SHORT COMMUNICATIONS 587

z C. H. STJELTER, M. DE LUCA, J. B. PETER AND P. D. t3OYER, Nature, 192 (1961) 43. z p. D. BOYER, M. DE LUCA, K. E. EBNER, D. E. HOLTgUIST AND J. B. PETER, J. Biol. Chem.,

237 (1962) PC 33o6. a j . B. PETER AND P. D. BOYER, J. Biol. Chem., 238 (I963) PC 118o. ¢ J. B. PETER, D. E. HULTQUIST, M. DE LUCA, G. tSZREIL AND P. D. BOYER, J. Biol. Chem.,

238 (1963) PC 1182. 5 B. CHANCE AND G. HOLLUNGER, J. Biol. Chem., 236 (1961) 1534. 6 p~. Vv-. ESTABROOK AND P. K. MAITRA, Anal. Biochem., 3 (1962) 369. 7 F. A. HOMMES, Biochem. Biophys. Res. Commun., 8 (1962) 248. s H. L~\¥ AND J. VALLIN, Biochim. Biophys. Acta, 63 (1963) 361. 9 R. M. BEHKI, J. E. BIAGLOW AND N. C. MELCHIOR, Abstr. Division of Biol. Chem. Am. Chem.

Soc., Chicago, Ill., S ep t embe r 1961, p. 9C. 10 B. CHANCE ANn T. ITO, Nature, 195 (I962) 15o. 11 B. C. PRESSMAN, J. Biol. Chem., 238 (I963) 4Ol. 12 j . B. CHAPPELL, J. Biol. Chem., 23g (1963) 4IO. 18 B. CHANCE AND G. HOLLUNGER, J, Biol. Chem., 238 (1963) 432.

Received April 29th, 1963

* P e r m a n e n t address : D e p a r t m e n t of Biochemis t ry , School of Medicine, Un ive r s i t y of Ni jmegen , N i jmegen (The Nether lands) .

** P re sen t address : I n s t i t u t e of Biological Chemis t ry , Un ive r s i t y of Copenhagen , Copen- hagen (Denmark) .

Biochim. Biophys. Acta, 81 (1964) 585-587

sc 63o16 Effect of imidazole on adenosine triphosphatase, adenosine triphosphate-

inorganic phosphate exchange reaction and oxidative phosphorylation

The preceding communication 1 has described the inhibitory effect of imidazole on the ATP-dependent succinate-linked reduction of NAD +. This inhibition has been inter- preted 1 as the interference by imidazole of the interaction of an endogenous coupling factor with the respiratory chain. To better assess the mode of action of imidazole, it was considered necessary to reinvestigate the original findings of BEHKI, BIAGLOW AND MELCHIOR 2 and those of Wtl AND RACKER 3 on the effect of imidazole on the for- ward reaction of oxidative phosphorylation. BEHKI et al.Z and Wu AND RACKER 3 have reported that imidazole significantly reduces the P/O ratio obtained during substrate oxidation by mitochondria. These results have been confirmed when submitochondrial particles derived from heart-muscle mitochondria are used as experimental materiah These results are illustrated in Fig. I. Although imidazole does reduce the P/O ratio when succinate or fl-hydroxybutyrate are used as substrates, the magnitude of inhi- bition and the sensitivity of the reaction is far less than that described x for the inhi- bitory effect of imidazole on the ATP-dependent succinate-linked reduction of NAD +. In agreement with the results of Wu AND RACKER 3 levels of imidazole of 25 mM or greater are required to demonstrate a 25-30% reduction of the P/O ratio. The P/O ratio observed tinting malate oxidation, however, is more significantly decreased by imidazole. 111 this case the inhibition pattern more closely resembles that obtained with the reverse reactions of oxidative phosphorylation. The difference in magnitude of imidazole inhibition between the three substrate systems tested is unexplainable but

Biochim. Biophys. Acta, 81 (i964) 587-590

5~8 SHORT COMMUNICATIONS

100

g ".,3

_c #

5 0

0 0

Malate

]3 - Hydroxybutyrate

; 1~ 1; 2b 2; [fmidazole] (rnM)

Fig. i. The effect of imidazole on the P/O ra t io ob ta ined wi th subnf i tochondr ia l par t ic les f rom beef heart . The basic reac t ion med ium conta ined io mM Ea~P]Pi (specific ac t iv i ty , 2 • lO 4 counts / min per / ,mole Pi), io mM t r i e thano lan l ine buffer (pH 7.4), 4 mM MgC12, 2 mM ATP, 12 mM glucose, 0. 5 mM EDTA, io un i t s of c rys ta l l ine hexok inase (EC 2 .7 . i . I ) (o.i mg), 2 mg bovine se rum albumin, and va ry ing concen t ra t ions of imidazole as indica ted . W h e n succ ina te was used as subs t r a t e io mM sodium succinate and i mg of par t ic le p ro te in were used. The P/O ra t io observed in the absence of imidazole was 0.8. W h e n NAD+-l inked subs t r a t e s were used, 0.25 mM NAD +, 2o mM f l -hyd roxybu ty ra t e , or 20 mM sodium mala t e and 20 mM sodium glu ta- m a t e were employed wi th 2 mg of par t ic le protein. The final reac t ion vo lume was 2.0 ml in all exper iments . A P/O ra t io of i . 7 was observed w i t h / ~ - h y d r o x y b u t y r a t e and a P /O ra t io of 1.3 was observed wi th g l u t a m a t e - m a l a t e in the absence of imidazole. The reac t ion was i n i t i a t ed by the add i t i on of the subs t r a t e to the reac t ion m i x t u r e equ i l ib ra ted a t 3 o°. 02 u p t a k e was measured po la rograph ica l ly in a closed reac t ion chamber and the reac t ion was t e r m i n a t e d a t the m o m e n t of anaerobios is by the add i t ion of perchlor ic acid. The ester i f icat ion of [.3~P~PI was de te rmined by ex t r ac t i on of the depro te in ized reac t ion m i x t u r e wi th molybd ic acid and i sobu tano l -benzene , according to LINDBERa AND ERNSTER 7, followed by a second ex t r ac t ion wi th i sobu tano l s a t u r a t e d

wi th H20 to remove the las t t races of [82PJPi,

cannot be attr ibuted to differences in the rate of respiratory activity. The inhibitory pat tern observed with succinate was the same when succinate respiration was de- creased (about 80%) by the addition of malonate to the level of malate oxidation.

BEHKI et al. 2 also reported on the effect of imidazole on the ATPase activity of mitochondria indicating that imidazole (lO-4O mmoles) caused a definitive increase in the ATPase activity 1. Examination of the ATPase activity of submitochondrial particles, either in the presence or absence of Mg 2+, showed a 25 % stimulation of the ATPase when 20 mM imidazole was present in the reaction medium. These results are presented in Table I.

Determination of the influence of imidazole on the ATP-E32P]Pi exchange reaction (Fig. 2) showed a pattern of inhibition analogous to that observed with the inhibitory effect of imidazole on the ATP-dependent succinate-linked reduction of NAD+. A 50% inhibition of the exchange reaction is observed with 8 mM imidazole.

The studies reported here, together with those described for the inhibitory effect of imidazole on the ATP-dependent succinate-linked reduction of NAD +, lead to the hypothesis that imidazole may act by competing in the energy-transfer reaction between a phosphorylated high-energy intermediate and its energy acceptor. Intro- duction of imidazole into the reaction medium may impede this reaction either by reacting directly to form an energy-transfer intermediate-imidazole complex or by disrupting the spatial organization required for the combination of the required coupling factor with its acceptor. This locus of inhibition would be compatible with

Biochim. Biophys. Acla, 81 (1964) 587-59 °

SHORT COMMUNICATIONS 589

T A B L E I

E F F E C T OF I M I D A Z O L E ON ATPAsE

A suspens ion of sub m i toc hond r i a l par t ic les f rom hea r t muscle 5 (7.2 mg of protein) was d i lu ted in a reac t ion vessel to i o ml wi th a m e d i u m con ta in ing 25 mM Tris buffer (pH 7.0) and 5o mM KC1 in 0.25 M sucrose. The reac t ion was i n i t i a t ed by the add i t i on of A T P to give a final concen- t r a t i o n of 5 mM. i - m l samples were w i t h d r a w n a t 3 ° sec, and a t i, 2, 4, and 6 min. The reac t ion was t e r m i n a t e d by add i t i on of the samples to I ml of lO% perchlor ic acid. Af ter cen t r i fuga t ion to remove the p r ec ip i t a t ed p ro te in the Pi concen t r a t ion was de t e rmined by the me thod of Fiske and S u b b a R o w as modif ied by KInG 6. Separa te e xpe r imen t s were carr ied out a t the var ious con- cen t r a t i ons of imidazole i nd i ca t ed e i ther in the presence or absence of MgC12 in the reac t ion medium. The ra te of A T P hydro lys i s was de t e rmined from the l inear slope of the curves re-

p re sen t ing e x t e n t of Pi formed as a func t ion of t ime. Temp. 25 °.

ATPase (#mole P~lmin per ml) Imida*ole

(raM) With Without 5 m M 3fg 2+ Mg ~+

o o.128 0.037 5 o.14o 0-038

IO o.163 o.o38 15 o.167 o.o45 2o o.16o 0.043 25 o.I7O - -

the site of participation of a "protein-bound activated histidine" which may result in the formation of the phosphohistidine described by BOYER et al. 4. The differences observed between the reactions participating in the reversal of oxidative phosphory- lation and the exchange reaction from those observed for the forward reactions of oxidative phosphorylation remain unexplained but may merely reflect differences in reactivity of the intermediates under the two conditions or differences in the rate- limiting reaction for the forward and reverse reactions of oxidative phosphorylation.

100

J~ z~ E

50 °/°y / /o

?

[ Im idazo le ] ( rnM)

Fig. 2. Effect of imidazo le on the [3~P]Pl A T P exchange reac t ion of submi tochondr i a l par t ic les f rom beef hear t . The reac t ion m i x t u r e con ta ined 4 ° mM [s2P]Pj (specific ac t iv i ty , 2- IO ~ counts• rain per /~mole Pj), 16 mM ATP, 16 mM MgCI~, 2 mM E D T A and i mg par t ic le p ro te in in a final vo lume of 0. 5 ml. This reac t ion was i n i t i a t ed by add i t i on of submi tochondr i a l par t ic les to the reac t ion m e d i u m equ i l i b ra t ed a t a c o n s t a n t t e m p e r a t u r e of 3 o°. The reac t ion was t e r m i n a t e d by add i t i on of perchlor ic acid af ter i o min. [a2P~P i incorpora t ion in to [3~PIATP was de t e rmined

b y the m e t h o d descr ibed in Fig. I.

Biochim. Biophys. Acta, 81 (1964) 587-590

59 ° SHORT COMMUNICATIONS

This work was supported in part by a United States Public Health Service Research Grant (RG 9956).

This study was carried out during the tenure of a Research Career Development Award (GM-K3-4III) to R.W.E. from the National Institutes of Health.

Department of Biophysics and Physical Biochemistry, Johnson Foundation, University of Pennsylvania,

Philadelphia, Pa. (U.S.A.)

T. E. CONOVER J. GONZE R. W. ESTABROOK

i F. HOMMES, R. W. ESTABROOK, t3. CHANCE AND U. F. RASMUSSEN, Biochim. Biophys. Acta, BBA 63009.

2 R. M. BEHKI, J. E. BIAGLOW AND N. C. MELCHIOR, Abstr. Division oJ Biol. Chem. Am. Chem. Soc., Chicago, Ill., September 1961, p. 9C.

a R. W u AND E. RACKER, in B. \VRIGHT, Control Mechanisms in Respiration and Fermentation, Ronald Press, New York, 1963, p. 265.

4 p. D. BOYER, D. E. HULTQUIST, J. ]3. PETER, G. KREIL, R. A. MITCHELL, M. DE LUCA, J. w . HINKSON, L. G. BUTLER AND R. W. MOYER, Federation Proc., 22 (1963) lO8O.

5 F. A. HOMMES, Biochem. Biophys. Res. Commun., 8 (1962) 248. 6 E. J. KING, Biochem. J., 26 (1932) 292. 7 0 . LINDBERG AND L. ERNSTER, Methods Biochem. Anal., 3 (1956) i.

Received November 4th, 1963 Biochim. Biophys. Acta, 81 (1964) 587-59 °

sc 63Ol 9 Weitere Befunde zum Mechanismus der mikrosomalen Ascorbins~ure-

abh~ngigen NADH2-Oxiclation

Nach Angaben von KRISCH UND STAUDINGER 1 ist die Ascorbins~ure als Elektronen- tibertr~ger an einer "mikrosomalen Atemkette" beteiligt. Sp~tere eigene Befunde waren mit dieser Annahme vereinbar z. Nur tiber die Effekte der Dehydroascorbin- s~ure traten Differenzen zwischen FRUNDER et al. 2 und STAUDINGER et al. a auf, die in Ref. 4 diskutiert sind.

Ftir eine Ascorbinsfiure-abh~ngige "mikrosomale Atemkette" sprach unter an- derem folgender BefundX: Die Q-Aufnahme der Mikrosomen ist in Gegenwart von Ascorbins~iure gering und wird durch NADH 2 mehrfach gesteigert. Im Gegensatz dazu fanden wir ohne und mit Zusatz von gereinigtem NADH 2 ann~hernd gleiche 02-Aufnahmen 4. Dieser Befund schloB aus, dab das NADH 2 in einer "mikrosomalen Atemkette" oxydiert wird. Man muBte daher nach anderen Ascorbins~ture-abh~tngigen 02- und NADH2-verbrauchenden Prozessen suchen.

Aus der Literatur 5 ist bekannt, daB AscorbinsRure eine nicht-enzymatische Lipid- peroxydbildung hervorruft. Wit untersuchten daher, (a) ob der in Gegenwart von Ascorbins~ure aufgenommene 02 vollst~ndig oder teilweise ftir Oxydationsprozesse an den Mikrosomenlipiden verbraucht wird, (b) ob dieser Prozess enzymatischer oder nicht-enzymatischer Natur ist und (c) welche Bedeutung das NADH2 bei dieser Reaktion besitzt.

Es wurden folgende Resultate erhalten (Tabelle I). (I) Pro Mol oxydiertes NADH 2 werden in intakten Mikrosomen etwa 1. 5 Mol 02

Biochim. Biophys. Acla, 81 (1964) 590-593