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On the nature of the compartmentation of the substrate-linked phosphorylation in rat-liver mitochondria
252
BIOCHIMICA
ET BIOPHYSICA
ACTA
ON THE NATURE OF THE COMPARTMENTATION OF THE SUBSTRATELINKED PHOSPHORYLATION IN RAT-LIVER MITOCHONDRIA
H. R. SCHOLTE
AND].
M.
TAGER
Laboratory of Biochemistry", University of Amsterdam, Amsterdam (The Netherlands) (Received April r4th, 1965)
SUMMARY I. During the oxidation of c-oxoglutarate or glutamate by rat-liver mitochondria in the presence of dinitrophenol and absence of added inorganic phosphate and phosphate acceptor, the endogenous inorganic phosphate is quantitatively converted to phosphoenolpyruvate. The depletion of inorganic phosphate results in a decline in the rate of respiration. 2. The conversion of inorganic phosphate to phosphoenolpyruvate during aoxoglutarate oxidation is inhibited by malonate. Under these conditions, a-oxoglutarate disappearance is stimulated. If malate is added as well as malonate, phosphoenolpyruvate formation is restored, and a-oxoglutarate disappearance is diminished. 3. In rat-heart sarcosomes, which contain no phosphopyruvate carboxylase (EC 4.I.I.3Z). the rate of oxidation of a-oxoglutarate in the presence of dinitrophenol and absence of phosphate acceptor does not show the marked decline observed with liver mitochondria. Inorganic phosphate does not disappear during the incubation.
INTRODUCTION
HUNTER 1 and JUDAH 2 discovered that the substrate-linked phosphorylation associated with the oxidation of a-oxoglutarate to succinate is not uncoupled by dinitrophenol. Thus dinitrophenol cannot replace PI in the mitochondrial oxidation of c-oxoglutarate" or of substrates that are oxidized via a-oxoglutarate, such as glutamate 4 - 9 , However, the mitochondria must be depleted of the appreciable amounts of Pi that they contain in order to show a maximum stimulation by added phosphate 2 , 3,l O. BORST AND SLATER3 have shown that rat-liver mitochondria contain sufficient endogenous PI to sustain a considerable respiration of a substrate such as glutamate in the presence of dinitrophenol. However, AZZONE AND ERNSTER ll found Abbreviation: PEP, phosphoenolpyruvate. • Formerly; Laboratory of Physiological Chemistry, Postal address: Amsterdam C (The Netherlands).
Biochim. Biophys. A eta,
IIO
(1965) 252-258
J. D.
Meyerplein 3,
SUBSTRATE-LINKED PHOSPHORYLATION
253
that, in the presence of dinitrophenol and absence of phosphate acceptor, the rate of oxidation of glutamate by rat-liver mitochondria rapidly declined unless Pi (> 0.1 mM) was added. The decline in respiration was accompanied by incorporation of trace amounts of 32P I into an organic form, which they identified as consisting mostly of ATP (95%). On the basis of these observations, AZZONE AND ERNSTERl l concluded that "The a-ketoglutarate-linked substrate level phosphorylation takes place in the mitochondria within a special compartment, yielding adenosine triphosphate which does not communicate freely with adenosine triphosphate originating from the respiratory chain phosphorylations". They explained the decline in respiration as being due to the removal of PI as "compartmentalized ATP" inaccessible to the dinitrophenol-induced ATPase. CHARLES et al.l 2 showed by direct analysis that the decline in respiration is brought about by the exhaustion of Pl. However, their results did not support the concept of a "compartmentalized ATP" inaccessible to the dinitrophenol-induced ATPase. They suggested instead that, in addition to the dinitrophenol-induced ATPase which returns esterified P to the system as P l , a slow side reaction occurs, which leads to the permanent disappearance of the Pl. This slow side reaction has now been identified. As has been known for some years, liver and kidney mitochondria from various species can synthesize PEP from oxaloacetate13-20, although this reaction has been reported to be very slow or absent in rat-liver mitoehondria11,19-21. In this paper it will be shown that the PI that disappears during the oxidation of glutamate or a-oxoglutarate by rat-liver mi.tochondria in the presence of dinitrophenol and absence of added phosphate acceptor is quantitatively converted not to ATP, as claimed by AZZONE AND ERNSTERll, but to phosphoenolpyruvate. METHODS Rat-liver mitochondria were isolated by the method of HOGEBOOM 22, as described by MYERS AND SLATER23 and rat-heart sarcosomes by the method of CLELAND AND SLATER24 . The standard reaction mixture contained 50 mM ReI, 25 mM Tris-H'Cl buffer (pH 7.5). 8 mM MgCI2, o.r mM 2,4-dinitrophenol, and 75 mM or 50 mM sucrose (derived from the liver- or heart-mitochondrial suspension, respectively), in a final volume of I ml. The reaction temperature was 25°. Oxygen uptake. mitochondrial protein and inorganic phosphate were determined by the experimental procedures given in ref. 12. The reaction was stopped by the addition of 0.1 ml 35 % HCI0 4 . After removal of the protein by centrifugation, the HCl0 4 was removed as KCI0 4 in the cold. Pi and PEP were determined in the neutralized extracts. PEP was determined by the method of KORNBERG AND PRICER25. Each cuvette contained 25 mM Tris-HCI buffer (pH 7-4), 0.5 mM ADP, 3.1 mM MgCl 2, 1.25 mM EDTA (pH 7.4), NADH, and an aliquot of the neutralized deproteinized reaction mixture in a final volume of 2 ml. Lactate dehydrogenase (EC I.l.I.27) was added and the reaction was started with pyruvate kinase (EC 2.7.1.40). ADP, lactate dehydrogenase and pyruvate kinase were obtained from Boehringer and Soehne. Oligomycin was kindly supplied by the Upjohn Chemical Company. Biocbim. Biophys. A cia,
IIO (1965) 252-258
254
H. R. SCHOLTE,
J.
M. TAGER
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Fig. 1. Time course of oxygen uptake. PI disappearance and PEP formation. during the oxidation of glutamate by rat-liver mitochondria in the presence of dinitrophenol and absence of added PI or phosphate acceptor. The reaction mixture contained the basic components, plus 9.5 IJ1g mitochondrial protein. Curve I (oxygen uptake), Curve 3 (PEP formation) and Curve 5 (PI concentration) in presence of 20 mM glutamate. Curve 2 (PI concentration) and Curve 4 (oxygen uptake) with no added substrate. RESULTS
Fig. I shows that during the oxidation or glutamate by rat-liver mitochondria in the presence of dinitrophenol and absence of added Pi or phosphate acceptor, PEP is formed. The PEP formation parallels the oxygen uptake. In the absence of glutamate, incubation with dinitrophenol leads to the liberation of Pi (cj. refs. 3 and IZ). 20
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20
40
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80
Time (min)
Fig. 2. The disappearance of PI and the formation of PEP during the oxidation of c-oxoglutarate in the presence of dinitrophenol and absence of added PI or phosphate acceptor. The reaction mixture contained the basic components plus 7.9 mg mitochondrial protein, and where present 20 mM c-oxoglutarate. Curve I (0-0), disappearance of PI during oxidation of a-oxoglutarate (calculated from Curves 5 and 6, the PI concentrations without and with added substrate, respecti vely): Curve 2 (6. - 6.), PEP formation in the presence of c-oxoglutarate; Curve 3 (e-e), oxygen uptake in presence of added substrate; Curve 4 (e-e), oxygen uptake in absence of added substrate. The initial PI concentrations in the Curves 5 and 6 varied slightly in the different incubations, as can be seen from the right-hanel curves. This is due to the fact that the incubations were begun at different times, and Pi was slowly released during storage of the mitochondria at 0° (30% during 50 min storage).
Biocliim, Biopbys. Acta, 110 (1965) 252-258
255
SUBSTRATE-LINKED PHOSPHORYLATION
25
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60
Time (min)
Fig. 3. Effect of oligomycin. arsenite, malonate, malonate + malate. and Pi on the oxygen uptake during oxidation of u-oxoglutarate by rat-liver mitochondria in the presence of dinitrophenol and absence of added phosphate acceptor. The same experiment as in Table 1. Curve 1, + \0 mM potassium phosphate buffer (pH 7.5); Curve 2, no further additions; Curve 3. +1.0 mM rnaicnate + 5 mM malate (no o-oxcglutarate) : Curve 4, + 20 mM malonate + 5 mM malate; Curve 5, + 20 roM malonate; Curve 6. + 21 J.!M oligomycin; Curve 7. no a:-oxoglutarate; Curve 8, + I mM arsenite.
When the amount of Pi found in the presence of glutamate is corrected for the amount liberated in its absence, it is evident that Pi is almost quantitatively converted to PEP during the oxidation of glutamate under these conditions. The relation between the disappearance of PI and the formation of PEP was also examined during the oxidation of a-oxoglntarate in the presence of dinitrophenol and absence of added Pi or phosphate acceptor. In Fig. 2 the Pi concentration as a function of time in the presence and absence of a-oxoglutarate (right hand curves) has been used to calculate the disappearance of Pi during the oxidation of a-axoglutarate. As shown in the left hand part of Fig. 2, there is a stoicheiometric conversion of inorganic phosphate to PEP. Table I and Fig. 3 show the effect of inhibitors. Oligomycin had little effect on the amount of PI utilized or the amount of PEP formed in 56 min (contrast ref. 19). However the oxygen uptake and c-oxoglutarate utilization were markedly decreased by oligomycin (see note added in proof in ref. rz). Arsenite inhibited the oxidation of c-oxoglutarate, formation of PEP and the disappearance of PI. Malonate also inBiochim. Biophys. Acta,
IlO
(1965) 252-258
H. R. SCHOLTE ,
J.
M. TA GER
TABLE!
+
E FF ECT OF O LI GOMY CIN, ARSE N ITE , M ALONATE, MA L O NATE M ALATE , AND I N OR GA NIC PH OSPHATE ON T HE OX I D AT IO N OF U - OXOG L UT ARAT E B Y RAT- L IVER M I T OCH O N D RI A IN THE P RES EN CE OF D IN ITROP H E N O L, AN D O N T HE DISAPP EARANC E O F I N O R G AN I C P H OSPH ATE AND THE FOR MATI ON
OF
PHOSP HOE NO L PYRU V AT E
T h e reaction mixt ure cont a ined the b as ic co mponen ts plus 9.9 rng mitochondrial protein and 20 mM o-oxoglutarate. R eactio n t ime was 56 min , The P i co nce ntra t ion in the reaction m ixture was 361 "M a t zero time . and 632 I'M after incu ba t.ion for 56 min in t h e absen ce of su bstra t e. The corresponding values fo r PEP were 13 and 30 ,uM, re spect iv ely. - LlPi = PI found aft er incubation for 56 min wit h out s ubstra te minlls that found after incubation for 56 m in with t h e addit ion s sh own in the table. LlP E P wa s calcula t ed similarly .
A dditions or omissi ons
N one + Oligomycin (:ZI 11M ) Arsenite (I roM) Malonate (20 mM) + Malon at e (20 mM) + malate (5 mM) No a -ox oglut arate ; Malonate (20 mM) mala t e (5 rnM) + PI (10 roM)
-LI P,
LJPEP
- A a-oxogluta rale
#M
11M
mM
5° 4 465 10 56 4 86
5.6
17·3
55 7 553
11.9
+ +
+
-LIO
uatom»
-74
0·9 12·3 13. 0
+
134 547
13. 0
94 476
28.9
3. 0 0.6
7·9 5. 1 14· 4 ..
_--_..
hibited t he forma tion of PEP and the disappearance of PI but stimulat ed the disappear ance of a-oxo glutara te. When malate was added as well as malonate there was a restoration of PEP formation an d P i disappearan ce, and a diminution of a-ox oglutarat e utilizat ion to t h e level found in t he presence of a-oxoglutarate alone . Wh en IO mM PI was added , a high rate of respirat ion was obtained, which remained linear wit h ti me ; the PEP formation was depressed slightly. In rat-heart sarcosomes, the oxidation of a-oxoglutarate in th e presence of dinitrophenol an d absence of added P I is low, but remains linear for at least 60 min (Fig. 4). Added PI stimulates the rate of oxidation. There is no disappearance of PI
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70
80
4 90
F ig. 4. The effect of added in organic phosphate on the oxid a t ion of a -oxogiutarat c b y rat-hea rt sarcosomes. T he re action m ixtur e co ntain ed the b asic: co mp onen t s p lu s 0.9 m g sar cosom a l protein and 10 rnM a-oxoglutaratc. Curve I, + 10 rrrM PI; Curve 2, + 0 . 1 rnM Pi ; Cu rve 3, no additions ; Curv e 4. no a -oxogilltarat e . At zer o tim e 65 pM P i was foun d in t he rea ction mi x t ure. Afte r go min t he a mo unts of Pl wer e 9 7 # M (Curve 4) . r 0 9 ~M (Curve 3) an d 187 pM (Curv e 2).
Bio chim, Biophy s. Acta, II O (1965) 25 2 -
25 8
257
SUBSTRATE-LINKED PHOSPHORYLATION
during a-oxoglutarate oxidation by rat-heart sarcosomes. These mitochondria contain no PEP carboxylase. DISCUSSION
ei al. 14 found that PEP is formed when a-oxoglutarate is oxidized by rabbit-liver mitochondria in the presence of Pi and dinitrophenol. NORDLIE AND LARDyl9 obtained the same results with guinea-pig liver mitochondria. Although PEP formation has been reported to be very slow or even absent in rat-liver mitochondrial 7,l 9- 21, it is clearly sufficiently rapid to account for the slow disappearance of Pi during the oxidation of glutamate or a-oxoglutarate by these mitochondria. From the results of this paper and those of CHARLES et al.l 2 , and the demonstration by HELDT ei al. 26 that GDP is the physiological phosphate acceptor for the substrate-linked phosphorylation step in rat-liver mitochondria, the following picture of the course of events during the oxidation of glutamate or a-oxoglutarate by ratliver mitochondria in the presence of dinitrophenol and absence of phosphate acceptor may be drawn. The Pi present in the mitochondria initially or liberated from acidstable phosphate compounds during incubation is esterified by the substrate-linked phosphorylation step of a-oxoglutarate oxidation (Eqn. I) MUDGE
Succinyl-CoA
+ GDP + PI'=' succinate + CoA + GTP
(I)
Most of the GTP reacts with ADP to form ATP (Eqn. 2) which is then hydrolyzed by the dinitrophenol-induced ATPase (Eqn, 3). Thereby, the PI esterified in Eqn. I is returned to the system GTP
+ ADP.=. GDP + ATP
ATP
+ H 20
(din itrophenol) ) ADP
+ Pi
Although most of the GTP reacts according to Eqn, 2, some reacts with the oxaloacetate (formed by the further oxidation of the succinate) to give PEP (Eqn. 4) GTP
+ oxaloacetate ee GDP + PEP + CO2
The PEP :a-oxoglutarate ratio was about 0.1 (Table I), indicating that about I in ro of the GTP molecules reacts according to Eqn. 4- CHARLES et al.l 2 found that the p:o ratio during oxidation of glutamate in the presence of dinitrophenol and absence of phosphate acceptor was o.or-o.ory, instead of the 0.33 to be expected if the ATP formed by the substrate-linked phosphorylation step were stable. Thus, under their conditions I in 20-30 molecules of GTP reacts according to Eqn. 4. The results reported in this paper confirm the conclusion by CHARLES et al. l 2 that there is no experimental basis for the proposition of AZZONE AND ERNSTER l !, recently reaffirmed by ERNSTER AND LEE 27 and adopted by NORDLIE AND LARDylD, that the ATP formed by the substrate-linked phosphorylation step of a-oxoglutarate oxidation is inaccessible to the dinitrophenol-induced ATPase. Biochim. Biophys. Acta, no (1965) 252-258
258
H . R. SCHOLTE,
J.
M. TAGE R
ACKNOWLEDGnIENTS
The authors wish to thank Professor E. C. SLATER for his encouragemen t and advice. This investigation was sup por ted in part by a grant from the Life In suran ce Medical Research Fund. R E F E R ENCE S I F . E. HU NTER, in W . D . M cELROY AND B. GLASS. Symp . on P hosp horu s Me tabolism, Vol. I, John s Hopkins Press, Baltimore, 1951 , p . 29 7. 2 J. D. J UDAH, B i ochem : ] ., 49 (1951) 27 1. 3 P . B ORST AND E . C . SLATER, Bioohim : Biophys. Acta, 48 (19 61) 36 2. 4 A. F . MULLER AND F. LEUTHARDT, Hel u. Cbim , A cta, 33 (r95 0) 268 . 5 H. A. KREBS "-ND D. B ELLAMY, Biochem, j., 75 (1960) 523. 6 P. BORST AND E. C . SLATER, B iochim. Biophys. Acta, 4r (19 60) 170. 7 J. B. CHAPPELL AND G. D. OREvrLLE, Nature, 190 (r96r) 502 . 8 E. A. JONES AND H . GUTFREUND, Biochem, j., 79 (r96r) 608. 9 P. BORST, Biochim, B iophys. Acta, 57 (1962) 256. 10 L. J. TEPLY, Arch. Biochem ., 2'f (1949) 3 83. I I G. F. AZZONE AND L. ERNST ER, J. Bioi . Chem ., 236 (1961) 1501. 12 R. CHARLES, J . M . TAGER AN D E . C . SLATER, Biochim . B iophys . A eta, 74 (196 3) 33 . 13 S. W . STANBURY AND G . H . MUDGE , ] . B io!. Chem., 2 10 (1954) 949 . 14 G. H. M UDGE, H . Vit o NEUBERG AND S . W . STANBURY, J . Bioi. Chern., 2 10 (195 4) 96 5. 15 W. BARTLEY, B iochem , ] .. 56 (19 54) 387. 16 W. BARTLEY ...ND Y . AVI-D o R. B iochem. J., 59 (r955) 194 · 1 7 R. S . BANDURSKI AND F . LIP MANN, J. B ioI. Ch8m., 2 19 (1956 ) 74 1. 18 P. S CHOLLMEYER AND M . K1.INGENBERG, B iochem , B i ophys . Res. Commun. , 4 (19 6 1) 43 . 19 R. C . N ORDLIE AND H . A . L ARDY, B iochem, Z. , 338 (19 6 3) 356. 20 R. C . NORDLIE AND H. A. LARDY, j. B ioi. Chern., 238 (196 3) 2259. 2 I L. M . CORWIN, J. Bioi. Ch8m. , 234 (1959) 1338. 22 G. H. HOGEBOOM, in S. P. COLOWICK AND N . O. K APLAN, M ethods of E nzy mol ogy , Vol. I , Aca d em ic Pr ess, New York, 195 5. p. 16 . 23 D. K. MYERS AND E . C. SLATER, B i ochem , ]., 6] (19 57) 55 8. 24 K. W . CLELAND AND E. C. SLATER, B iochem, J., 5 3 (1953) 547 · 2 5 A. K ORNBERG AND "V. E . PRIC ER, j. B ioi . Chem ., 193 (1951) 48 1. 26 H. W. HELDT, H . J Aeons AND M. KL I NGENBERG, B iochem . B iop by s. Re s . Commlln ., 17 (19 64) 130 . 2 ] L . ERNSTER AND C.-P. LEE , Ann. Rev. B iochem .• 33 (1964) 77 8.
Biochim . B iop hy s. A cta, lID (1965) 252-258
BIOCHIMICA
ET BIOPHYSICA
ACTA
ON THE NATURE OF THE COMPARTMENTATION OF THE SUBSTRATELINKED PHOSPHORYLATION IN RAT-LIVER MITOCHONDRIA
H. R. SCHOLTE
AND].
M.
TAGER
Laboratory of Biochemistry", University of Amsterdam, Amsterdam (The Netherlands) (Received April r4th, 1965)
SUMMARY I. During the oxidation of c-oxoglutarate or glutamate by rat-liver mitochondria in the presence of dinitrophenol and absence of added inorganic phosphate and phosphate acceptor, the endogenous inorganic phosphate is quantitatively converted to phosphoenolpyruvate. The depletion of inorganic phosphate results in a decline in the rate of respiration. 2. The conversion of inorganic phosphate to phosphoenolpyruvate during aoxoglutarate oxidation is inhibited by malonate. Under these conditions, a-oxoglutarate disappearance is stimulated. If malate is added as well as malonate, phosphoenolpyruvate formation is restored, and a-oxoglutarate disappearance is diminished. 3. In rat-heart sarcosomes, which contain no phosphopyruvate carboxylase (EC 4.I.I.3Z). the rate of oxidation of a-oxoglutarate in the presence of dinitrophenol and absence of phosphate acceptor does not show the marked decline observed with liver mitochondria. Inorganic phosphate does not disappear during the incubation.
INTRODUCTION
HUNTER 1 and JUDAH 2 discovered that the substrate-linked phosphorylation associated with the oxidation of a-oxoglutarate to succinate is not uncoupled by dinitrophenol. Thus dinitrophenol cannot replace PI in the mitochondrial oxidation of c-oxoglutarate" or of substrates that are oxidized via a-oxoglutarate, such as glutamate 4 - 9 , However, the mitochondria must be depleted of the appreciable amounts of Pi that they contain in order to show a maximum stimulation by added phosphate 2 , 3,l O. BORST AND SLATER3 have shown that rat-liver mitochondria contain sufficient endogenous PI to sustain a considerable respiration of a substrate such as glutamate in the presence of dinitrophenol. However, AZZONE AND ERNSTER ll found Abbreviation: PEP, phosphoenolpyruvate. • Formerly; Laboratory of Physiological Chemistry, Postal address: Amsterdam C (The Netherlands).
Biochim. Biophys. A eta,
IIO
(1965) 252-258
J. D.
Meyerplein 3,
SUBSTRATE-LINKED PHOSPHORYLATION
253
that, in the presence of dinitrophenol and absence of phosphate acceptor, the rate of oxidation of glutamate by rat-liver mitochondria rapidly declined unless Pi (> 0.1 mM) was added. The decline in respiration was accompanied by incorporation of trace amounts of 32P I into an organic form, which they identified as consisting mostly of ATP (95%). On the basis of these observations, AZZONE AND ERNSTERl l concluded that "The a-ketoglutarate-linked substrate level phosphorylation takes place in the mitochondria within a special compartment, yielding adenosine triphosphate which does not communicate freely with adenosine triphosphate originating from the respiratory chain phosphorylations". They explained the decline in respiration as being due to the removal of PI as "compartmentalized ATP" inaccessible to the dinitrophenol-induced ATPase. CHARLES et al.l 2 showed by direct analysis that the decline in respiration is brought about by the exhaustion of Pl. However, their results did not support the concept of a "compartmentalized ATP" inaccessible to the dinitrophenol-induced ATPase. They suggested instead that, in addition to the dinitrophenol-induced ATPase which returns esterified P to the system as P l , a slow side reaction occurs, which leads to the permanent disappearance of the Pl. This slow side reaction has now been identified. As has been known for some years, liver and kidney mitochondria from various species can synthesize PEP from oxaloacetate13-20, although this reaction has been reported to be very slow or absent in rat-liver mitoehondria11,19-21. In this paper it will be shown that the PI that disappears during the oxidation of glutamate or a-oxoglutarate by rat-liver mi.tochondria in the presence of dinitrophenol and absence of added phosphate acceptor is quantitatively converted not to ATP, as claimed by AZZONE AND ERNSTERll, but to phosphoenolpyruvate. METHODS Rat-liver mitochondria were isolated by the method of HOGEBOOM 22, as described by MYERS AND SLATER23 and rat-heart sarcosomes by the method of CLELAND AND SLATER24 . The standard reaction mixture contained 50 mM ReI, 25 mM Tris-H'Cl buffer (pH 7.5). 8 mM MgCI2, o.r mM 2,4-dinitrophenol, and 75 mM or 50 mM sucrose (derived from the liver- or heart-mitochondrial suspension, respectively), in a final volume of I ml. The reaction temperature was 25°. Oxygen uptake. mitochondrial protein and inorganic phosphate were determined by the experimental procedures given in ref. 12. The reaction was stopped by the addition of 0.1 ml 35 % HCI0 4 . After removal of the protein by centrifugation, the HCl0 4 was removed as KCI0 4 in the cold. Pi and PEP were determined in the neutralized extracts. PEP was determined by the method of KORNBERG AND PRICER25. Each cuvette contained 25 mM Tris-HCI buffer (pH 7-4), 0.5 mM ADP, 3.1 mM MgCl 2, 1.25 mM EDTA (pH 7.4), NADH, and an aliquot of the neutralized deproteinized reaction mixture in a final volume of 2 ml. Lactate dehydrogenase (EC I.l.I.27) was added and the reaction was started with pyruvate kinase (EC 2.7.1.40). ADP, lactate dehydrogenase and pyruvate kinase were obtained from Boehringer and Soehne. Oligomycin was kindly supplied by the Upjohn Chemical Company. Biocbim. Biophys. A cia,
IIO (1965) 252-258
254
H. R. SCHOLTE,
J.
M. TAGER
20 3
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....a 0
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3-
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a.
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0
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4
5 20
10
30
40
Time (min)
Fig. 1. Time course of oxygen uptake. PI disappearance and PEP formation. during the oxidation of glutamate by rat-liver mitochondria in the presence of dinitrophenol and absence of added PI or phosphate acceptor. The reaction mixture contained the basic components, plus 9.5 IJ1g mitochondrial protein. Curve I (oxygen uptake), Curve 3 (PEP formation) and Curve 5 (PI concentration) in presence of 20 mM glutamate. Curve 2 (PI concentration) and Curve 4 (oxygen uptake) with no added substrate. RESULTS
Fig. I shows that during the oxidation or glutamate by rat-liver mitochondria in the presence of dinitrophenol and absence of added Pi or phosphate acceptor, PEP is formed. The PEP formation parallels the oxygen uptake. In the absence of glutamate, incubation with dinitrophenol leads to the liberation of Pi (cj. refs. 3 and IZ). 20
400
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20
40
60
80
Time ('T1in)
20
40
.6
50
80
Time (min)
Fig. 2. The disappearance of PI and the formation of PEP during the oxidation of c-oxoglutarate in the presence of dinitrophenol and absence of added PI or phosphate acceptor. The reaction mixture contained the basic components plus 7.9 mg mitochondrial protein, and where present 20 mM c-oxoglutarate. Curve I (0-0), disappearance of PI during oxidation of a-oxoglutarate (calculated from Curves 5 and 6, the PI concentrations without and with added substrate, respecti vely): Curve 2 (6. - 6.), PEP formation in the presence of c-oxoglutarate; Curve 3 (e-e), oxygen uptake in presence of added substrate; Curve 4 (e-e), oxygen uptake in absence of added substrate. The initial PI concentrations in the Curves 5 and 6 varied slightly in the different incubations, as can be seen from the right-hanel curves. This is due to the fact that the incubations were begun at different times, and Pi was slowly released during storage of the mitochondria at 0° (30% during 50 min storage).
Biocliim, Biopbys. Acta, 110 (1965) 252-258
255
SUBSTRATE-LINKED PHOSPHORYLATION
25
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s: 0
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7
5
a 20
30
40
50
60
Time (min)
Fig. 3. Effect of oligomycin. arsenite, malonate, malonate + malate. and Pi on the oxygen uptake during oxidation of u-oxoglutarate by rat-liver mitochondria in the presence of dinitrophenol and absence of added phosphate acceptor. The same experiment as in Table 1. Curve 1, + \0 mM potassium phosphate buffer (pH 7.5); Curve 2, no further additions; Curve 3. +1.0 mM rnaicnate + 5 mM malate (no o-oxcglutarate) : Curve 4, + 20 mM malonate + 5 mM malate; Curve 5, + 20 roM malonate; Curve 6. + 21 J.!M oligomycin; Curve 7. no a:-oxoglutarate; Curve 8, + I mM arsenite.
When the amount of Pi found in the presence of glutamate is corrected for the amount liberated in its absence, it is evident that Pi is almost quantitatively converted to PEP during the oxidation of glutamate under these conditions. The relation between the disappearance of PI and the formation of PEP was also examined during the oxidation of a-oxoglntarate in the presence of dinitrophenol and absence of added Pi or phosphate acceptor. In Fig. 2 the Pi concentration as a function of time in the presence and absence of a-oxoglutarate (right hand curves) has been used to calculate the disappearance of Pi during the oxidation of a-axoglutarate. As shown in the left hand part of Fig. 2, there is a stoicheiometric conversion of inorganic phosphate to PEP. Table I and Fig. 3 show the effect of inhibitors. Oligomycin had little effect on the amount of PI utilized or the amount of PEP formed in 56 min (contrast ref. 19). However the oxygen uptake and c-oxoglutarate utilization were markedly decreased by oligomycin (see note added in proof in ref. rz). Arsenite inhibited the oxidation of c-oxoglutarate, formation of PEP and the disappearance of PI. Malonate also inBiochim. Biophys. Acta,
IlO
(1965) 252-258
H. R. SCHOLTE ,
J.
M. TA GER
TABLE!
+
E FF ECT OF O LI GOMY CIN, ARSE N ITE , M ALONATE, MA L O NATE M ALATE , AND I N OR GA NIC PH OSPHATE ON T HE OX I D AT IO N OF U - OXOG L UT ARAT E B Y RAT- L IVER M I T OCH O N D RI A IN THE P RES EN CE OF D IN ITROP H E N O L, AN D O N T HE DISAPP EARANC E O F I N O R G AN I C P H OSPH ATE AND THE FOR MATI ON
OF
PHOSP HOE NO L PYRU V AT E
T h e reaction mixt ure cont a ined the b as ic co mponen ts plus 9.9 rng mitochondrial protein and 20 mM o-oxoglutarate. R eactio n t ime was 56 min , The P i co nce ntra t ion in the reaction m ixture was 361 "M a t zero time . and 632 I'M after incu ba t.ion for 56 min in t h e absen ce of su bstra t e. The corresponding values fo r PEP were 13 and 30 ,uM, re spect iv ely. - LlPi = PI found aft er incubation for 56 min wit h out s ubstra te minlls that found after incubation for 56 m in with t h e addit ion s sh own in the table. LlP E P wa s calcula t ed similarly .
A dditions or omissi ons
N one + Oligomycin (:ZI 11M ) Arsenite (I roM) Malonate (20 mM) + Malon at e (20 mM) + malate (5 mM) No a -ox oglut arate ; Malonate (20 mM) mala t e (5 rnM) + PI (10 roM)
-LI P,
LJPEP
- A a-oxogluta rale
#M
11M
mM
5° 4 465 10 56 4 86
5.6
17·3
55 7 553
11.9
+ +
+
-LIO
uatom»
-74
0·9 12·3 13. 0
+
134 547
13. 0
94 476
28.9
3. 0 0.6
7·9 5. 1 14· 4 ..
_--_..
hibited t he forma tion of PEP and the disappearance of PI but stimulat ed the disappear ance of a-oxo glutara te. When malate was added as well as malonate there was a restoration of PEP formation an d P i disappearan ce, and a diminution of a-ox oglutarat e utilizat ion to t h e level found in t he presence of a-oxoglutarate alone . Wh en IO mM PI was added , a high rate of respirat ion was obtained, which remained linear wit h ti me ; the PEP formation was depressed slightly. In rat-heart sarcosomes, the oxidation of a-oxoglutarate in th e presence of dinitrophenol an d absence of added P I is low, but remains linear for at least 60 min (Fig. 4). Added PI stimulates the rate of oxidation. There is no disappearance of PI
~
10
'E" ...
e
0
'"
2
3
:l..
~
~
6
E D.
4
'"
2
:J
0
40
50 Time
30
60
70
80
4 90
F ig. 4. The effect of added in organic phosphate on the oxid a t ion of a -oxogiutarat c b y rat-hea rt sarcosomes. T he re action m ixtur e co ntain ed the b asic: co mp onen t s p lu s 0.9 m g sar cosom a l protein and 10 rnM a-oxoglutaratc. Curve I, + 10 rrrM PI; Curve 2, + 0 . 1 rnM Pi ; Cu rve 3, no additions ; Curv e 4. no a -oxogilltarat e . At zer o tim e 65 pM P i was foun d in t he rea ction mi x t ure. Afte r go min t he a mo unts of Pl wer e 9 7 # M (Curve 4) . r 0 9 ~M (Curve 3) an d 187 pM (Curv e 2).
Bio chim, Biophy s. Acta, II O (1965) 25 2 -
25 8
257
SUBSTRATE-LINKED PHOSPHORYLATION
during a-oxoglutarate oxidation by rat-heart sarcosomes. These mitochondria contain no PEP carboxylase. DISCUSSION
ei al. 14 found that PEP is formed when a-oxoglutarate is oxidized by rabbit-liver mitochondria in the presence of Pi and dinitrophenol. NORDLIE AND LARDyl9 obtained the same results with guinea-pig liver mitochondria. Although PEP formation has been reported to be very slow or even absent in rat-liver mitochondrial 7,l 9- 21, it is clearly sufficiently rapid to account for the slow disappearance of Pi during the oxidation of glutamate or a-oxoglutarate by these mitochondria. From the results of this paper and those of CHARLES et al.l 2 , and the demonstration by HELDT ei al. 26 that GDP is the physiological phosphate acceptor for the substrate-linked phosphorylation step in rat-liver mitochondria, the following picture of the course of events during the oxidation of glutamate or a-oxoglutarate by ratliver mitochondria in the presence of dinitrophenol and absence of phosphate acceptor may be drawn. The Pi present in the mitochondria initially or liberated from acidstable phosphate compounds during incubation is esterified by the substrate-linked phosphorylation step of a-oxoglutarate oxidation (Eqn. I) MUDGE
Succinyl-CoA
+ GDP + PI'=' succinate + CoA + GTP
(I)
Most of the GTP reacts with ADP to form ATP (Eqn. 2) which is then hydrolyzed by the dinitrophenol-induced ATPase (Eqn, 3). Thereby, the PI esterified in Eqn. I is returned to the system GTP
+ ADP.=. GDP + ATP
ATP
+ H 20
(din itrophenol) ) ADP
+ Pi
Although most of the GTP reacts according to Eqn, 2, some reacts with the oxaloacetate (formed by the further oxidation of the succinate) to give PEP (Eqn. 4) GTP
+ oxaloacetate ee GDP + PEP + CO2
The PEP :a-oxoglutarate ratio was about 0.1 (Table I), indicating that about I in ro of the GTP molecules reacts according to Eqn. 4- CHARLES et al.l 2 found that the p:o ratio during oxidation of glutamate in the presence of dinitrophenol and absence of phosphate acceptor was o.or-o.ory, instead of the 0.33 to be expected if the ATP formed by the substrate-linked phosphorylation step were stable. Thus, under their conditions I in 20-30 molecules of GTP reacts according to Eqn. 4. The results reported in this paper confirm the conclusion by CHARLES et al. l 2 that there is no experimental basis for the proposition of AZZONE AND ERNSTER l !, recently reaffirmed by ERNSTER AND LEE 27 and adopted by NORDLIE AND LARDylD, that the ATP formed by the substrate-linked phosphorylation step of a-oxoglutarate oxidation is inaccessible to the dinitrophenol-induced ATPase. Biochim. Biophys. Acta, no (1965) 252-258
258
H . R. SCHOLTE,
J.
M. TAGE R
ACKNOWLEDGnIENTS
The authors wish to thank Professor E. C. SLATER for his encouragemen t and advice. This investigation was sup por ted in part by a grant from the Life In suran ce Medical Research Fund. R E F E R ENCE S I F . E. HU NTER, in W . D . M cELROY AND B. GLASS. Symp . on P hosp horu s Me tabolism, Vol. I, John s Hopkins Press, Baltimore, 1951 , p . 29 7. 2 J. D. J UDAH, B i ochem : ] ., 49 (1951) 27 1. 3 P . B ORST AND E . C . SLATER, Bioohim : Biophys. Acta, 48 (19 61) 36 2. 4 A. F . MULLER AND F. LEUTHARDT, Hel u. Cbim , A cta, 33 (r95 0) 268 . 5 H. A. KREBS "-ND D. B ELLAMY, Biochem, j., 75 (1960) 523. 6 P. BORST AND E. C . SLATER, B iochim. Biophys. Acta, 4r (19 60) 170. 7 J. B. CHAPPELL AND G. D. OREvrLLE, Nature, 190 (r96r) 502 . 8 E. A. JONES AND H . GUTFREUND, Biochem, j., 79 (r96r) 608. 9 P. BORST, Biochim, B iophys. Acta, 57 (1962) 256. 10 L. J. TEPLY, Arch. Biochem ., 2'f (1949) 3 83. I I G. F. AZZONE AND L. ERNST ER, J. Bioi . Chem ., 236 (1961) 1501. 12 R. CHARLES, J . M . TAGER AN D E . C . SLATER, Biochim . B iophys . A eta, 74 (196 3) 33 . 13 S. W . STANBURY AND G . H . MUDGE , ] . B io!. Chem., 2 10 (1954) 949 . 14 G. H. M UDGE, H . Vit o NEUBERG AND S . W . STANBURY, J . Bioi. Chern., 2 10 (195 4) 96 5. 15 W. BARTLEY, B iochem , ] .. 56 (19 54) 387. 16 W. BARTLEY ...ND Y . AVI-D o R. B iochem. J., 59 (r955) 194 · 1 7 R. S . BANDURSKI AND F . LIP MANN, J. B ioI. Ch8m., 2 19 (1956 ) 74 1. 18 P. S CHOLLMEYER AND M . K1.INGENBERG, B iochem , B i ophys . Res. Commun. , 4 (19 6 1) 43 . 19 R. C . N ORDLIE AND H . A . L ARDY, B iochem, Z. , 338 (19 6 3) 356. 20 R. C . NORDLIE AND H. A. LARDY, j. B ioi. Chern., 238 (196 3) 2259. 2 I L. M . CORWIN, J. Bioi. Ch8m. , 234 (1959) 1338. 22 G. H. HOGEBOOM, in S. P. COLOWICK AND N . O. K APLAN, M ethods of E nzy mol ogy , Vol. I , Aca d em ic Pr ess, New York, 195 5. p. 16 . 23 D. K. MYERS AND E . C. SLATER, B i ochem , ]., 6] (19 57) 55 8. 24 K. W . CLELAND AND E. C. SLATER, B iochem, J., 5 3 (1953) 547 · 2 5 A. K ORNBERG AND "V. E . PRIC ER, j. B ioi . Chem ., 193 (1951) 48 1. 26 H. W. HELDT, H . J Aeons AND M. KL I NGENBERG, B iochem . B iop by s. Re s . Commlln ., 17 (19 64) 130 . 2 ] L . ERNSTER AND C.-P. LEE , Ann. Rev. B iochem .• 33 (1964) 77 8.
Biochim . B iop hy s. A cta, lID (1965) 252-258