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Unspecific permeation and specific exchange of adenine nucleotides in liver mitochondria
312
PRELIMINARY NOTES
SCHUURS AND SIMPSON12. Both systems exhibit rapid rise and fall profiles, with peak content (or activity) occurring early in the logarithmic stage of growth. It has also been reported that the highest K + levels13 and peak activity of amino acid incorporation by ribosomal extracts6,14 occur at the beginning of logarithmic growth in Escherichia coli. Thus it seems reasonable to suggest that the variation of cellular K + during growth reflects the activities of protein synthesis and that during the early log phase, K + is taken up by the cells in response to the requirements of the protein-synthesizing machinery. This work was supported in part by a contract with the U.S. Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, New York, and in part by a training grant from the National Institutes of Health.
Department of Radiation Biology, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. (U.S.A.)
W . B A R R I E G. J O N E S A S E R ROTHSTEIN F R E D SHERMAN J . N E W E L L STANNARD
I 2 3 4 5 6 7 8 9 IO ii 12 13 14
L. J. WICKERHAM, U.S. Dept. Agr. Tech. Bull., (1951) No. lO29. E. J. CONWAY AND F. DUGGAN, Biochem. J., 69 (1958) 265. M. LUBIN AND H. L. ENNIS, Bioehim. Biophys. Acta, 80 (1964) 614. T. \V. CONWAY, Proc. Natl. Acad. Sci. U.S., 51 (1964) 1216. G. J. SPYRIDE, Proc. Natl. Acad. Sci. U.S., 51 (1964) 122o. 1VI. 1R. LAMBORG AND P. C. ZAMECNIK, Biochim. Biophys. Acta, 42 (196o) 206. A. TlSSlgRES, D, SCHLESSINGER AND F. OROS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 145o. T. NAKAMOTO, J. :E. ALLENDE AND T. W. CONWA¥, Federation Proc., 22 (1963) 524 . M. LUBIN, Bioehim. Biophys. Acta, 72 (1963) 345. M. LOBIN AND H. L. ENNIS, Federation Proc., 22 (1963) 302. D. SCHLESSlNGER, Biochim. Biophys. Acta, 80 (1964) 473. J. M. LUCAS, A. H. W. M. SCHUURS AND ]V[. L. SIMPSON, Biochemistry, 3 (1964) 959S. G. SCHtlLTZ AND A. K. SOLOMON, J. Gen. Physiol., 45 (1961) 360. W. DOERFLER, W. ZILLIG, E. FUCHS AND M. ALBERS, Z. Physiol. Chem., 33 ° (1962) 96.
Received February 26th, 1965 Biochim. Biophys. Acta, lO4 (1965) 31o-312
PN 21 085
Unspecific permeation and specific exchange of adenine nucleotides in liver mitochondria* It was previously communicated that the endogenous adenine nucleotides of mitochondria act as primary substrate in the forward and reverse reactions of oxidative phosphorylation, these being followed by the reaction of the exogenous adenine nucleotides 1. Early investigations indicated a relationship between endogenous and exogenous adenine nucleotides ~-4. A newly developed "centrifugal-filtration" technique permitted investigation of the uptake of and exchange between exogenous and * T h e s e d a t a h a v e been r e p o r t e d in p a r t a t t h e 6 t h I n t e r n a t i o n a l Congress of B i o c h e m i s t r y , New York, 1964, in t h e S y m p o s i u m on Metabolism and its Control.
Bioehim. Biophys. Acta, lO4 (1965) 312-315
313
PRELIMINARY NOTES
endogenous adenine nucleotides in the mitochondria s. The results are summarized here. (I) The penetration of mitochondria by adenine nucleotides (ATP, ADP and AMP) was found to be extremely rapid, reaching completion in the shortest time measurable (20 sec). The amount of permeated adenine nucleotides was found to be proportional to the external concentration over a large range (o.5-1o mM). The permeable space thus measured amounted in most cases to approx. 75 % of the aqueous phase of mitochondria. The permeated adenine nucleotides can be rapidly washed out. (2) By employing 14C-labelled adenine nucleotides the exchange of the exogenous with the endogenous adenine nucleotides was differentiated from the uptake of adenine nucleotides by permeation. The rate of exchange is still rapid, but considerably slower than the permeation and can be measured at lower temperatures with the rapid "centrifugal-filtration" technique 5. The exchange rates were found to vary widely among the adenine nucleotides studied. As an example, the following rates (taken at 8 ° during the initial linear period of up to 30 sec) are given, expressed as per cent of total adenine nucleotides exchanged per minute : ATP 46 %, ADP 80 To, AMP 2 %. loo] O~ C
+
.ot
0
t.d
o 1
3
5
16 2b ' 4'o ' 6'o ' so' ' lbo
Incubation time (min)
Fig. I. T i m e course of t h e e x c h a n g e of e n d o g e n o u s a d e n i n e n u c l e o t i d e s b y a d d e d [14CJATl% [14C~ADP a n d ~I~C~AMP. T h e % e x c h a n g e refers to t h e t o t a l of t h e e n d o g e n o u s a d e n i n e nucleotides. I n c u b a t i o n c o n d i t i o n s : 4 m g p r o t e i n of r a t liver m i t o c h o n d r i a p e r ml, i m M s u c c i n a t e , 500/zM e x t e r n a l a d e n i n e nucleotide, 5 °, 0.25 M saccharose m e d i u m , i m M E D T A , 20 m M trie t h a n o l a m i n e - H C 1 , p H 7.2. + - - + , A D P ; O - - O , A T P ; O - - O , AMP.
All three forms of the endogenous adenine nucleotides a r e - - w i t h i n an error of 5 To--completely exchangeable, independent of the type of added adenine nucleotides (Fig. I). In the case of added ADP over 80 % exchange is reached in 30 sec at 28 ° and in 4 min at 5 °. The rate of the exchange has a marked temperature dependence, being most pronounced (threefold increase) between I ° and 7 °. (3) The preferential exchange with ADP raises the question whether ADP is an exclusive substrate, ATP and AMP being transformed to ADP before they are exchanged. Parallel experiments with [14C~ATP and [~zPIATP, especially in the presence of dinitrophenol, show t h a t most of the ATP is exchanged as an intact molecule (Table I). Here an example of the individual exchange of the three endogenous adenine nucleotides is given. Table I also demonstrates that on uncoupling, for Biochim. Biophys. Acta, lO 4 (1965) 312-315
314 TABLE
PRELIMINARY
NOTES
I
EVIDENCE FOR THE TRANSLOCATION OF A T P AS AN INTACT MOLECULE AND THE EFFECT OF UNCOUPLING OF OXIDATIVE PHOSPHORYLATION R a t l i v e r m i t o c h o n d r i a w e r e i n c u b a t e d w i t h E8-14C~ATP o r [7-32P A T P i n t h e " c e n t r i f u g a l l a y e r f i l t r a t i o n ''2. C o n d i t i o n s : i n c u b a t i o n t i m e 3 ° sec, 8 °, 3 m g m i t o c h o n d r i a l p r o t e i n p e r s a m p l e , i mM succinate, no phosphate added, with o.o8 mM dinitrophenol. Exogenous and endogenous a d e n i n e n u c l e o t i d e s w e r e e h r o m a t o g r a p h e d w i t h s i m u l t a n e o u s r e c o r d i n g a t 265 m # a n d 2 8 o m/x. T h e r a d i o a c t i v i t y of t h e f r a c t i o n s w a s m e a s u r e d a n d t h e a m o u n t of e x c h a n g e d a d e n i n e n u c l e o t i d e s was calculated.
Additions
Individual exchange of endogenous adenine nucleotides A TP m#moles/ mg protein
A DP mlzmoles / mg protein
%
A MP ml~moles / mg protein
°/o
~14C]ATP
2.44
[3ZpIATP
1.88
57.o
2.33
56. 7
o.8
4o.7
o.92
14. 4
o/o
Total exchange mt~rnoles/ o~, mg protei1*
16.9
5.57
42.o
--
2.8
IS. 4
example by dinitrophenol, the translocation by ATP is greatly stimulated and reaches the high rate of ADP exchange. (4) While atractyloside has no effect on the permeation of the adenine nucleotides into the mitochondria, it drastically inhibits the exchange between endogenous and exogenous adenine nucleotides. The exchange is inhibited more than 9 ° % with each of the three added adenine nucleotides at 5oo ~M by 5o/~M atractyloside. With 3.6 mM ADP the exchange is inhibited to 5o % by atractyloside. Despite the addition of atractyloside the permeated adenine nucleotides can be rapidly washed out, whereas the exchanged adenine nucleotides remain bound. (5) The exchange reaction is highly specific for the adenine nucleotides. Practically no exchange could be measured with adenosine, adenosine diphosphate ribose, UDP, GDP, and IDP. The reported results show that the entrance of adenine nucleotides into the mitochondria involves at least two steps: an unspecific, possibly diffusion-limited, permeation and a specific exchange between the endogenous and exogenous adenine nucleotides. It can be assumed that the exchange is catalyzed by a specific adenine nucleotide exchange enzyme*. Besides its specifity, the exchange reaction has also other characteristics suggesting an enzyme-catalyzed reaction : the comparatively low rate, the temperature and concentration dependence and the existence of a specific inhibitor (atractyloside). The adenine nueleotide exchange enzyme is apparently in the main pathway for the oxidative phosphorylation of exogenous adenine nucleotides, since at low temperature the endogenous adenine nucleotides are phosphorylated prior to the exogenous adenine nucleotides 1, and the exchange occurs at about the same rate as the oxidative phosphorylation of external adenine nucleotides. The function of the exchange enzyme could be to facilitate and control the transport of the exogenous adenine nucleotides to the enzymes of oxidative phosphorylation which are enclosed in a certain compartment of the mitochondria. * The terms exchange 5 and translocation a are used synonymously, since at the moment it c a n n o t b e d e c i d e d w h e t h e r t h e m e c h a n i s m is a m e m b r a n e - l i n k e d t r a n s l o c a t i o n o r a n e x c h a n g e between various phases.
Biochim. Biophys. Aela, lO 4 (1965) 3 1 2 - 3 1 5
315
PRELIMINARY NOTES
SIEKEVITZ AND POTTERs attributed this role to the adenylate kinase. The exchange enzyme also appears to control the specifity of oxidative phosphorylation for adenine nucleotides, since disruption of the mitochondrial compartment removes its function and brings out the reactivity of the phosphorylative enzymes with other nucleotides such as IDP, UDP, etc. e, which was desbcried earlier in sonic particles by LGw et ald. The results are not in agreement with the concept of an "impenetrable unit" of endogenous adenine nucleotides which act as a primary substrate in the oxidative phosphorylation followed by a transphosphorylating "mesomerase" as postulated by BRIERLEY AND GREEN8. The reported exchange makes superfluous any transphosphorylation between endogenous and exogenous adenine nucleotides which has been implicated in various studies on the endogenous mitochondrial adenine nucleotides ~,",10,s. The inhibition of adenine nucleotide "binding" as described by BRUNI et al. n has to be re-interpreted in view of the present results, since atractyloside does not inhibit a net uptake of adenine nucleotides, but the exchange reaction. Whereas under the present conditions the compartment of endogenous adenine nucleotides appears to be filled, an additional uptake of large amounts of adenine nucleotides is observed parallel with the accumulation of Ca 2+ and phosphate in the presence of ATP 12. I t has been communicated by us* that mainly ADP, not ATP accumulated, in agreement with the preference for ADP in the exchange reaction. This could now be confirmed by CARAFOLI et al. ~3. This research was supported by a grant from the Bundesministerium ffir wissenschaftliche Forschung, Bad Godesberg.
Institut fi~r Physiologische Chemie der Philipps-Universitdt, Marburg (Germany)
E. PFAFF M. KLINGENBERG
H. W. HELDT I H. W. HELDT, H. JACOBS AND M. KLINGENBERG, Biochem. Biophys. Res. Commun., 18 (i965) 174. 2 P. SIEKEVITZ AND V. R. POTTER, J. Biol. Chem., 215 (1955) 237. 3 B. C. PRF~SSMAN, J. Biol. Chem., 232 (1958) 967. 4 B. C. PRESSMAN, Federation Proc., 17 (1958) 291. 5 M. KLINGENBERG, E. PFAFF UND A. KRGGER, in B. CHANCE, Rapid Mixing and Sampling Techniques in BiochemistYy, Academic Press, New York, 1964, p. 333. 6 M. KLINGENBERG, u n p u b l i s h e d experiments. 7 H. LGw, I. VALLIN ANn n . ALM, in B. CHANCE, Energy-linked Functions of Mitochondria, Academic Press, New York, 1963, p. 5. 8 G. BRIERLEY AND D. E. GREEN, Proc. Natl. dcad. Sci. U.S., 53 (1965) 739 A. KEMP, JR. AND E. C. SLATER, Biochim. Biophys. Acla, 92 (1965) 178. io A. BRUNI AND G. AZZONE, Biochim. Biophys. dcta, 93 (1964) 462. I I A. BRUNI, S. LUClANI AND A. R. CONTESSA, Nature, 2Ol (1964) 1219. 12 E. CARAFOLI AND A. L. LEHNINGER, Biochem. Biophys. Res. Commun., 16 (1964) 66. 13 E. CARAI~OLI, C. S. ROSSI AND A. L. LEHNINGER, personal c o m m u n i c a t i o n .
Received March 8th, 1965 * C o m m u n i c a t e d at S y m p o s i u m held b y Professor B. CHANCE at Malvern, U.S.A., in A u g u s t 1964.
Biochim. Biophys. Mcta, lO4 (1965) 312-315
PRELIMINARY NOTES
SCHUURS AND SIMPSON12. Both systems exhibit rapid rise and fall profiles, with peak content (or activity) occurring early in the logarithmic stage of growth. It has also been reported that the highest K + levels13 and peak activity of amino acid incorporation by ribosomal extracts6,14 occur at the beginning of logarithmic growth in Escherichia coli. Thus it seems reasonable to suggest that the variation of cellular K + during growth reflects the activities of protein synthesis and that during the early log phase, K + is taken up by the cells in response to the requirements of the protein-synthesizing machinery. This work was supported in part by a contract with the U.S. Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, New York, and in part by a training grant from the National Institutes of Health.
Department of Radiation Biology, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. (U.S.A.)
W . B A R R I E G. J O N E S A S E R ROTHSTEIN F R E D SHERMAN J . N E W E L L STANNARD
I 2 3 4 5 6 7 8 9 IO ii 12 13 14
L. J. WICKERHAM, U.S. Dept. Agr. Tech. Bull., (1951) No. lO29. E. J. CONWAY AND F. DUGGAN, Biochem. J., 69 (1958) 265. M. LUBIN AND H. L. ENNIS, Bioehim. Biophys. Acta, 80 (1964) 614. T. \V. CONWAY, Proc. Natl. Acad. Sci. U.S., 51 (1964) 1216. G. J. SPYRIDE, Proc. Natl. Acad. Sci. U.S., 51 (1964) 122o. 1VI. 1R. LAMBORG AND P. C. ZAMECNIK, Biochim. Biophys. Acta, 42 (196o) 206. A. TlSSlgRES, D, SCHLESSINGER AND F. OROS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 145o. T. NAKAMOTO, J. :E. ALLENDE AND T. W. CONWA¥, Federation Proc., 22 (1963) 524 . M. LUBIN, Bioehim. Biophys. Acta, 72 (1963) 345. M. LOBIN AND H. L. ENNIS, Federation Proc., 22 (1963) 302. D. SCHLESSlNGER, Biochim. Biophys. Acta, 80 (1964) 473. J. M. LUCAS, A. H. W. M. SCHUURS AND ]V[. L. SIMPSON, Biochemistry, 3 (1964) 959S. G. SCHtlLTZ AND A. K. SOLOMON, J. Gen. Physiol., 45 (1961) 360. W. DOERFLER, W. ZILLIG, E. FUCHS AND M. ALBERS, Z. Physiol. Chem., 33 ° (1962) 96.
Received February 26th, 1965 Biochim. Biophys. Acta, lO4 (1965) 31o-312
PN 21 085
Unspecific permeation and specific exchange of adenine nucleotides in liver mitochondria* It was previously communicated that the endogenous adenine nucleotides of mitochondria act as primary substrate in the forward and reverse reactions of oxidative phosphorylation, these being followed by the reaction of the exogenous adenine nucleotides 1. Early investigations indicated a relationship between endogenous and exogenous adenine nucleotides ~-4. A newly developed "centrifugal-filtration" technique permitted investigation of the uptake of and exchange between exogenous and * T h e s e d a t a h a v e been r e p o r t e d in p a r t a t t h e 6 t h I n t e r n a t i o n a l Congress of B i o c h e m i s t r y , New York, 1964, in t h e S y m p o s i u m on Metabolism and its Control.
Bioehim. Biophys. Acta, lO4 (1965) 312-315
313
PRELIMINARY NOTES
endogenous adenine nucleotides in the mitochondria s. The results are summarized here. (I) The penetration of mitochondria by adenine nucleotides (ATP, ADP and AMP) was found to be extremely rapid, reaching completion in the shortest time measurable (20 sec). The amount of permeated adenine nucleotides was found to be proportional to the external concentration over a large range (o.5-1o mM). The permeable space thus measured amounted in most cases to approx. 75 % of the aqueous phase of mitochondria. The permeated adenine nucleotides can be rapidly washed out. (2) By employing 14C-labelled adenine nucleotides the exchange of the exogenous with the endogenous adenine nucleotides was differentiated from the uptake of adenine nucleotides by permeation. The rate of exchange is still rapid, but considerably slower than the permeation and can be measured at lower temperatures with the rapid "centrifugal-filtration" technique 5. The exchange rates were found to vary widely among the adenine nucleotides studied. As an example, the following rates (taken at 8 ° during the initial linear period of up to 30 sec) are given, expressed as per cent of total adenine nucleotides exchanged per minute : ATP 46 %, ADP 80 To, AMP 2 %. loo] O~ C
+
.ot
0
t.d
o 1
3
5
16 2b ' 4'o ' 6'o ' so' ' lbo
Incubation time (min)
Fig. I. T i m e course of t h e e x c h a n g e of e n d o g e n o u s a d e n i n e n u c l e o t i d e s b y a d d e d [14CJATl% [14C~ADP a n d ~I~C~AMP. T h e % e x c h a n g e refers to t h e t o t a l of t h e e n d o g e n o u s a d e n i n e nucleotides. I n c u b a t i o n c o n d i t i o n s : 4 m g p r o t e i n of r a t liver m i t o c h o n d r i a p e r ml, i m M s u c c i n a t e , 500/zM e x t e r n a l a d e n i n e nucleotide, 5 °, 0.25 M saccharose m e d i u m , i m M E D T A , 20 m M trie t h a n o l a m i n e - H C 1 , p H 7.2. + - - + , A D P ; O - - O , A T P ; O - - O , AMP.
All three forms of the endogenous adenine nucleotides a r e - - w i t h i n an error of 5 To--completely exchangeable, independent of the type of added adenine nucleotides (Fig. I). In the case of added ADP over 80 % exchange is reached in 30 sec at 28 ° and in 4 min at 5 °. The rate of the exchange has a marked temperature dependence, being most pronounced (threefold increase) between I ° and 7 °. (3) The preferential exchange with ADP raises the question whether ADP is an exclusive substrate, ATP and AMP being transformed to ADP before they are exchanged. Parallel experiments with [14C~ATP and [~zPIATP, especially in the presence of dinitrophenol, show t h a t most of the ATP is exchanged as an intact molecule (Table I). Here an example of the individual exchange of the three endogenous adenine nucleotides is given. Table I also demonstrates that on uncoupling, for Biochim. Biophys. Acta, lO 4 (1965) 312-315
314 TABLE
PRELIMINARY
NOTES
I
EVIDENCE FOR THE TRANSLOCATION OF A T P AS AN INTACT MOLECULE AND THE EFFECT OF UNCOUPLING OF OXIDATIVE PHOSPHORYLATION R a t l i v e r m i t o c h o n d r i a w e r e i n c u b a t e d w i t h E8-14C~ATP o r [7-32P A T P i n t h e " c e n t r i f u g a l l a y e r f i l t r a t i o n ''2. C o n d i t i o n s : i n c u b a t i o n t i m e 3 ° sec, 8 °, 3 m g m i t o c h o n d r i a l p r o t e i n p e r s a m p l e , i mM succinate, no phosphate added, with o.o8 mM dinitrophenol. Exogenous and endogenous a d e n i n e n u c l e o t i d e s w e r e e h r o m a t o g r a p h e d w i t h s i m u l t a n e o u s r e c o r d i n g a t 265 m # a n d 2 8 o m/x. T h e r a d i o a c t i v i t y of t h e f r a c t i o n s w a s m e a s u r e d a n d t h e a m o u n t of e x c h a n g e d a d e n i n e n u c l e o t i d e s was calculated.
Additions
Individual exchange of endogenous adenine nucleotides A TP m#moles/ mg protein
A DP mlzmoles / mg protein
%
A MP ml~moles / mg protein
°/o
~14C]ATP
2.44
[3ZpIATP
1.88
57.o
2.33
56. 7
o.8
4o.7
o.92
14. 4
o/o
Total exchange mt~rnoles/ o~, mg protei1*
16.9
5.57
42.o
--
2.8
IS. 4
example by dinitrophenol, the translocation by ATP is greatly stimulated and reaches the high rate of ADP exchange. (4) While atractyloside has no effect on the permeation of the adenine nucleotides into the mitochondria, it drastically inhibits the exchange between endogenous and exogenous adenine nucleotides. The exchange is inhibited more than 9 ° % with each of the three added adenine nucleotides at 5oo ~M by 5o/~M atractyloside. With 3.6 mM ADP the exchange is inhibited to 5o % by atractyloside. Despite the addition of atractyloside the permeated adenine nucleotides can be rapidly washed out, whereas the exchanged adenine nucleotides remain bound. (5) The exchange reaction is highly specific for the adenine nucleotides. Practically no exchange could be measured with adenosine, adenosine diphosphate ribose, UDP, GDP, and IDP. The reported results show that the entrance of adenine nucleotides into the mitochondria involves at least two steps: an unspecific, possibly diffusion-limited, permeation and a specific exchange between the endogenous and exogenous adenine nucleotides. It can be assumed that the exchange is catalyzed by a specific adenine nucleotide exchange enzyme*. Besides its specifity, the exchange reaction has also other characteristics suggesting an enzyme-catalyzed reaction : the comparatively low rate, the temperature and concentration dependence and the existence of a specific inhibitor (atractyloside). The adenine nueleotide exchange enzyme is apparently in the main pathway for the oxidative phosphorylation of exogenous adenine nucleotides, since at low temperature the endogenous adenine nucleotides are phosphorylated prior to the exogenous adenine nucleotides 1, and the exchange occurs at about the same rate as the oxidative phosphorylation of external adenine nucleotides. The function of the exchange enzyme could be to facilitate and control the transport of the exogenous adenine nucleotides to the enzymes of oxidative phosphorylation which are enclosed in a certain compartment of the mitochondria. * The terms exchange 5 and translocation a are used synonymously, since at the moment it c a n n o t b e d e c i d e d w h e t h e r t h e m e c h a n i s m is a m e m b r a n e - l i n k e d t r a n s l o c a t i o n o r a n e x c h a n g e between various phases.
Biochim. Biophys. Aela, lO 4 (1965) 3 1 2 - 3 1 5
315
PRELIMINARY NOTES
SIEKEVITZ AND POTTERs attributed this role to the adenylate kinase. The exchange enzyme also appears to control the specifity of oxidative phosphorylation for adenine nucleotides, since disruption of the mitochondrial compartment removes its function and brings out the reactivity of the phosphorylative enzymes with other nucleotides such as IDP, UDP, etc. e, which was desbcried earlier in sonic particles by LGw et ald. The results are not in agreement with the concept of an "impenetrable unit" of endogenous adenine nucleotides which act as a primary substrate in the oxidative phosphorylation followed by a transphosphorylating "mesomerase" as postulated by BRIERLEY AND GREEN8. The reported exchange makes superfluous any transphosphorylation between endogenous and exogenous adenine nucleotides which has been implicated in various studies on the endogenous mitochondrial adenine nucleotides ~,",10,s. The inhibition of adenine nucleotide "binding" as described by BRUNI et al. n has to be re-interpreted in view of the present results, since atractyloside does not inhibit a net uptake of adenine nucleotides, but the exchange reaction. Whereas under the present conditions the compartment of endogenous adenine nucleotides appears to be filled, an additional uptake of large amounts of adenine nucleotides is observed parallel with the accumulation of Ca 2+ and phosphate in the presence of ATP 12. I t has been communicated by us* that mainly ADP, not ATP accumulated, in agreement with the preference for ADP in the exchange reaction. This could now be confirmed by CARAFOLI et al. ~3. This research was supported by a grant from the Bundesministerium ffir wissenschaftliche Forschung, Bad Godesberg.
Institut fi~r Physiologische Chemie der Philipps-Universitdt, Marburg (Germany)
E. PFAFF M. KLINGENBERG
H. W. HELDT I H. W. HELDT, H. JACOBS AND M. KLINGENBERG, Biochem. Biophys. Res. Commun., 18 (i965) 174. 2 P. SIEKEVITZ AND V. R. POTTER, J. Biol. Chem., 215 (1955) 237. 3 B. C. PRF~SSMAN, J. Biol. Chem., 232 (1958) 967. 4 B. C. PRESSMAN, Federation Proc., 17 (1958) 291. 5 M. KLINGENBERG, E. PFAFF UND A. KRGGER, in B. CHANCE, Rapid Mixing and Sampling Techniques in BiochemistYy, Academic Press, New York, 1964, p. 333. 6 M. KLINGENBERG, u n p u b l i s h e d experiments. 7 H. LGw, I. VALLIN ANn n . ALM, in B. CHANCE, Energy-linked Functions of Mitochondria, Academic Press, New York, 1963, p. 5. 8 G. BRIERLEY AND D. E. GREEN, Proc. Natl. dcad. Sci. U.S., 53 (1965) 739 A. KEMP, JR. AND E. C. SLATER, Biochim. Biophys. Acla, 92 (1965) 178. io A. BRUNI AND G. AZZONE, Biochim. Biophys. dcta, 93 (1964) 462. I I A. BRUNI, S. LUClANI AND A. R. CONTESSA, Nature, 2Ol (1964) 1219. 12 E. CARAFOLI AND A. L. LEHNINGER, Biochem. Biophys. Res. Commun., 16 (1964) 66. 13 E. CARAI~OLI, C. S. ROSSI AND A. L. LEHNINGER, personal c o m m u n i c a t i o n .
Received March 8th, 1965 * C o m m u n i c a t e d at S y m p o s i u m held b y Professor B. CHANCE at Malvern, U.S.A., in A u g u s t 1964.
Biochim. Biophys. Mcta, lO4 (1965) 312-315