A were added after the addition of either DPNII (0.5 mix), potassium succinate (7 mal), or choline chloride (7 m&I), and the rate of oxygen upt,ake was determined as a fllnction of the antim)-tin ;\ coucclltration. Temperature, 2‘2”.

by 50% the rate of DPNH oxidation in mitoc*hondria and submitochondrinl particles mere similar to those report’ed previously (20). I~~i~i~~o?~ Dy anti,mycirL A. When t.he DPKH, choline, or succinat8c oxidasc activities of the submitochondrial particle preparat’ions were tit’rated with ant’imycin A, a sharp decline in the rat’e of oxidation of each substrate was observed when t#he inhibitor cow cerkation was increased to a criGca1 level (Fig. 5). This result strongly suggests t’hat the antimyrin A-sensitive site is common to the terminal respiratory chain pathway available for the oxidation of each substrate. Similar results were obtained when t)hc oxidation of either P-hydroxybutyrate, choline, or sucrinate by liver mitochondria was titrated with antimycin A. ~7~~~~~~~~~ by cyanitle. I’otassium cyanide (1 rnn,f), when added t.o the react,ion mixture under t,he condit,ions specified in F’ig. 1, w:w

found to induce an essentiall\- complete inhibition of P-hydroxybutyrate, choline, and suwinat,e oxidation by liver mitochondria. When sublnito~horldrial parti&s wrc used, n slow c,ynnidc resistant. respicition (Qo., value = 5-S) was observrtl. Tllcx l~aturo of tlw c.yariiClc-1’csist:lilt, respiration is unlinown, but it was c>vidently mcdiatcd by a comporicrd (~oniinon to the three oxidnt ion pathways \mcler study, since it XL+ observed during the oxidation of each of the three substrates used. IrzfEuewr oj TTR. Ziegler is) has introdwcd tho we of the lipid-soluble, iron chclnting c*ompound TTB w :~n inhibitor of i.he sn~cirlatc-ubicluinone (C,j) rcductase region of Ihe sucrinatc oxidnw qstcm. Since his studies wcrc confined to heart muscle preparations, which do not IX~I~Iai 11a choline 0xid:ltion system (31)) soiw .
3%

TYLER, GONZE, AN~I ~sT~4~nooK

FIG. 6. Influence of TTB on the oxidation of added substrates by liver mitochondria. In each experiment, 0.1 ml of the same stock suspension of mitochondria (7.2 mg protein) was added to 2.9 ml of sucrose-TRA-phosphate buffer. Further additions made at the times indicated were: potassium succinate (7 mM), choline chloride (7 mM), sodium P-hydroxybutyrate (10 mM), mCI-CCP (0.6 ,uM), and ethanol (0.6%). Temperature, 22”.

trated by l’ig. BC and Fig. GD demonstrate the insensitivity of the ,&hydroxybutyrate oxidase system t)o TTB. The experiment of Fig. GD provides a cont,rol for that, shown in the other experiments. The TTB was added as a solution in ethanol (Fig. Mu), and an equivalent amount of ethanol alone was added in the experiments of Kg. AD. Succinate failed to restore oxygen upt,ake in the TTB- and nmytal-treated mitochondria, but did restore oxygen upt’ake in the ethanoland amytal-treated system. The selective inhibition of suwinate oxidase by TTB was confirmed by the result,s of experiment,s performed with submitochondrial particles. In the latter rxperiment,s, a 50% inhibition of suwinate oxidation was produced by the addibion of about 3 PM TTB. When the TTB concentration was increased to 0.1 m&I, the succinate oxidation rate was inhibited by more than 90 ?, but no significant effect was observed on the oxidation rate of either DPNH or caholine. In other experiments with phosphorylating liver mitochondria, a stjimulat,ion of the rate of oxidation of DI’Xlinked substrates by 50 FM TTB was obscrwd. This effect was evidently due to an

uncoupling a&ion of TTB on t#he particles, thus causing a loss of rcspirat’ory control and an increased oxidation rate. Inhibitor-resistant omdation of choline. Kimura et al. (5) have described tjhc condtions necessary for the development of an inhibitor-resistant oxidabion of rholine in t,hc presence of excess axide, antimyc4n A, quinoline oxide, and, to a more limited cxtent, cyanide. In order to obtain more irlformation about t,his phenomenon, some measuremenls were made of the oxygen uptake of liver mitochondria incubated under their (5) rea&ion caondit,ions. The data from these cxperimentjs are prcsentcd i tl Table I. The findings of Iiimlwa ct al. (5) were confirmed since, when mitochondria were incubated in phosphate buffer contai IIing added cytochrome c, t’he percentage iIthibition of choline oxidation by a high COIIcent,ration of antimycin A (GS!i,) was significantly less than t,hnt observed during succinate oxidation (93 5,). However, it is clear from an examination of the data of Table I that t,his result, does not reflect a true difference in t,he antimyc%~ A sensitivity of t,lle choline and suwinate oxidation sgs-

I?;IlIBITION

OF CHOLI?;E

TABLE

TABLE I Urr.%KE BY PHOSPHATE-TREATED

OXHGEN

IKFLV’ENCE

MIT~CH~NDRIA

OS

The 3-ml reaction mixture contained 8.3 mg mitochondrial protein suspended in potassium phosphate buffer (0.050 M, pH, 76). Further additions were made after a 5-min incubation of the mitochondria as follows: cytochrome c, 2 X lo-” M; antimycin A, 7.5 pg; DPNII, 0.6 mM; choline chloride, 10 mu; and potassium succinate, 10 mM. Temperature, 36’. O; Inhibition by Cytochrome c antimycin ___~A (cyt. c added) Minus Plus Qoz Valut?

Additions --__

None Ant,imycin 12 DYNH Antimycin A, DPNH Choline Choline, Antimycin A Succinste Succinate, Antimycin A

12 13 56 23 47 12 53 15

15 15 80 84

Nil

4i

15 267 15

379

OSII)ASE

68 93

PMS

II CIIAIS

OF RESPIRATORY AND

FERRICY~NI~E

INHIBITORS

REDUCTASE

ACTIVITIES

Inhibitors and substrates were used at the following concentrations: amytal (3 m-\r) ; rotenonc (5 fill) ; TTB (0.1 1x1~) ; potassium succinate (10 mu); choline chloride (7 mlt); and DPNH (0.2 mM). P&IS activities were assayed polarographically in 3 ml of sucrose-TRh-phosphate buffer containing KCN (1.5 mlz) and submitochondrial part,icles (2.6 my prot,ein). The reaction was started by the addition of PMS (0.5 mnl). Ferricyanide rcductase activity was assayed spectrophotometrically at 420 rnp in 3 ml of potassium phosphate buffer (0.05 M, pII 7.2), containing 3 pg ant,imycin, 7.5 mg bovine serum albumin, and submitochondrial part,icles (3.2 mg protein). The reaction u-as started by the addition of potassium ferricyanide (0.15 mu). Both assays were conducted at 22”. Succinate

Inhibitor

-

Electronacceptor’~

-___-__ PhfS

Ferricyanide -

(1&a, = ~1 02 uptake/hour/mg

protein.

terns, but is due instead to the presence of an inhibitor-resistant oxidation of endogenous substrate. The latter oxidation was largely unaffected by a number of respiratory chain inhibitors, including amytal (2 mnr), malonate (10 mM), rotenone (10 PM), TTB (0.1 m&r), and antimycin A (0.9 pg per milligram protein). The only inhibitor which had a marked effect on the endogenous substrate oxidat*ion rate was 1 mM potassium cyanide, which inhibited the rate by about GO% . Kimura et al. (5) also found cyanide to be a more effective inhibitor than antimycin A for this inhibitor-resistant oxidation. One probable pathway responsible for the oxygen uptake observed under these condit’ions is the so-called “external” DPXH oxidation pathway (22, 23), which is known to be insensitive t,o inhibitors of the phosphorylating respiratory chain. The maximum capacity of the “ext.ernal” pathway, under t,he conditions used, is illustrated by the rate observed in the presence of DPNH, added cytochrome c, and antimycin A. The rate of oxidation via this pathway was in considerable excess of the rate of the inhibitor-resistant oxidation of endogenous subst,rate, and the activity of the “external” pathway was therefore easily

Choline

Succinate

DPNH

None Amyt al Rotenone TTB None Amytal Rotenone TTB NOM?

Amytal Rotenone TTB

0.18 O.O(i 0.1s 0.19 0 .23 0.21 0.24 0.18 -

0.03 0.00 0.03 0.01 0.05 0.05 0.05 0.01 1.52 1.50 1.48 1.56

n Rates are expressed as pmoles of substrate consumed/min/mg protein.

sufficient to account for the endogenous oxidation rate observed, provided that an endogenous DPNH-generating system was operative under these conditions. Studies with artijcial electron acceptors. In general, the influence of respiratory chain inhibitors on the reduction of P1\IS or ferricyanide by added substrat,es teas consistent with t.hat obtained when oxygen served as terminal electron acceptor. Thus cholinePMS and choline-ferricyanide reductase activities were amytal-sensitive but rotenoneinsensit)ive, and the comparable activities with succinate as substrate were inhibited by TTB but not by amytal or rotenont (Table

TYLER,

380

GONZE, AND ESTABROOK

‘P

Roten

itochondria

In Buffer

+0.6pM mCI-CCP

l”/“l’ll”I”l”l’rl’ 400 430 460

490

548 i

520 550

580

610

Wavelength (mp)

FIG. 7. Spectrophotometric tracings illustrating the effect of succinate and choline on the oxidation-reduction state of cytochrome b in rat liver mitochondria. In the experiments shown in the upper part of the figure, mitochondria (7 mg protein) were preincubated for 5 minutes in sucrose-TRA-phosphate buffer containing 0.6 PM mCI-CCP, and placed in a 3-ml cuvette of a dual wavelengt,h spectrophotometer set at 562-575 mp. Further additions were made as indicated in the figure. At the times indicated by the letters A and B, samples were withdrawn and spectra representing the differences in absorption between the treated samples and that of the preincubated mitochondria containing no inhibitors or substrates were recorded at liquid nitrogen temperat)ure.

II). The observed inhibit ion of choline-PI1 IS xctivity by nmytal is in disagreement, with a previous report (Y), in which much lower control act’ivities were recorded. The unexpec%ed finding was made that t hc caholincferricayanide rcductasc activity was inhibitjet by TTB (Tahlc II), whcrens the more act,ive choline oxidation system was unaffected (Fig. 6). Spectwsmpy

of the ct&ochm~/e

col~~ponents.

Packer et al. (9) presented data on the cyt’ocahromcs reduced in the aerobic steady stat,e during choline oxidation by liver mi toc*hondria. It was stated in their paper that ident>ical caytorhromc specka were observed with mitochondria made anaerobic by treatment with eit,her choline or succinate. A possible

source of error in conclusions drawn from these experiments arises from the presence of endogcnous substrates in the mitochoudrial preparation. For example, the spwtrum obtained with mitochondria in t,hc prwenw of c+oline may represent the sum of those pigments reduced spec4ically by c~holine, and the pigments reduwd by either endogcnow succinate or DPS-linked substjrafes or both. In the present experiments, an nttcnlpt was made to ensure the specific reduction of respiratory pigments by choline by prctrcating the mitochondrin with mnlonate and rot,enone to inhibit, the reducing action of any endogenous suwinate or DPSH. Conversely, the reduction of respiratory pigments by succinate was performed with mitochondria pretrcat’ed with amytal and

rotenone, t.o inhibit the redwing a(4011 of endogenous choline and DPSH. The reduction of cytochrome b by caholinc and by succinate in the prewnw of suitable inhibitors was measured in a dual WLWlength spectrophotometcr with X2 minus 575 1np as measuring mavelc1lgths. Ais shown by t’hc spect’rophotometric tracings prosenlcd in the upper part of Fig. 7, the addt.ion of suwinatc wused an upwwl deflw tion of the trwing, indicating the reduction of cytochrome b to t’he steady state level. Ai rapid and complete redwtion of cytochromc occwrred when the mitochondrial suspension bwame anaerobic*. When caholine was atldttd instead of succinatc, no significant redwtion of c*ytorhrome b was observed in the aerobic st.eady st,nt,e, in agreement wit.h a previous report ((3), but upon reaching the anaerobic state an exlcnsivc redwt.ion ownwd, show ing that almost eclual amounts of cytochromc b were r~~lucwl by suwinste or by choline. This result ~1s confirmed by rwording the complcie caylochrome spectrum of the anaerobic mit ochondrin at liquid nitrogen tcnperaturc in :I wavelength st~anning spetatrophotoniet.er (Fig. 7, lower tracings). Tlw low t,einperature spectra show that, the same amount’s of cytochrome b, cl , and c were rem duced by either substrate. l’rom this result it may be conclndcd that both t’hc cytochrome b and the c*ytochrome cl cw~~poncnts of the choline and the suwinatc oxidation systems are fully interlinked. Similar studies wit,h subll1itoc.holidrinl liver particle prep a&ions showed that bc+n-ten GOand 100 ’ ; of the cytochroine b of these prcparatiow which was reduc~iblc by swc*inato was nlw redwiblc by cMine.

The ohservntion that the c*holinc oxidasc pathway of liver and kidney mi tochondria is insensitive to rotc~none, hut. is ,sensitivc lo relnt.ively low c~once~~lrations of an1yt:ti, suggests that the roteno11c-se11sitive and amytnl-sensitive components of the respiratory chain :w not’ identical. It would appear that the rotcnone-sensit,ivc site is located exclusively in the DPSH oxidase pathway, whereas amytal is a less specific inhibitor in the flnvoprotein region, sinw at suitable -

c.o11t:e11fratioilsit. has bwii found to inhibit several different oxidation pathways which cont~airi spwific flavoprot eins. In addition, amytal has bcw shown t)o inhibit 11ot only DI’SH oxidation and c*holinc oxidation but, also succ:inate oxidation (20, 24) and the initi:ll reaction of proline oxidation (Z). 12 fur1 her dist,inction between thcb ac%ions of ;unytal and roi~e11onc~ on tlicx respiratory (*hai11 lies in t.hc differenw in the t,ime required for a full inhibitory effect to bc cstablishcd after the addition of inhibit,or t)o t,hc enzyme system. In the wsc of amytal, t,hc att~aitiment of t,he final degree of inhibition of oxygen llptdie and the conc~omitnnt change in cytochrome redwtion upon the addition of the inhibitor are nearly instnntaneous (sea Figs. 18, 213, and t.hc lower trace of 3). In (‘ot1trasl tho rcspo11sicto the addition of rotenone undw similar cwnditions is achieved in a rclutivcly slow m:mncr (see Pigs. IA, 2A, and the upper t,raw of E‘ig. 3). This phc~nomenon is similar to the time lag observed by Estabrook (26) during the inhibition of oxygcii upt*akc by :uitimyc+~ A, which, like rotBcllon~~,is a stoichiometric: inhibitor of t,hc rcspir:Jory cnhxin. The dwelopment. of :I c.ynnidc-i11smsitivc oxidation of caholinc:in rat liver preparations wltic*h c~ontnincd other fully c*panidr-scnsitivc oxidation systems was described many years ago (27, 30). Rcc~ently, Kimura et nl. (5) have dcti1ic~l 1hc c:onditions recluired fo1 a11appawnt inhibitor-rcsist:1t1~ oxidation of choline by rut liver 111ito(:hondria incub:Lt,(~d in 1hc pwscnw of cx(*css axide, nnt.imyc:i1i, c~uinolinc oxidc1, or, to :L more limited cxtcnt, cyanide. They c:o~wludcd that. the choline and swcinatc oxidation chains wcrc interlinkctl at. the lcvc~l of cyt,oc:hron1c c1 , but not; at’ the lcvcl of c*ytoc:hromc b. This cwtrclusion was h:~scd largely upon the im:d lo post,ulate the prcsenw of an autooxitIix:~bl~ c~omponent in t.he c*holinc oxitlat,io11 syst cm, ilot present in 11~ suwinat(~ systcn1, whit+ LV:LS rcsponsihlc for the i1ll1ibitor-resist antI oxidation ohscrvctl in th(> prownw of c*holit1cb.The autooxidizable c~on1poncnt was thought, t#o bo located at, the level of c:yt~oc*lrromcb, since the inhibition of c:holinc oxidation by amytal at 1liv flnvoprot~oin level was aompletc, whereas ~hc inhibition by antimyc411, at, :L

ss2

TYLER,

(:ON%E, ANf) EYTABROOK

site between cyt’ochromes b and ~1, was incomplete. It is evident from t,he results obtained during the present study that their conclusion is open t,o serious objections. In the first place, the results prescnt,cd in the present, paper show clearly that, t)hc apparent, inhibitor-resistant oxidation of choline observed under t,hcir conditiow is due to an inhibitor-resistant oxidation of endogenous substrate. Since t’ho sucackatc oxidation rate is murh higher than the choline oxkkion rate in the absence of inhibitors. whoi the same amount of inhibitor-resistant respiration is expressed as a perucntagc of t,he activity of t,he uninhibited systems, the apparent inhibitor-resistant oxidation of choline appears to be far more significant than that of succinatc. Indeed, Kimura et al. (5) observed similar inhibitor hit,rutions with cyanide, antimycin, or quinoline oxide when the rate of sucoinate oxidation was inhibited by malonate to a level commensurate with the rate of choline oxidation. However, they apparently failed to appreciak t.hst, an oxidation of endogenous substrntc also could be responsible for their observations. As in this c*nse, it is misleading to compare t,he degree of inhibition of various respiratory chain act,ivit,ies on a percent,age inhibition basis only, if the aotivit’ies of t’hc noninhibitcd systems differ considerably. Since t)he cxperimcnts summarized in Table I were of relatively short duration, compared with those of Kimura et al. (5), another series of experiments was carried out where it was found that the initial rate of oxidabion of endogenous substrate was maintained for at, least 30 minutes. Minnaert (28) observed that t,he oxidation of endogenous substrate in rat. liver mitochondria continued for periods of up t,o 3 hours. Thus there seems lit’tle doubt that the oxidation of endogenous sub&rates was occurring throughout the 20-minute incubation periods used in the experiments of Kimura et al. In support of the present’ explanation of the previous results (5), it’ may bc noted that, with submitochondrial particle preparations, which lack endogenous sub&ate, the inhibition of either choline or sucoinate oxidation by antimycin was essentially complete. The observdion (5) that the apparent

inhibitor-resistant oxidation of choline was small or insignificant in aget1 or frozentharved mitocholtdri:~ is readily untlcrstootl, since t.hcsc treat8ments would bc cxpcct cd to deplete the mit~oc;hondria of endogcnous suhstr:it,es and the (fro-factors required for theii oxidation. Ckher experimental evidence which argues against, the conclusions of Kimura et al. is provided by spectroscopic studies. Acac*ording to the present resu1t.q up011 attaining anaerobiosis the cytochrome h of rat liver nlitochondria is fully reducible by either choline or succinatc. This result implies that, the caytochrome 0 component’s of the respiratory chains supporting the oxidation of choli nc and of succinate are fully interlinked. A further pkr*e of evidence cited by Kimurn et al. (5), in support of their c~onc:lusion that, the respirat,ory chains of t,hc choline and the sucknate oxidat)ion systlems were not directly interc*onnccted at the Ievel of cyt)ochrome 11,was t’heir failure to detctct, t,he anaerobic oxidation of choline by fumaratc. As they point,ed out, the rate of the anaerobic cross renct,iorls bet’u-een the SUcinate and DPNH oxidases of heart, or hhe succinate and cu-glycerophosphat,e oxiduses of brain is orlly about 2 % of the aerobic oxidation rate of the slower of the two intcracking oxidases. A permeability barrier 1.0 choline exists in fresh mitochondria (5, 17), which is abolished by swelling agents or uw coupling agents under aerobic* caonditions (17). The inabilit,y of the mitoc~hondria to swell when incubat,ed under t.hc anaerobic conditions employed in the cross reacation experiments of Kimura et al. might be expected to reduce the rate of entry of choline into the mitochondria, and I hereby reduce the rate of interaction of t,he c*holinc and sucGiate systems to an insignificant level. Reocntly Rianchi and Azzone (19) have demonstrated t’he anaerobic reduc*tion of oxaloacetate to malatc in rat liver mitochorldris t,reated with choline. Their data are consistent with the hypothesis that the reducing equiva1ent.s arising during the single step oxidation of choline to bet!aine nldehyde can bring about the reduct)ion of pyridine nucleotide, through an anaerobic cross mu-

tion ~wt~we~l tlw (*holine a11d DPNH oxidat#iou systeins. In :~grerment with the results of Iiimura ei al. (.i), when liver rnit.ochondrin wew incubat~ed in phosphate: buffer a cwrlsidcrable stimulation of succinate oxidatiorl by added cytochrome c ~;ns obstrvc~tl (T:lble I). Tiimur:l et al. c~oncluded from this result that miIochondri:t prepared in 0.25 31 SUNY~SC’ arc partially tlehGcwt, in cyt.oc*hronw c. Howcvcr, it, is lmown from the I\-ork of Ch:tppell (13) and others that1 liver mitochondria iwub:\tcd under rather different c*onditions WJI wtnlyacl ai1 cxtreinely rapid oxidation of succinatcx which is not depcndcnt~ 011 the addition of cyt,ochrome C. Thus t.hc requirc1w1It for added cytochronw c, in order to obtain optimal rates of siwinatc oxidation by mitochondria suspe1~1et1 in hypotonic phosphate buffer, is most probably a r~onsc~ucrwe of the nmrlml extent of mitochorldrial swelling and loss of cndogerlous c:yl,ochrome c which owurs under these csorlditions, and does not, indkate a true deficiency of cytochrornc c in the mitochondria prior to incubatioll. The present studies on the action of TTH on Ihc respiratory enzyme systems of rat, liver mit,oc:hondria ~IearIy show t.hat the compound exerts a selective and powerful inhibitory eff cct on the suacinat~c oxidation system. These rcsulk confirm the previous cowlusion (8) t’hat TTB is a specific: inhibitor of succinate oxidation. It has hccn suggcst,cd (8) t,hat TTH &s by forming A strong c~omplcx with enzyme-bound 1m11hemc iron, which can no longer undergo tmhc altematc oxidation and reduction which is considered essential for clecstron transfer to owur during suwinatc oxidation. Sinw SLUcimte oxida,tion is specifically inhibited by TTH, the hypothesis implies cithcr that a connnon nowheme iron protein is not fumtional during t.hc transfer of reducing ccluivxlents in t’hc other pathways of Ihc rcspirat,ory chain (e.g., DPSH oxidaso and (*holint! ox&se) ; or that other fact#orx, for exumplc t,hc accessibilitly of the inhibitor to nonheme iron groups, are responsible for i’hc sclwtive inhibition of suwinate oxida.tion by TTB. Studies made in this laboratory have shown t,hnt the TTB-sensit-ive silt> is funct~ionnl in

9.

10. II. 12. 13.

11. 15. IG. IT. 18. 19. 20.