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Uncoupling effect of amino compounds on choline oxidation in vitro
SHORT COMMUNICATIONS
187
BBA 63198 Uncoupling effect of amino compounds on choline oxidation in vitro Following initial reports b y WELLS 1# that 2-amino-2-methylpropanol and dimethylethanotamine exerted an inhibitory effect on choline oxidase in vitro, RUSSELL, GREENE AND MULFOR3~ demonstrated that uncoupling of phosphorylations from choline oxidation also occurred. I t was of interest to investigate effects of other amino compounds, particularly since the uncoupling effects could not be demonstrated with/5-hydroxybutyrate or succinate oxidations. Table I shows that the amino alcohols tested uncoupled the phosphorylations associated with choline oxidation. Though not shown for all cases, neither oxidation rates nor the phosTABLE I EFFECT OF AMINO COMPOUNDS ON THE OXIDATIVE PHOSPHORYLATION RI~ACTIONS OF CHOLINE, S U C C I N A T E AND fl-HYDROXYBUTYRATE O 3 c o n s u m p t i o n was m e a s u r e d m a n o m e t r i c a l l y b y s t a n d a r d W a r b u r g procedures. Mitochondria preparation, reaction conditions and concentrations of reactants, excepting the substrates, were the s a m e as those given previously s-s. Reaction time was 3 ° min. Mitochondria protein concent r a t i o n s were 2, 4 a n d 9 m g for the oxidations of succinate, / 5 - h y d r o x y b u t y r a t e and choline, respectively, to m a k e the O~ c o n s u m p t i o n s for all s u b s t r a t e s similar. Protein concentrations were m e a s u r e d b y the biuret reaction 9 and Pi was determined b y t h e m e t h o d of LOHMANN AND JENDRASSIKTM. All a m i n o c o m p o u n d s were free bases except choline and betaine, which were HC1 salts (Distillation P r o d u c t s Industries, Rochester, N.Y., U.S.A.) and all were neutralized to p H 7-4 prior to use. The concentrations of the following amino c o m p o u n d s were 5 ° mM : ethanolamine; m o n o m e t h y l e t h a n o l a m i n e ; d i m e t h y l e t h a n o l a m i n e ; 3-aminopropanol; 2-amino-2-methylp r o p a n o l ; and betaine. Betaine aldehyde was 3 ° raM. The c o n c e n t r a t i o n s of suceinate and /5h y d r o x y b u t y r a t e were 20 mM and t h a t of choline was 30 mM. Control values are above the amino c o m p o u n d added. All d a t a were calculated f r o m duplicate, independent e x p e r i m e n t s with duplicate r u n s within each e x p e r i m e n t as a m i n i m u m .
Substrate
Inhibitor
P, 02 P]O uptake uptake (l~moles) (l~moles)
Choline
None Ethanolamine None Monomethylethanolamine None Dimethylethanolamine None 3-Aminopropanol None 2-Amino-2 - m e t h y l p r o p a n o l None Betaine aldehyde None Betaine
3. I I 2.17 3.38 2.1i 3.18 1.69 3.66 2.55 3-47 0.53 4.08 o.oo 2.79 2.21
2.98 2.74 2.35 2.18 2.15 1-94 2.81 2.47 3-31 1.9I 2.80 2.07 1.64 z.67
1.04 o.8o 1.44 0.97 1.48 o.87 1.3 ° 1 .o 3 I.O 5 o.z8 1.46 o.oo 1.7 ° 1.32
None Betaine aldehyde None Betaine
2.97 3-97 6. 4I 6.14
1.92 2.31 3.51 3.71
1.55 1.73 1.8o 1.66
None Betaine aldehyde None Betaine
6.14 6.o 3 9-44 8-76
2.65 2.62 3-7 o 3.39
2.32 2.30 2.55 2-58
Choline Choline Choline Choline Choline Choline Succinate Succinate /5-Hydroxybutyrate fl-Hydroxybutyrate
Biochim. Biophys. Acta, 128 (1966) I 8 7 - I 8 9
188
SHORT COMMUNICATIONS
p h o r y l a t i o n s of succinate or f l - h y d r o x y b u t y r a t e were affected b y the a m i n o comp o u n d s tested. The m o s t m a r k e d uncoupling effect was o b t a i n e d with b e t a i n e a l d e h y d e which is not oxidized a n d is the p r o d u c t of choline o x i d a t i o n b y m i t o chondria 4 v. The absence of uncoupling effect b y b e t a i n e a l d e h y d e with succinate and f i - h y d r o x y b u t y r a t e is also shown, which typifies the lack of effect of a m i n o c o m p o u n d s on these substrates. A n o t h e r difference between t h e choline oxidase s y s t e m and succinate a n d ~ - h y d r o x y b u t y r a t e was the e x t r e m e s e n s i t i v i t y of choline o x i d a t i o n to uncoupling b y 2,4-dinitrophenol. A t I/~M 2,4-dinitrophenol, phosp h o r y l a t i o n s were c o m p l e t e l y inhibited. Since t h e o x i d a t i o n of choline was slow relative to succinate o r / / - h y d r o x y b u t y r a t e oxidations, it was of interest to d e t e r m i n e the effect of a m i n o alcohols on choline p e r m e a b i l i t y into mitochondria. I t was p r e v i o u s l y n o t e d t h a t choline perm e a b i l i t y into t h e m i t o c h o n d r i a l m e m b r a n e was restricted 11. I n i t i a l consideration, however, was given to the effect of choline c o n c e n t r a t i o n on the rate of 0 2 consumption. Fig. I shows t h a t s a t u r a t i o n of choline oxidase required approx. 3o mM choline, which was the c o n c e n t r a t i o n level required b y amino c o m p o u n d s to uncouple a n d m a y reflect a general i m p e r m e a b i l i t y of amino c o m p o u n d s into mitochondria. o
o o~ 2.c
s
d_- 1£
5 E :t.
10
20 310 410 5'0 610 Choline concentration (mM)
Fig. i. The experimental conditions were similar to those in Table I except for the varied concentrations of choline. O---O, Pi uptake; © (2), the number of ¢,atoms of 0 2 taken up during the same 3o-min time period. Endogenous values for the Pi and 0 2 uptakes were negligible. Consideration was n e x t given to the effect of the a m i n o c o m p o u n d s on choline p e r m e a b i l i t y into m i t o c h o n d r i a as m e a s u r e d b y the [MeJaC~choline which associated w i t h the p a r t i c u l a t e fraction of rnitochondria in the presence a n d absence of various amino compounds. Table I I shows only the highest concentrations of the a m i n o c o m p o u n d s tested, since the lower levels were also less in their effect. These d a t a were consistent with the observations of WILKEN a n d co-workers12, ]3 t h a t t h e presence of A T P before the a d d i t i o n of [MeJaC~choline g r e a t l y d i m i n i s h e d choline u p t a k e b y mitochondria. U n d e r the conditions of Table I I , the a d d i t i o n of choline a n d A T P were simultaneous in order t h a t conditions be similar to those used for o x i d a t i v e p h o s p h o r y l a t i o n measurements. I t was also observed, t h a t the u p t a k e of labeled s u b s t r a t e was as efficient a t 0 ° as at 3 o°, again in a g r e e m e n t w i t h WILKEN a n d co-workers~2, ~3, and the effect of amino c o m p o u n d s at the lower t e m p e r a t u r e were similar to those shown. These d a t a a n d others (ref. 3 a n d u n p u b l i s h e d observaBiochim. Biophys. dcta, 128 (1966) 187 189
189
SHORT COMMUNICATIONS TABLE II INHIBITION
OF
[Me-x4C]CHOLINEU P T A K E
BY AMINO ALCOHOLS
The procedures for the isolation and m e a s u r e m e n t of radioactive c o m p o n e n t s w a s the same as WILKEN, KAGAWA AND LARDY12, except t h a t ethanol was used for extraction. The i n c u b a t i n g m i x t u r e s and e x p e r i m e n t a l conditions were the s a m e as those given in Table I except t h a t hexokinase was o m i t t e d and i n c u b a t i o n s were limited to 2o rain. The average protein in reaction mixt u r e s was 8. 7 mg, a n d the values given were based on t h e entire reaction mixture. The specific a c t i v i t y of the [Me-laC]choline was 6. lO s counts/rain per #,mole. E a c h of the amino c o m p o u n d s added h a d a final concn. Of 4 ° mM.
[ Me-14C] choline added (tzmoles)
Radioactivity (counts/rain) in mitochondrial fraction Choline
Choline + 2-amino2-methylpropanol
Choline + 3-aminopropanol
Choline + ethanolamine
4° 20 IO 6
5618 2825 1637 11o6
4348 2415 1284 877
4878 2735 1362 915
4878 2638 1299 932
tions) indicated that the effect of amino alcohols on choline uptake was very small, ranging from 3 to 23 %, by comparison with the inhibitions of phosphate uptake at the same inhibitor concentrations (see Table I). Along with the above, the relatively minor effect of the amino compounds on the 02 consumption argues against permeability changes as a major role in the inhibitory effects of the amino alcohols. The possibility remains open that the amino compounds may interact with phosphate intermediates of the choline oxidase system. This investigation was supported by Research Grant AM-o8747, and Training Grant 2 T1 GM 492, National Institutes of Health, Public Health Service. The authors are grateful to Dr. M. J E L L I N E K of St. Louis University School of Medicine for the pure betaine aldehyde used in these experiments.
Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kan. (U.S.A.) I 2 3 4 5 6 7 8 9 IO II 12 13
K E N N E T H T. N . Y U E PERCY
J.
DWIGHT
RUSSELL
J.
MULFORD
I. C. WELLS, J. Biol. Chem., 207 (1954) 575. 1. C. WELLS, J. Biol. Chem., 217 (1955) 631. P. J. RUSSELL, S. T. GREENE AND D. J. MULFORD, Biochim. Biophys. Acta, 98 (1965) 445J. R. CHRISTENSEN AND L. J. DANIEL, Federation Proc., 12 (1953) 189. H. A. ROTHSCHILD AND E. S. G. BARRON, J. Biol. Chem., 209 (1954) 511. M. JELI.INEK, D. R. STRENGTH AND S. A. THAYER, J. Biol. Chem., 234 (1959) 1171. K. T. N. YUE, P. J. RUSSELL AND D. J. MULFORD, Biochim. Biophys. Acta, lO8 (1966) 191. G. H. HOGEBOOM, W. C. SCHNEIDER AND O. E. PALLADE, J. Biol. Chem., 172 (1948) 619. R. W. ROBINSON AND C. G. HOGDEN, J. Biol. Chem., 135 (194 o) 707 . I~. LOHMANN AND L. JENDRASSIK, Biochem. Z., 178 (1926) 419. G. R. WILLIAMS, J. Biol. Chem., 235 (196o) 1192. D. R. WILKEN, R. KAGAXVAAND H. A. LARDY, J. Biol. Chem., 240 (1965) 1843. T. KAGAWA, D. R. WILKEN AND H. A. LARDY, J . Biol. Chem., 24 ° (1065) 1836.
Received February 8th, 1966 Revised manuscript received June t3th, 1966 Biochim. Biophys. Acta, x28 (I966) 187-189
187
BBA 63198 Uncoupling effect of amino compounds on choline oxidation in vitro Following initial reports b y WELLS 1# that 2-amino-2-methylpropanol and dimethylethanotamine exerted an inhibitory effect on choline oxidase in vitro, RUSSELL, GREENE AND MULFOR3~ demonstrated that uncoupling of phosphorylations from choline oxidation also occurred. I t was of interest to investigate effects of other amino compounds, particularly since the uncoupling effects could not be demonstrated with/5-hydroxybutyrate or succinate oxidations. Table I shows that the amino alcohols tested uncoupled the phosphorylations associated with choline oxidation. Though not shown for all cases, neither oxidation rates nor the phosTABLE I EFFECT OF AMINO COMPOUNDS ON THE OXIDATIVE PHOSPHORYLATION RI~ACTIONS OF CHOLINE, S U C C I N A T E AND fl-HYDROXYBUTYRATE O 3 c o n s u m p t i o n was m e a s u r e d m a n o m e t r i c a l l y b y s t a n d a r d W a r b u r g procedures. Mitochondria preparation, reaction conditions and concentrations of reactants, excepting the substrates, were the s a m e as those given previously s-s. Reaction time was 3 ° min. Mitochondria protein concent r a t i o n s were 2, 4 a n d 9 m g for the oxidations of succinate, / 5 - h y d r o x y b u t y r a t e and choline, respectively, to m a k e the O~ c o n s u m p t i o n s for all s u b s t r a t e s similar. Protein concentrations were m e a s u r e d b y the biuret reaction 9 and Pi was determined b y t h e m e t h o d of LOHMANN AND JENDRASSIKTM. All a m i n o c o m p o u n d s were free bases except choline and betaine, which were HC1 salts (Distillation P r o d u c t s Industries, Rochester, N.Y., U.S.A.) and all were neutralized to p H 7-4 prior to use. The concentrations of the following amino c o m p o u n d s were 5 ° mM : ethanolamine; m o n o m e t h y l e t h a n o l a m i n e ; d i m e t h y l e t h a n o l a m i n e ; 3-aminopropanol; 2-amino-2-methylp r o p a n o l ; and betaine. Betaine aldehyde was 3 ° raM. The c o n c e n t r a t i o n s of suceinate and /5h y d r o x y b u t y r a t e were 20 mM and t h a t of choline was 30 mM. Control values are above the amino c o m p o u n d added. All d a t a were calculated f r o m duplicate, independent e x p e r i m e n t s with duplicate r u n s within each e x p e r i m e n t as a m i n i m u m .
Substrate
Inhibitor
P, 02 P]O uptake uptake (l~moles) (l~moles)
Choline
None Ethanolamine None Monomethylethanolamine None Dimethylethanolamine None 3-Aminopropanol None 2-Amino-2 - m e t h y l p r o p a n o l None Betaine aldehyde None Betaine
3. I I 2.17 3.38 2.1i 3.18 1.69 3.66 2.55 3-47 0.53 4.08 o.oo 2.79 2.21
2.98 2.74 2.35 2.18 2.15 1-94 2.81 2.47 3-31 1.9I 2.80 2.07 1.64 z.67
1.04 o.8o 1.44 0.97 1.48 o.87 1.3 ° 1 .o 3 I.O 5 o.z8 1.46 o.oo 1.7 ° 1.32
None Betaine aldehyde None Betaine
2.97 3-97 6. 4I 6.14
1.92 2.31 3.51 3.71
1.55 1.73 1.8o 1.66
None Betaine aldehyde None Betaine
6.14 6.o 3 9-44 8-76
2.65 2.62 3-7 o 3.39
2.32 2.30 2.55 2-58
Choline Choline Choline Choline Choline Choline Succinate Succinate /5-Hydroxybutyrate fl-Hydroxybutyrate
Biochim. Biophys. Acta, 128 (1966) I 8 7 - I 8 9
188
SHORT COMMUNICATIONS
p h o r y l a t i o n s of succinate or f l - h y d r o x y b u t y r a t e were affected b y the a m i n o comp o u n d s tested. The m o s t m a r k e d uncoupling effect was o b t a i n e d with b e t a i n e a l d e h y d e which is not oxidized a n d is the p r o d u c t of choline o x i d a t i o n b y m i t o chondria 4 v. The absence of uncoupling effect b y b e t a i n e a l d e h y d e with succinate and f i - h y d r o x y b u t y r a t e is also shown, which typifies the lack of effect of a m i n o c o m p o u n d s on these substrates. A n o t h e r difference between t h e choline oxidase s y s t e m and succinate a n d ~ - h y d r o x y b u t y r a t e was the e x t r e m e s e n s i t i v i t y of choline o x i d a t i o n to uncoupling b y 2,4-dinitrophenol. A t I/~M 2,4-dinitrophenol, phosp h o r y l a t i o n s were c o m p l e t e l y inhibited. Since t h e o x i d a t i o n of choline was slow relative to succinate o r / / - h y d r o x y b u t y r a t e oxidations, it was of interest to d e t e r m i n e the effect of a m i n o alcohols on choline p e r m e a b i l i t y into mitochondria. I t was p r e v i o u s l y n o t e d t h a t choline perm e a b i l i t y into t h e m i t o c h o n d r i a l m e m b r a n e was restricted 11. I n i t i a l consideration, however, was given to the effect of choline c o n c e n t r a t i o n on the rate of 0 2 consumption. Fig. I shows t h a t s a t u r a t i o n of choline oxidase required approx. 3o mM choline, which was the c o n c e n t r a t i o n level required b y amino c o m p o u n d s to uncouple a n d m a y reflect a general i m p e r m e a b i l i t y of amino c o m p o u n d s into mitochondria. o
o o~ 2.c
s
d_- 1£
5 E :t.
10
20 310 410 5'0 610 Choline concentration (mM)
Fig. i. The experimental conditions were similar to those in Table I except for the varied concentrations of choline. O---O, Pi uptake; © (2), the number of ¢,atoms of 0 2 taken up during the same 3o-min time period. Endogenous values for the Pi and 0 2 uptakes were negligible. Consideration was n e x t given to the effect of the a m i n o c o m p o u n d s on choline p e r m e a b i l i t y into m i t o c h o n d r i a as m e a s u r e d b y the [MeJaC~choline which associated w i t h the p a r t i c u l a t e fraction of rnitochondria in the presence a n d absence of various amino compounds. Table I I shows only the highest concentrations of the a m i n o c o m p o u n d s tested, since the lower levels were also less in their effect. These d a t a were consistent with the observations of WILKEN a n d co-workers12, ]3 t h a t t h e presence of A T P before the a d d i t i o n of [MeJaC~choline g r e a t l y d i m i n i s h e d choline u p t a k e b y mitochondria. U n d e r the conditions of Table I I , the a d d i t i o n of choline a n d A T P were simultaneous in order t h a t conditions be similar to those used for o x i d a t i v e p h o s p h o r y l a t i o n measurements. I t was also observed, t h a t the u p t a k e of labeled s u b s t r a t e was as efficient a t 0 ° as at 3 o°, again in a g r e e m e n t w i t h WILKEN a n d co-workers~2, ~3, and the effect of amino c o m p o u n d s at the lower t e m p e r a t u r e were similar to those shown. These d a t a a n d others (ref. 3 a n d u n p u b l i s h e d observaBiochim. Biophys. dcta, 128 (1966) 187 189
189
SHORT COMMUNICATIONS TABLE II INHIBITION
OF
[Me-x4C]CHOLINEU P T A K E
BY AMINO ALCOHOLS
The procedures for the isolation and m e a s u r e m e n t of radioactive c o m p o n e n t s w a s the same as WILKEN, KAGAWA AND LARDY12, except t h a t ethanol was used for extraction. The i n c u b a t i n g m i x t u r e s and e x p e r i m e n t a l conditions were the s a m e as those given in Table I except t h a t hexokinase was o m i t t e d and i n c u b a t i o n s were limited to 2o rain. The average protein in reaction mixt u r e s was 8. 7 mg, a n d the values given were based on t h e entire reaction mixture. The specific a c t i v i t y of the [Me-laC]choline was 6. lO s counts/rain per #,mole. E a c h of the amino c o m p o u n d s added h a d a final concn. Of 4 ° mM.
[ Me-14C] choline added (tzmoles)
Radioactivity (counts/rain) in mitochondrial fraction Choline
Choline + 2-amino2-methylpropanol
Choline + 3-aminopropanol
Choline + ethanolamine
4° 20 IO 6
5618 2825 1637 11o6
4348 2415 1284 877
4878 2735 1362 915
4878 2638 1299 932
tions) indicated that the effect of amino alcohols on choline uptake was very small, ranging from 3 to 23 %, by comparison with the inhibitions of phosphate uptake at the same inhibitor concentrations (see Table I). Along with the above, the relatively minor effect of the amino compounds on the 02 consumption argues against permeability changes as a major role in the inhibitory effects of the amino alcohols. The possibility remains open that the amino compounds may interact with phosphate intermediates of the choline oxidase system. This investigation was supported by Research Grant AM-o8747, and Training Grant 2 T1 GM 492, National Institutes of Health, Public Health Service. The authors are grateful to Dr. M. J E L L I N E K of St. Louis University School of Medicine for the pure betaine aldehyde used in these experiments.
Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kan. (U.S.A.) I 2 3 4 5 6 7 8 9 IO II 12 13
K E N N E T H T. N . Y U E PERCY
J.
DWIGHT
RUSSELL
J.
MULFORD
I. C. WELLS, J. Biol. Chem., 207 (1954) 575. 1. C. WELLS, J. Biol. Chem., 217 (1955) 631. P. J. RUSSELL, S. T. GREENE AND D. J. MULFORD, Biochim. Biophys. Acta, 98 (1965) 445J. R. CHRISTENSEN AND L. J. DANIEL, Federation Proc., 12 (1953) 189. H. A. ROTHSCHILD AND E. S. G. BARRON, J. Biol. Chem., 209 (1954) 511. M. JELI.INEK, D. R. STRENGTH AND S. A. THAYER, J. Biol. Chem., 234 (1959) 1171. K. T. N. YUE, P. J. RUSSELL AND D. J. MULFORD, Biochim. Biophys. Acta, lO8 (1966) 191. G. H. HOGEBOOM, W. C. SCHNEIDER AND O. E. PALLADE, J. Biol. Chem., 172 (1948) 619. R. W. ROBINSON AND C. G. HOGDEN, J. Biol. Chem., 135 (194 o) 707 . I~. LOHMANN AND L. JENDRASSIK, Biochem. Z., 178 (1926) 419. G. R. WILLIAMS, J. Biol. Chem., 235 (196o) 1192. D. R. WILKEN, R. KAGAXVAAND H. A. LARDY, J. Biol. Chem., 240 (1965) 1843. T. KAGAWA, D. R. WILKEN AND H. A. LARDY, J . Biol. Chem., 24 ° (1065) 1836.
Received February 8th, 1966 Revised manuscript received June t3th, 1966 Biochim. Biophys. Acta, x28 (I966) 187-189