THIC EIWE’J’ 011’ 2,4-1~3NJ’19tO~‘IIIc:T\TOI~ ON 1‘1112 E’OHMATlON 0% I’HOS~‘I-10lr:NO~,PY~1VAT~~ IiY IJWIZ. MITOCHONJX1TA” RY GILHERT II. MUDGE, HANS W. NEUI0~IIG,t AND S. W. STANIlUIrYt (Prom the Department of Medicine, College of Physicians and Surgeons, Columbia University, and the Presbyferian liospital, New York, hTew York) (Itcccivcd for publication, Pobruary 13, 1954) The apparent stimulation of aerobic phosphorylation by lINIB1 has been obscrvcd Gth preparations of rabbit liver mitochondria; some of the characteristics of this action have been reported in the preceding paper (2). The cxpcrimcntal conditions under which this is observed arc somewhat different from those which have been employed in many studios on aerobic phosphorylation, and certain aspects warrant emphasis: (1) the essential components include the mitochondrial suspension, substrate (usually (Y- ketoglutaratc), and orthophosphatc, \vith incubation under aerobic condi- tions; (2) the system contains no fluoride or added phospha~tc acceptor, i.e. no adcnylic acid, hcxosc, or hcxokinasc; and (3) this action of IINl’ is polyphasic and critically dcpcndcnt upon its concentration, for, as the amount of 1IN.l’ is increased, phosphorylatJion is sequentially stIimulatcd, inhibited, and then stimulated. In the prcscnt study dot&d cvidcncc is prcscntcd Tvhich identifies I’l(:Y as the major product of phosphorylation at all the concentrations of IINP examined. Somo of tho implications of the net conversion of substrate to PIG 1611 be discussed. EXPEHIMENTAL The experimental details have been described previously (2) a.nd arc summarized briefly. Mitochondrial suspensions were prcpa.red in sucrose from rabbit liver and \vcrc washed three times in either 0.150 or 0.075 M Na.Cl. Incubation \vas carried out in Warburg vesselswith oxygen in the * This study was supported by a grant from the llockcfellcr Foundation to llr. John V. Taggart. A preliminary report was presented before the meeting of the American Society of lliological Chemists, Chicago, April 6-10, 1953 (1). t National Research Council Fellow in the Medical Sciences. 1 I~cllow of the llockefeller Foundation on leave of absence from the Department of Medicine, University of Manchester, England. * The following abbreviationsare used:DNI’, 2,4-dinitrophcnol; PING’, phospho- enolpyruvate; TCA, trichloroacctic acid; AMP, adenosincmonophosphate; ADl’, adenosincdiphosphatc; ATI’, adcnosinctriphosphatc; IIRS, hydrazinc-rcactivc sub- stance; l’:O, micromolcs of orthophosphate cstcrificd per microatom of oxygen con- sumed; PO, phosphate as dcterminod without hydrolysis by the method of lziskc and Subbarow, considered identical with orthophosphatc. 9G5 by guest on February 4, 2020 http://www.jbc.org/ Downloaded from
RY GILHERT II. MUDGE, HANS W. NEUI0~IIG,t AND S. W. STANIlUIrYt (Prom the Department of Medicine, College of Physicians and Surgeons, Columbia
University, and the Presbyferian liospital, New York, hTew York)
(Itcccivcd for publication, Pobruary 13, 1954)
The apparent stimulation of aerobic phosphorylation by lINIB1 has been obscrvcd Gth preparations of rabbit liver mitochondria; some of the characteristics of this action have been reported in the preceding paper (2). The cxpcrimcntal conditions under which this is observed arc somewhat different from those which have been employed in many studios on aerobic phosphorylation, and certain aspects warrant emphasis: (1) the essential components include the mitochondrial suspension, substrate (usually (Y- ketoglutaratc), and orthophosphatc, \vith incubation under aerobic condi- tions; (2) the system contains no fluoride or added phospha~tc acceptor, i.e. no adcnylic acid, hcxosc, or hcxokinasc; and (3) this action of IINl’ is polyphasic and critically dcpcndcnt upon its concentration, for, as the amount of 1IN.l’ is increased, phosphorylatJion is sequentially stIimulatcd, inhibited, and then stimulated.
In the prcscnt study dot&d cvidcncc is prcscntcd Tvhich identifies I’l(:Y as the major product of phosphorylation at all the concentrations of IINP examined. Somo of tho implications of the net conversion of substrate to PIG 1611 be discussed.
EXPEHIMENTAL
The experimental details have been described previously (2) a.nd arc summarized briefly. Mitochondrial suspensions were prcpa.red in sucrose from rabbit liver and \vcrc washed three times in either 0.150 or 0.075 M Na.Cl. Incubation \vas carried out in Warburg vessels with oxygen in the
* This study was supported by a grant from the llockcfellcr Foundation to llr. John V. Taggart. A preliminary report was presented before the meeting of the American Society of lliological Chemists, Chicago, April 6-10, 1953 (1).
t National Research Council Fellow in the Medical Sciences. 1 I~cllow of the llockefeller Foundation on leave of absence from the Department
of Medicine, University of Manchester, England. * The following abbreviations are used: DNI’, 2,4-dinitrophcnol; PING’, phospho-
enolpyruvate; TCA, trichloroacctic acid; AMP, adenosincmonophosphate; ADl’, adenosincdiphosphatc; ATI’, adcnosinctriphosphatc; IIRS, hydrazinc-rcactivc sub- stance; l’:O, micromolcs of orthophosphate cstcrificd per microatom of oxygen con- sumed; PO, phosphate as dcterminod without hydrolysis by the method of lziskc and Subbarow, considered identical with orthophosphatc.
gas phtsc, at 25’, and in most cxpcriments for 40 to 50 minutes. ‘l’hc slarulard s@?m contained 1 ml. of the mitochondrial suspension (a,bout 7 mg. of N), 30 PM of sodium a-kctoglutsratc, 10 PM of orthophosphatc, 60 PM of ‘l’ris(hydroxymcthyl)aminomcthane hydrochloride bufTcr (@I 7.4), 75 E.IM of KCl, and 5 ~IH of MgC12, final volume3 ml., and alkali in the cen- ter well.
In cvcry cxpcrimcnt the effects of three critical concentrations of lIN1’ have been cxamincd: low I>Nl’ (1O-6 M), medium lIN3’ (5 X 1O-6 M), and high IINI’ (1.5 x 30” M). The a,mount of added orthophosphatc was calculated to include that contributed by the mitochondrial suspension.
Incubation for 65 minutes according to tho standard procedure. Each cup ini- tially contained 10.7 p&s of orthophosphatc. With an authentic sample of l’Kl’, hydrolysis by alkaline iodine was 60 per cent of theory. The results above indicate a similar degree of hydrolysis, by this method, of the ester formodduring incubation.
After incubation the reaction mixture was decanted into chilled 50 per cent ‘I‘CA and then diluted to volume. Phosphate was measured by the method of P’iskc and Subbarow (3).
ll~drolysis Charackrislics oj Phosphate Kster--.-,Whcn the reaction mixture wa,s analyzed for orthophosphatc after 40 or more minutes of rtcrobic incu- bation, approxirnatcly one-half of the 10 PM of added orthophosphatc had disqpcarcd (Table 1). With the low and high concentrations of 1)Nl’ an increased amount of phosphate ester was formed, while at the medium con- ccntration the a.mount of phosphate csterificd was the same as, or somc- times less than, that in the control cup. llydrolysis of the 1’CA filtrate in 1 N IICl at 300” gradually libomtcd orthophosphatc, with complotc rc- covcry of added orthophosphatc, within 40 to GO minutes of hydrolysis. (‘l’hc 40 minute interval was routinely cmploycd.) It was also found that the cstcrs formed at the low and at the high concentrations of l)Nl’ have
0. II. MUDGE, 11. 1-V. NEUIIGRG, AND S. \I\‘. STANlWllY 937
similar hydrolysis characteristics (Fig. I), and that the rate of hydrolysis is idontica.1 with that of an a.uthcntic sa.mplc of 1’151’ prepared by the m&hod of l%wr and Fischer (4).
As is indicated in l‘ablc 1, the phosphate cstcr was completely hydro- lyzed by mercuric chloride in a neutral or slightly alkaline solution (5). ‘There was partia,l hydrolysis in alkaline iodine, by the method of Lohmann and Mcycrhof (5). These two proccdums ha,ve been regarded as specific tests for PJW. The esters formed at the several concentrations of IIN wcrc hydrolyzed in a similsr manner by each of these methods (l’ablc 3).
IO II
z 7,
8 l-
L I I J 0 20 40 GO
MINUTES HYDROLYSIS
I N HCL 100°C
Pm. 1. llydrolysis of ester phosphate. h&L incubated according to tbc standard procedztro for 40 miwtcs. As indicated by the arrow, 8.4 PM of PO wcrc added per cup. ‘I’hc cxpcrimcnt was run with ten diffcrcnt concentrations of IINI’, and the hydrolysis cheractcristics were dctennincd for those two cups, rcprcscnting low and high DNI’, from which similar amounts of 1’0 were recovered. In a scparatc expcri- merit ester phosphate formod without l)Nl’ had similar hydrolysis characteristics.
Appearance of IIydrazine-lleaclivc Substance on II~drolysis of Phosphate I&r--Acid hydrolysis of the phosphate ester liberated approximately cquivalcnt amounts of orthophosphatc and oarbonyl group (Fig. 3, as estimated by the formation of 2,4-dinitrophcnylhydrazonc according to the method of IGxlemann and Ilaugcn (6). Similar reactions were ob- served after incubation at each of the critical concentrations of l>Nl’ (Table II). This constitutes presumptive cvidcncc tha.t diffcrcnt amounts of the same cstcr arc formed at each concentration of I>NI’. When mito- chondria were incubated with either citrate or glutamate as substrate, an incrcascd amount of phosphate was cstcrificd at the high lcvcl of J>NJ’, and this ha.d the same characteristics as the compound formed with m-k&o-
gluk~~tc, in that equimolar nmounts of phosphate and carbony group WCPC libcrrrtcd by hydrolysis, 6th by heating in acid or by tlrc! mercury prOCCdUrC.
CONTROL 2,4-DNP
/A HRS
MINUTES HYDROLYSIS
I N HCL loo0 C
FIQ. 2. Liberation of orthophosphatc and hydrazine-reactive substance (NRS) on hydrolysis of 3’CA filtrate of reaction mixture. Incubation according to the slandard procedure for 65 minutes. 12.8 PM of 1’0 added J)er cup; 5.1 and 3.0 PM of 1’0 recovcrcd in the control and at 1.5 X 10-a M DNL’, respectively. Quantitabivc csti- mat,ion of IJJtS under these conditions is subject to the following considerations. The molar extinctBion coeff~cicnts of the hydrazones of pyruvato and cY-kotogluta~rate are 12,100 and 8050, rcspcctively, at 520 mp in the Heckman DU spcctrophotomctel (mtio 1.60). The ratio of optical densities on an cquimolar basis in the Coleman junior spcctrol)botoxucter, which WRS routinely used, was 1.5G. IJowcvcr, when the hydrazones were carried through the ethyl acetate extraction procedure (method of Friedemann and IIaugen), the relative recoveries reduced t,hc above ratio to 1.1. Also, when either a-kotoglutarate or pyruvate was subjected to acid hydrolysis for 40 minutes, there was a loss of approximately 20 per cent ~16 dctcrmined by mcawre- mcnt of the hydrazone subsequently prepared. Purthcrmore, the koto acid liberated from the phosphate cstcr during hydrolysis is itself subjected to the decomposing action of the hydrolytic procedure, but for varying intervals of limo, as shown by the curve in Fig. 1. Any correction for the above variables will itself bc varied by the relative proportions of a-ketoglutarate and lW’ in the reaction mixture. Ilc- c~,usc of these consideration6 a prccisc correction factor could not bc obtained, but it was found practical to rcfcr lll& to an internal standard of unhydrolyzcd cu-kcto- glutaratc and I J1lStO Co a hydrolysed pyruvatc standard. (l’lic! subscriJlt6 dcnotc the duration in minutes of acid-heat hydrolysis.) l’hc ma.gnit8udc of thcsc errors in no way invalidates t.ho conclusions t,hat have been drawn.
&sulk of Paper Chro?nalographic Analysis----Further cvidencc for the formation of I’IW was obtained by paper chromatography of the 2,4-diui- t.rophenylllydraaoncs prepared from the TCA filtrates of t,he reaction mix- tures. A butanol-water-ethanol solvcut system gave satisfactory results
Q. Il. MUDGB:, II. W. NP:UHE:RG, AND S. IV. Fj’l’ANlWI~Y 9G9
and was routinely employed . ‘l’hc hydrazonos of a-ketoglutaratc and oxalrtcctato consistently had slightly lower RF values than those rcportcd by others (7--g). Howcvcr, this facilitated the separation of thcsc com- pounds from the hydmxone of pyruvatc. The basic cxpcrimcntal obscrva- tions wcrc also confirmed by the USC of butanol-ammonia and tertiary amyl alcohol-ethanol-water as solvents (7, S), but tho chromatograms were less satisfactory.
TABLE II Balance Bxpcriment Showing Meiabolism of a-Ketogluiarate in Relation to Oxidation
Incubation for 55 minutes according to the standard procedure. ISach cup initially contained 11.6 PM of orthophosphatc and 30 PM of cu-kctoglutaratc. Reaction mix- ture hydrolyzed in 1 N IICl for 40 minutes at 100°. In oalculation of *he ratio of the final lint, the amount of PM’ formed has bcon estimated as the increase in ortho- phosphate following hydrolysis. In twenty-six similar experiments the averago P:O ratios wcrc 0.14,0.16,0.08, and 0.17 for the control, low, medium, and high DNP, respectively.
As is shown in Table 111, the hydrazones obtained from the unhydrolyzcd filtrate had Rp vctlucs similar to those of cu-kctoglutaratc; the intensity of the spots varied approximately with the amount of unoxidized substrate, After acid hydrolysis for 40 minutes, a new spot appcnred with an RF value corresponding to the hydraEone of pyruvatc. With the addition of IINP, at each of tho critical concentrations cxtlmincd, tho hydrazones obtainod after hydrolysis had identical RF values, all of which were the smw as that of pyruvatc.
In order to avoid the presence of reactive carbonyl groups in the unhy-
Paper Chromatography of 8,4-l~initrophenylhydrazones
Expcrintcnt No.
1. After incubation with or-ketoglu- tarate; standard procedure
2. After incubation with different substrfLtos
3. Hydrolysis of o-k&o acid standards
r-Kctoglutarate standard Oxalacctato standard Pyruvatc standard
n-Kctoglutaratc standard Oxalacctato standard Pyruvatc standard a-Kctoglutaratc as sub-
strate
Citrate as substrate
Glutamate as substrate
cu-Kotoglutarate
Oxalacctatc
I’yruvate
_____._ -_--------__ -
:onccntration of 2,4-I)NP
M
qone
o-6
i x 10-b
.5 x 10-a
.5 x 10-a
1.6 x 10-a
!.5 x 10-a
0 40
0 40
0 40 0
40
0 40 a 0
& 0
40 IIg
4: Ik 0
40 a?
0 40 Jk
0.14 0.14
0.20 0.19 None
0.21 0.14 Nom 0.18 0.11 0.12
0.12 0.11 0.12 0.11 0.10 0.11 Nom
0.11 0.14 0.12 0.15
0.14
0.40
0.42
0.42
0.43
0.42
0.37
0.26 0.37
0.35 0,35
0.33 0.85
0.85
0.34 0.32 0.33
----- Ascending chromatograms on Whatman paper No. 1 for 18 hours. The solvent
system was the organic phase of a mixture of butanol-water-ethanol, 50:40:10 parts by volume. The 2,4-dinitrophenylhydrazoncs were prepared in the usual mannor (6) and then extracted with acidified petroleum other to remove DNP. ‘l’hc hy- draxoncs wore again taken up in ethyl acctatc, the solvent evaporated on a stcatn bath in a stream of nitrogen, and the residual hydraxonos dissolved in a minimal amount of 0.2 M sodium phosphate (pli 7.25).
* Ilydrolysis procedures are abbreviated as follows: unhydrolyeed, 0; heat-mid for 40 minutes, 40; mercuric chloride, Hg.
G. II. MUDGIC, II. IV. NlXJI1E:HQ, AND S. 1%‘. S’l’ANJlUItY 971
drolyz;cd spccimcn, citrate and glutamate were added as substrate (lc:xpcri- mcnt 2, ‘J’ablc HI). With citrate a very faint spot was dctcctcd bcforc hydrolysis, having an RF va,luc corresponding to that of cY-kctoglutarate. With glutamate, no significant amounts of hydraaonc wcrc dctcctcd cithcr by paper chromatography or by quantitative spcctrophotomctry. With both substrates, howcvcr, the chromatogram of the hydraxone appcarjng after hydrolysis corresponded to that of pyruvate.
The hydrazoncs were also identified by tbcir absorption spectra bctwccn 340 and 640 mp with a lscckman IIU spectrophotomcter. ‘J’he hydrnxoncs were cluted from the chromatograms in 30 per cent NazCOs a,nd their co101
dcvclopcd with I .5 N NaOJI. Standards of c-u-kcto acids were treated jn a similar mamrcr. l’hc hydrazoncs which wcrc formed after acid hydroly- sis, with RF values corresponding to that of pyruvate, had absorption spectra, with a peak at 445 mp, which were identical with that of the pyruvatc standard. ‘J’he hydrazoncs of oxalacctatc and pyruvatc hnvc similar absorption spectra, but they were readily diffcrcmiatcd by their widely dissimilar Rp values. In an cxpcrimcnt with the critical concentra- tions of IINI’, both the RF values and absorption spectra of the hydraxoncs appearing after hydrolysis wcrc identical with those of the control without IINI’, and all values corresponded to those for pyruvate.
In an attempt to identify the phosphate compounds directly, the reaction mixtures wcrc centrifuged without precipitation of the protcjn, and the supcrnatant solution was chromatographcd in mcthanol-wa.tcr-conccn- trated NJ140JT (GO:30:10) according to the method of Bandurski and Axelrod (10). No phosphate spots were detected other than those attrib- utable to orthophosphatc and 1’151’.
lhwium-Alcohol li’ractionation--The reaction mixture from sevclsl large scale expcrimcms, incubated with 1.5 X 1O-s M IINJ’, was subjcctcd to barium-alcohol fractionation (11). Jn the cxpcrimcnt depicted in Table JV, the cstcr phosphate was dmost quantitatively recovered in the barium- soluble, alcohol-insoluble fraction, which was further purified as the silvcr- barium salt of I’JSl’ according to the procedure of Jlolnnann and Mcyerhof (5). Owing to the losses sustained in the crystallization proccdurcs, only a small a.mount of fine crystalline white needles wa.s obtained after two rccrystallizations. ‘J’hc nature of the silver-barium salt was confirmed by elemental analysis (see Table IV). Some of the crystals wcrc also sub- jected to acid hydrolysis for 40 minutes, and the 2,4-dinitrophcnylllydra- ZOJE was prepared from the hydrolysate. The hydrazone of pyruvic acid was idcntificd by pa,pcr chromatography and by analysis of the absorpt,Jon spectrum after clution from the paper. No other hydraxoncs were dc- tcctcd .
l’ossiblc P’owzafion of O1A.w l’hosphaic JSsicrs- ~--III view of t~hc mcclkanisnk
postulated by Kalckar (12) and by liipmnnn (13) that pl~os~~hocnolox~lacc- tatc might, bo a precursor of I’IW, and, since ~xa~la~~tat~ might, bc dccar- boxylatcd during the preparative procedures, standards of the scvcra1 cu-kcto acids MTT~ subjcctcd to acid and t,o mercury hydrolysis (ICxpcrimcnt, 3, ‘J’ablc 111). After acid hydrolysis, the hydraeonc prcparcd from oxdace-
l’AR1.E IV Ilariwwl lcohol Praclionalion with Crystallization of Silver-Barium
step No.
-
Orthophosphatc added TCA filtrate after incubation Extraction 3 times with 5 ~01s. ethyl ace-
,‘ alcohol-insoluble l’pt. of Step G redissolved in 0.1 n IINOs;
amorphous gpt. after addition of 150 ~JM AgNOk and 0.5 vol. methanol
Amorphous nnt. of Stc~ 7 redissolved and crystalheed twice; final recovery of Ag-Ha salt as fine white needles
1’0
PM
233 28 30
32 I 0 1
0
Pa
6-f
202 202
63 2
130 113
19
1’40-0
174 172
31 1
130 112
19
Aerobic incubation was carried out according to the slandard procedure on a 24- fold scale in large vcsscls with 1.6 X 10-a nr TINP. Phosphate contributed by t,hc enzyme is not included in the calculations of Stop 1, Stop 3 removed virtually all tho 1INl’ and residual a-ketoglutaratc. Steps 4, 5, and 6 wcrc carried out at 1’11 3.2. l~~lcn~cnt~l analysis of 3.5 mg. of crystalline matcrinl, vacuum ether-dried: found, C 8.76,11 1.61,1’7.13 per cent; theory, C S.07, II 1.35, PG.95 ncr cent. ‘J’hcory, calculated for the silver-barium salt of $kos~~hocnol~kyruvatc with 2 molts of water of crystallization lrcr mole of ~rhosplkate ester.
tatc had an ICF value corresponding Co that of pyruvate, but, after mercury hydrolysis thcrc was no cha.ngc in the IiF wduc of the hydrazonc. This would indicate that oxdscetate is stable during the prcparat,ion of the hydrazonc from the mercury hydrolysate. In the cxpcrimciit,s previously described, the hydrazone of only pywv~tc appeared Rftw both types of hydrolysis. ‘J’hcrcforc, there was no iict, accumulation of phosphoc~~ol- oxal~cctate during incubat)ion, but its formation in trace amount~s is not excluded.
Jnorganic pyrophosphrttc has been previously demonstrated as one of the products of oxidativc phoq,horylat.ion in studies with’insolublc pa’liclcs
G. II. MUI)GE:, II. W. NIOUIWIG, ANIl S. W. STANIWRY 973
from liver (14). An attempt was therefore made to detect the accumula’- tion of this compound under the conditIions of the present cxpcrimcnts. The reaction mixlure was subjected to enzymatic hydrolysis with yosst in- organic pyrophosphatasc (I 5). No pyrophosphatc was dctectcd after in- cubaCon cithcr with or without IINI’.
‘I‘hc peculiar pattern of phosphor~~la.t,ion under the influcncc of IINl’ suggested the possibility that IIN]‘, or a degradation product, might be phosphorylatcd at one or more of the concentrations studied. ‘J’hc amount of DNI’ added was significantly loss, on a stoichiomctric basis, tlmn the quantity of phosphate ester that was formed, and secondly, at each of tlrc critical concentrations oxamincd, IINI’ was rccovcred quantit8ativcly when the reaction mixture was analyacd by a specific extraction proccdurc (16). Also, the absorption spectra of DNI’ bcforc and after incubat,ion wcrc identical. ‘J’hus, there was no ovidonce of metabolic conversion or dccom- position of l>Nl’. J,ikcwisc, it,s polyphasic action cannot be cxplaincd by dotoctablc diffcrenccs in itIs metabolic fate.
IMzw~c Study---.ln the balance cxpcrimcnt of Table II, ccrlain features may bc cmphasixcd which pertain to the metabolic fate of a-kctoglutarate under those conditions. All cvidcncc indicat,cs that the carbon atoms of 1’151’ arc derived from the wketoglutarate added as substrate: first, thcrc is no other known precursor pros& in sulffrcient quantity within the mitochondria; secondly, there is no other non-mitoclrolldri~l source of carbon atoms in the reaction mixt,urc; and thirdly, when cu-kctoglutaratc is added in limiting quantities, thorc is a parallel reduction in PIW formation, Calculated on the basis of tho oxidativc reactions of the citric acid cycle, the conversion of 1 molt of cr-kctoglutaratc to 1 molt of I%1 would rcquirc 3 atoms of oxygen, which would give a I’:0 ratio of 0.33. Since no othcl phosphate acceptors have boon eddcd (except those cndogcnous to the mitochondria), it is unlikely that otther phosphorylations arc of quantita- tive significance, and this ratio may therefore be accepted as the theoretical maximum. The obscrvcd I’:0 ratios wcrc about 0.15. In the balances measured by the method of l~riodcmann and Ilaugon, about 40 per cent of the cu-kctoglutaratc was convortod to 1’151’ as the metabolic end-product. Ipor example, at the low 3)NI’ conoontration in Table IT, 30 PM of cu-kcto- glutaratc were added and 5 PM rcmaincd after incubation. Of the 25 FM .which had disappeared, about 10 PM were rccovcred as I’IW. It is to bc noted that the above accounting applies to the low and high l)Nl’ as well as to the control.
Reactions of n nacrobic GZ~/coZ~sis--- ..‘J’hc folmat)ion of PICI’ under tIhc prcs- cnt, conditions neccssit,atIcd FI considorat#ion of the reactions of anacrohic glycolysis. IVhcn frurtosc-I ,G-diphosphatc was acldcd as suhst,rat,c, no 1’151’ WAS formed. lodoacctatc (Pig. 3) failed to modify the I)N1’-st,imu-
l&d formation of I’IW. Fluoride, an inhibitor of cnolasc, failed to inhibit PEP formation except at concentrations grcatcr than 0.044 M, at which respiration was also dcprcssed. The stimulatory effects of fluoride at
Fxa. 3. 3QTcct of additional inhibitors on DNP-stimulated phosphorylation. Standard procedure: incubation for 40 to 50 minutes. Solid line, controls; dash line, additional inhibitor as indicated. Iodoacelate (0.901 M); initial 1’0 12.9 NIH per cup; iodoacetatc slightly dcpressod respiration in the control cup, but had no effect on DNP-stimulated respiration. Fluoride (0.011 nr); initial 1’0 11.2 j.fM per cull; the in- creased phosphorylation at about 6 X IO-’ M DNP was associated with respiratory stimulation ((2), Table V). Arsenate (0.016 11); initial 1’0 11.4 PM per cup; oxygon consumption (in microatoms): no arsenate, control, 40; 1.5 X 10-a M IINP, 08; with arsenate, control, 24 ; 1.6 X 10-a M DNP, 58. ICtTect of magnesium; control cups con- tained the standard medium; experimental cups identical except for omission of MgCln (5 PM per cup); initial 1’0 11.3 PM per cup; at about IO-4 M DNP, the grcatcr inhibition of phosphorylation in the absonco of added MgCll was associated with a parallel depression of respiration; in the control and at other concentrations of DNP, tho oxygen consumption was the same with as without added MgCln.
medium IXW’ concentrations have been dcscribcd proviously (2). Arsc- nattc was examined because of its action in producing a net dephosphory- lation of I’ICP in a system in which this reaction can bc accomplisbcd by the intcrmcdiatc formation of glyccraldchydc phosphate (17). As rcportcd by Craw and Ilipmann (18), studies on arsenate in a mitochondrial system arc complicated by the formation of arscnitc and the resultant dcprcssion
of a-ketoglutarate oxidation. This &cot on respiration has boon con- firmod by us. Tho markod inhibition by arsonato of I’KI’ formation for the control and Zow concentrations of DNI’ (Fig. 3) was associated with a significant doprossion of rospiration. IIowovor, at higA lcvcls of IIN]’ both respira.tion and phosphorylation wore stimulatod oven in tho prosonce of arsonato. Although oxporiments with double inhibitors cannot bc ro- gardod as co~~clusivc in doaling with an onsymo system as complex a,s the mitochondrial preparations, the results arc uniformly nogativo in tcrm$
TABLE v
Eflect of Phosphate Acceptor on Products of Phosphorylation
Cups with no addod phosphate acceptor contained the components described in the sfandard procedure, except that orthophosphatc was initially placed in the sido arm. Cups with the hexose acceptor contained the same components, but with the addition to eaoh oup of 100 PM of fructose, 3 &M of AMP, 33 &M of Nalj”, and 0.02 n-d. of yeast hoxokinasc; final volume. 3 ml. Incubation in oxygen at 25” for 56 minutes. After 5 minutes equilibration the stop-cooks were closed and orthophosphate tippcd in from the side arm. Rach cup initially contained 10.6 JAM of orthophosphato (side arm) and 1.6 PM from tho mitochondrial suspension. In addition, the mitochondria contained 0.7 PM of mercury-labile phosphate.
of implicating any compounds of the snacrobic glycolytic cycle as prcour- sors of J’IW in this system.
Inflect of Added Phosphate Acceptor--Hunter (19) and Judah (20) have proviously roportod that tho A P/A a-kotoglutarato ratio is about 0.40 fol tho IINP-rosistant aerobic phosphorylation; a similar ratio 1va.s obtained by us (soo ‘J’ablc II). Since no phosphato acceptors wcro routinely addod, further oxpcrimcntIs wcrc dono to dctermino the ofrcct of AMP, hcxosc, hoxokinase, and fluorido (Table V). Although ma.ximal I’: 0 ratios, in the proscncc of a hoxoso acceptor, arc most readily dcmonstratod in a tnito- chondrial systom with 50 PM or moro of orlhophosphato por cup, only a limitod amount (10 PM per cup) was added in theso cxpcrimonts, bocausc such an amount facilitated the dotoction of mercury-labile phosphato and
also bccausc the increased formation of PI31 Rt high lcvcls of IINl’ is inhibited by high concentrations of o~%hophosphat~c (2). With ibc hcxosc scccptor, shnost all of the orLhophosphato cstcrifrcd was mercury-sta~blc and msy bc considcrcd mostly hexose phosphate (14). As ihc conccnt~ra- tion of IINI’ wss iiicrcascd, thcrc was liLt.lc change in the smount of phos- phate estcrificd, but the quant,ity of I’lCl (mercury-labile phosphat~c) in- crcascd markedly, and at IO--3 M IINI’ approximntcly 75 per cent of the orLhophosphate that ha,d dissppcared was idcntificd as IW’.
1151’ was originally isolated by lrohmami and hlcycrhof (5) as RJI int.cr- mediate of anaerobic glycolysis, snd it has been snbscqucntly dcmon- stratcd as R product of scrobic mct~abolism (12, 21).2 Under t)hc prcscnt. condit,ions the net formation of I’IW via the cnolasc rcbion from precw- sors in the glycolytic cycle may be cxcludcd for reasons previously clis- cussed. In isolated systems, the synthesis of I’IW has been sl~own t(o occur via the following reactions (22, 23):
It has also been proposed tbst I’IW might arise through the formaCon of an addition product of orthophosphatc snd fumarntc, with subscqucnt oxidation of the phosphomrllstc and decarboxylrttion of phosphocnoloxnl- acctste (13). I’hosphomalatc has previously been found to 1~ mctaboli- tally inert in CL mitochondrial system (24), an olx‘crvrttion which may be cited as suggcstivc, but not conclusive, evidcncc against its r61e RS RII
intcrmediatc. Current evidence on wkctoglutaratc oxidation indicates that the mcch-
anisms of phosphorylation at the substrate lcvcl3 RI’C fundsmcnt~s.lly differ-
* Leloir aud Mufios (21) used a liver euzymc prcparntiou haviug au adcnylic nud cytochromc rcquiremcnt which forrncd 3151’ with added succinatc, funrarate, .and rnalate as well as citrate. Kalckar (12) used au aqueous extract of kiducy which formed 3W from added malate; the rcquiremcrrts for cofactors wcrc not stated. The results of t,hc present study, without nddcd cofactors, differ from t,hr. nbovc iu that significnnt yields of I’IW wcrc not obtnincd with added succinnte, fumnrntc, or malnte. In view of t~hcsc diffcrcnccs, the 161~ of cofactors and other expcrimcnlnl vnrinblcs requires furlhcr examination in the case of these substrates.
* l’hc t.crm “substrntc phosphorylatiou” hns bccu gcnorally used to dcuotc n phosplrorylatiou nssocinted with the oxidation of substrate per SC, in coutrndist.iuc- tion to pbosphorylatiou nccompanying the rcnctions of clcctron transport via the cocnxyme system. Ju the prcscnt study, n diffcrcut t’ypc of aerobic substrate phos- pborylatiou has bccu dcmoustrated; nnmcly, t.hat the end-product of phosphoryln- tion (PM’) is dcrivcd from oxidizable substrate (ol-ketoglutaratc). !l’ho possibility
0. 11. MlJlK4lC, 11. W. NEUI~I4tlG, AND 6. \I’. S’J’ANRURY 977
cnt from those at the lcvcl of electron transporl. The latter arc uncoupled by 3)NI’ (20, 25), wl~creastl~c former are not. Substrate phosphorylations that are I’INP-resistant have been observed under a number of conditions. Those include three types of expcrimcnts with mitochondrid preparations: first, the aerobic oxidation of a-kctogluta.rate with added hcxosc trap (19,20); second, tho anaerobic dismutation of a-kctoglutaratc with addod hcxoso trap (19) ; and third, as in the prcscnt studies, the aerobic oxidation of wkctoglutarats without added phosphate acceptor. Jn addition, studies on purified preparations of wkctoglutaratc dchydrogcnase have demon- strated a IJNP-resistant phosphorylation associa.tcd with the formation of ATI’ (2G, 27).
In view of the above observations a reasonable soqucncc for the IINP- resistant formation of PIW might bc postulated as follows: the generation of ATJ’ at the substrate phosphorylation of ar-kctoglutarato oxidation and its subscqucnt reaction with either oxalacctatc or pyruvatc as in &actions I and 2, The equilibrium of Reaction 2 is far to the left (23), whoreas the equilibrium of Reaction 1 is far mom favorable to the formation of PEP (22). The cnzymcs involved iu thcsc reactions have to date not been iso- lated from rabbit liver mitochondria, and thus they cannot be definitely implicated in the synthesis of PET’ in the present system.
Howcvw, thcrc arc scvcral observations which fail to bc cxpla,incd by the reaction sequence postulated above and which thcrcforc suggest that the phosphorylations may not bc mcdiatcd via the adcnylic system. First, high yields of PEP arc obtained with a-kotoglutaratc as substrate, but not with succinatc (2). Although a DNP-resistant phosphorylation would not bc anticipated with succinatc, virtually no PEP wa,s formed with succinate oxidation, cvcn in the abscncc of JINP. As has bwn dcmon- stratcd previously with a system in which hcxosc scrvcs as phosphate accep- tor, succinate oxidation genoratcs suffrcicnt ATP to yield I’:0 ratios of about 2.0 (14). Thus, the failure of I’IW synthesis with succinatc, but in the abscncc of IMP, is not readily reconciled with tho hypothesis that the reaction is mediated via ATP. Secondly, the scqucnccs postulated above fail to cxpldn the results obtained with the added phosphate ac- ceptor system; namely, ,thc prcfcrcntid formcltion of PEP, ra.thor than hcx- osc phosphate, at the high concentration of IDNI’. If both phosphoryl- ations wrc mcdia.tcd via ATJ’, this finding could be at,tributed cithcr to DNP inhibition of transfer of phosphate from ATI’ to hcxosc, or to stim- ulation by DNI’ of transfer of phosphate from ATI’ to I’JW. No infor- mation is available regarding the latter possibility, but the effect of IINP
rclnains, however, that the phosphnto precursors of PEP coulcl thcn~sclvcs bc forwad ns R result of phosplrorylation occurring at either the wM,rnto or olcctro~~ tran~por(. level of a-ketoglutarato oxidation.
on hcxokinasc has been rccxamincd. In simple assays performed without added mitochondria, 1.5 X IO-8 M TINI’ had no disccrniblc effect on the hcxokinasc reaction. When studied in the mitoohondrial preparation by the addition of ATP, the assay of hcxokinasc activity was jnconclusive ow- ing to the simultaneous stimulation of ATJ’ase, which can bc considered to be in competition with hcxokinaso for the terminal phosphntc of ATI’. It has been shown previously that hcxosc phosphate is formed via the hcx- okinasc reaction during the anaerobic dismutation oi cu-kctoglutarate in mitochondrial prcpa.rations. Under thcsc conditions no 1’151’ is formcd. lluntcr (39) rcportcd that DNI’, in concentrations up to 1.2 X 1O--s M, dots not lower the A P/A a-kotoglutaratc ratio, and this finding ha.s been confirmed by us. This would suggest that, under these conditions, DNJ’ has no cffcct on the gcncration of ATP, nor dots it interfere significantly with the hcxokinasc reaction. In so far as these findings arc a,pplicablc to the aerobic mitochondria.1 system, they indicate that high conccntra- tions of I)NP do not inhibit hexokinase activity, and thercforc that the prcfcrcntial formation of IYICP, rather tha,n of hcxose phosphate, could not bc cxplaincd on this basis. As alternative hypotheses, this phcnomcnon might bc attributed either to a specific stimulation of phosphate transfcl from ATP to PI31 or to the formation of l’l”P via phosphorylatcd intcrmc- diates other than the adcnylic system.
SUMMARY
l~hosphocnolpyruvatc has been idcntificd 1s the phosphate compound formed during the aerobic incubation of rabbit liver mitochondria with a-kctoglutaratc and orthophosphatc. On addition of increasing conccn- trations of IINI’ the a.mount of I’JSP formed varies in a polyphasic pattern. High yields of I’El’ aro obtained with ol-kctoglutaratc, or its immcdiatc precursors, as substrate; ncgligiblc I’ICP formation results from the oxida- tion of succinatc, fumarato, and mdatc. The product of one type of J)NI’-resistant substrate phosphorylation ha.s thus been idcntificd as I’RI’. Some of the possible metabolic pathways of this phosphorylation have been discussed .
The authors are indcbtcd to Franctes Dulbcrg and Robert ICycr for their very capable technical assistance.
AdcZenduna-Since this paper was submitted for publication, Utter cl al. (28) have reported that inosinctriphosphatttc is a precursor of I’M’ in the oxalacctatc carboxyl- aso reaction, and that ATP may be involved only indirectly in a t~ansl)hosl,ho~yla- tion to form inosinctriphosphate. Tt has also been reported by Sanadi and Aycngar (29) that the phosphoryla,tion coupled with the oxidation of a-kctoglutarate is medi- atcd via guanosine polyphosphates. Both of these observations are pertinent to
U. II. MUI)GIG, 11. 1%‘. NICUI~EI~G, AND 6. W. S’l’ANI3UItY 979
the prcscnt problem in that they identify phosphorylatcd intermcdiatcs, other than t,hosc of the adenylic system, abich might bc involved in the IINP-r&&ant phos- phorylations loading to the formation of 1’15P. Ilowwcr, at the prcscnt time, thcrc is no cvidcnoc regarding the offcct of IINI’ on any specific trallslr~Iosl)horyIa.tion involving the above intcrmcdiatcs.
l3IlIIJOGItAI’IIY
1. Mudgc, G. II., Stanbury, S. W., and Neubcrg, 11. W., Pcderation Z’roc., 12, 101 (1953).
2. Stanbury, S. W., a.nd Mudgc, G. II., J. Hiol. Chem., 210, 949 (1954). 3. Fiske, C. II ., and Subbarow, Y., J. Hiol. Chem., 66, 375 (1925). 4. 1%x, N., and Fischer, II. 0. I,., J. Zjiol. Chem., 180, 145 (1949). 5. Lohmann, I<., and Mcycrhof, O., Hiochem. %., 278, 60 (1934). 6. Pricdcmann, 1‘. 15.) and Ilaugen, G, 15., J. Hiol. Chem., 147, 415 (1943). 7. Altmann, S. hf., Cook, IL M., and Datta, S. I’., Hiochcm. J., 49, p. Xii (1951). S. Cavallini, I)., Frontali, N., and l’oschi, G., Nature, 163, 568 (1949). 9. Scligson, I)., and Shapiro, B., Anal. Chem., 24, 754 (1952).
10. Bandurski, IL S., and Arclrod, I%., J. Zliol. Cnena., 193, 405 (1951). 11. J&age, G. A., in Umbrcit, W.. W., Burris, II. II., and Stauffcr, J. I’., Manometric
tcchniquos and tissue motabolism, Minneapolis, 1% (1949). 12. Kalckar, II., Biochent. J., 83, 631 (1939). 13. l,ipmann, IQ., Advances in Z#wgpnoZ., 1, 99 (1941). 14. Cross, It. J., l’aggart, J. V., Covo, G. A., and Green, I>. IL, J. iliol. Chem., 177,
655 (1949). 15. Maml, !I’., Ztiochem. J., 38, 345 (1944). 16. Mudgc, G. II., and l’aggart, J. V., Am. J. Z’hysiol., 161, 173 (1950). 17. Mcyorhof, O., and Junowics-Kocholaty, IL., J. HioZ. Chcm., 146, 443 (1942). 18. Crane, It. I<., and Idpmann, P., J. Viol. Chcm., 201, 235 (1953). 19. IIuntcr, I?, IL, Jr., in McElroy, W. I)., and Glass, IL, Phosphorus metabolism,
I<altimorc, 1, 297 (1951). 20. Judah, J. I>., IZiochent. J., 49, 271 (1951). 21. Lcloir, I,. I”,, and Mutioe, J. hf., J. Biol. Chem., 163, 63 (1944). 22. Utter, hl. V., and Kurahashi, K., J. Am. Ch:hem. Sot., 76, 758 (1953). 23. TJardy, JI. A., and %icglcr, J. A., J. Riol. Chem., 169, 343 (1945). 24. Yricdkin, hf., in McElroy, W. II., and Glass, IS., Phosphorus metabolism, ISalti-
more, 1, 38s (1951). 25. liarkulis, S. S., and IIchningcr, A. I,., J. HioZ. Chem., 193, 697 (1951). 26. Ilift, II., Oucllet, I,., 18ttlcfield, J. W., and Sanadi, I>. It., J. Riol. Chem., 204,
666 (1953). 27. Kaufman, S., Gilvarg, C., Cori, O., and Ochoa, S., J. Riol. Chem., 203,869 (1953). 28. Utter, M. V., Kurahashi, I<., and Rose, I. A., J. Riol. Chent., 207, SO3 (1954). 29. Sanadi, I). lt., and Ayongar, l’., Federation Proc., 13, 287 (1954).
