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The effect of fluorocitrate on the synthesis of amino acids from ammonia and α-keto acids in rat liver homogenate
.4RCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
69, 634-643 (19%)
The Effect of Fluorocitrate on the Synthesis of Amino Acids from Ammonia and a-Keto Acids in Rat Liver Homogenate A. E. Braunstein
and R. M. Azarkh
From the Institute of Biological and Medical Chemistry, .Icademy of Medical Sciences of the Union of Soviet Socialist Republics, Noscow, IrS2T.R.
Received January 15, 1957
The importance of indirect amination by the joint action of glutamica dehydrogenase and aminopherases in t,he biosynt,hesis of amino acids from their keto analogs and ammonia had first been suggested in 19X8-N by Euler and by Braunstein. In this laboratory and elsewhere, much txperiment’al evidence has been obtained in support of the predominant role of this mechanism, or ‘%ransreamination” (1, 2), in the conversion of ammonia to amino acids by animals (l-7)) microorganisms, and plants. It is almost generally accepted, at present, that glutamic acid is the primary product of assimilation of inorganic nitrogen in animals, plants, and most bacterial species. But several authors still hold the opinion that alanine and aspartic acid, and possibly other amino acids, are formed from their keto analogs by direct amination, rather than via glut’amic. acid, in the tissues of animals (8-10) and of plants (II). It has recently been shown in this laboratory that the rates of amination of keto acids, except a-ketoglutaric acid, by surviving liver or kidney tissue of rats and birds are greatly lowered when the activit,y of aminopherases in the tissue is reduced, either as a result of ppridoxme deficiency (6) or by the blocking of pyridoxal enzymes with rarbonyl reagents (1, 2). Transamination is depressed to very low levels in the liver of rats depleted in pyridoxal phosphate 11y repeated administration of heavy doses of isoniazid (1). Homogenates of such livers readily aminate ketoglutarate, but they synthesize almost no alanine and only small amounts of aminodicarboxylic acids from pyruvate and ammonia : 634
FLUOROCITRATE AND AMINO ACID SYNTHESIS IN LIVER
635
their capacity to form alanine is restored by supplementation of the incubation mixtures with purified glutamic-alanine aminopherase (1). it has also been shown that steric inversion of the n-isomers of essential mine acids is impaired in the living pyridoxine-deficient rat, owing to Jure of reamination of the cr-keto acids (4, 5). In full accord with the theory of transreamination, it could be demonitnbted (1,2) that the synthesis of amino acids from pyruvate or oxalacete in mammalian or avian liver and kidney tissue preparations is o,j.eatly depressed if the oxidative formation of ketoglutarate via the D citric acid cycle is blocked with the aid of appropriate specific inhibitors, e.g., with fluoroacetate (14), cocaine (12), or mesotartrate (13). The direct amination of ketoglutarate is not impaired, as a rule, under these conditions. In such experiments the amination of pyruvate and oxalacetate could be restored, as expected, by supplementation of the incubation samples with glutamate or with such intermediates of the citric acid cycle, the conversion of which to ketoglutarate had not been blocked (1, 2). We have extensively used fluoroacetate in experiments on the synthesis of amino acids in liver homogenates. The effects of fluoroacetate were considered as due to the enzymic formation of fluorocitrate, resulting in specific inhibition of the conversion of citric acid to ketoglutarate [owing to blocking of aconitase (15)]. Doubt has been cast on this interpretation by a recent investigation from R. A. Peters’ laboratory (16), tending to show that in liver fluoroacetate is not transformed into fluorocitrate. We have therefore made some further experiments on the amination of keto acids in rat liver homogenates, using concentrates of biosynthetic fluorocitric acid (14) or pure synthetic fluorocitrate.’ These experiments are reported below. Their results were quite similar to those obtained formerly with fluoroacetate; they confirm the view that the synthesis of amino acids from ammonia and keto acids, other than ketoglutarate, in animal tissues proceeds by way of transreamination. EXPERIMENTAL
General Procedure and Analytical
Methods
Determinations were made of the increase in total NH2 nitrogen and in individual amino acids upon incubation of stabilized rat liver homogenates with a-keto acids and ammonium carbonate. The livers were taken from well-fed young male albino rats weighing SO-120 g. 1 We wish to express our gratitude gift of synthetic sodium fluorocitrate.
to Professor R. A. Peters for the generous
Gxi
BRAUNSTEIN
AND
AZARKH
As formerly found by the authors, the rates of a.mino acid synthesis in rat tissues are markedly affected by the age and origin (strain) of the animals, by their nutritional state (a generous supply of protein and vitamins rather than of carbohydrate [cf. (17)l seems to be especially important.), and by seasonal influencer (low rates of amination in spring and early summer). In experiments with homogenates it is important to st,abilize the mitochondri:l (i.e., the enzyme systems of the respiratory tricarboxylic cycle) wit,h ATP, Mg+. I<+, and ethylenediamine tetraacetate (EDTA), and carefully to adjust the pH of the incubation mixture (optimal range: pH 7.0-7.2). The experimental procedure was as follows: the rat, livers were cooled in iced 1.15% KC1 solution and homogenized 2 min. in a Potter-Elvehjem glass homogcsizer with 6.5 vol. of buffer solution (pH 7.2) containing0.1 M K phosphate. 1.15% KCl, and 4 X low3 M disodium EDTA. Theexperimental samples contained, in a total volume of 3 ml. : 1.5 ml. homogen ate (= 200 mg. liver tissue), 2 X 1OW M ATP, 2 X lo-” M MgS04 , 3 X lo+ dl (KH4)&03 The keto acids (Ka pyruvate or Sa ketoglutarate) were added at :I level of 80 pmoles per sample. Before the addition of ammonium carbonate anti keto acid the samples were left in cont,act wit,h fluorocitrat.e (resp. with plain bllf fer) at room temperature for 5-10 min. The samples were incubated with shaking in 0% for 60-80 min. at 3X”, and fiard with trichloroacetic acid. Total amino N was determined spectrophotometrically by the ninhydrin filtrates, madr method of Cocking and Yemm (18) in aliquots of the deprot,einized alkaline, freed of NH8 in z~acuo, and suitably diluted (1:50). Unidimensional paper chromatograms (run in water-saturated phenol, with HCl in the atmosphere) of the undiluted filtrates were used for t.he quantitative estimation of &nine, ~Zutamate, and aspartate, according to Giri and Rao (191. with separate calibration graphs for each amino acid. The amounts of amino N and of the individual amino acids formed from thr added keto acids were calculed by subtraction of the respective values determined in corresponding cont,rol samples incubated with all supplements except keto acid. The analytical results are expressed as micromoles/g. fresh liver tissue. The concentrate of enzymic fluorocitrate was obtained by incubation of fluoro acetate and fumarate with guinea pig kidney homogenate, fractional precipitation of Pb salts, and elution of the tricarboxylate zone from paper chromatograms (14). The concentrate probably contained less t,han 1% fiuorocitric acid. PRESEKTATION
In
AND
Discussion
OF E:XPERIMEXTAL
REYXLTS
the experiments with enzymic fluorocitrate (ICE), arbit,rary (0.2-1.0 ml.) of the aqueous solut,iolls of the tricarhoxylic acid fraction eluted from paper chromatograms were used. Their content of fluorocitrate (FC) and of citrate has not been determined; but when cornparing t,he effects of FCE with those of synthetic fluorocit,rate (KX), the presence of significant amounts of citrate in t,he former preparatiorl should be taken into account. amounts
FLUOROCITRATE
AND
AMINO
ACID
SYNTHESIS
IN
LIVER
637
The effects of different concentrations of FCS (2-15 pg. per test sample) on the synthesis of amino acids from ammonia and pyruvate or ketoglutarate in rat liver homogenate are charted in Fig. 1, the curves representing the percentage degree of inhibition of total NH2 N formation. It will be seen that the amination of pyruvate is inhibited to the extent of 46 % by 2 pg. FCS and of 75 % by 10 pg. FCS, no further increase of inhibition being obtained with higher concentrations of fluorocitrate. With cr-ketoglutarate, a very slight inhibition (lo-11 %) of amino acid synthesis is observed, which is constant at all tested FCS concentrations (cf. Table I, Expt. 3). This slight inhibition effect is probably due to the increased rate of oxidation of ketoglutarate in the FC-poisoned homogenate (see below). In the following experiments FCS was employed at a level of 10 pg. per test sample. Table I summarizes the data of a number of experiments concerning the effects of FCE and FCS on the amination of pyruvate by stabilized rat liver homogenate. It can be seenthat the total NH2 N formation from NH, and pyruvate is reduced by 62-87 % in the presence of FCE or FCS; the synthesis of alanine is inhibited to a lesser extent (usually 45-65 %), owing to the total suppression by FC of the formation of glutamate and aspartate (or to the conversion of these amino acids to alanine). Experiments l-3 show that a supplement of 20 pmoles glutamic acid causes a substantial increas of NH2 N and alanine formation (and some additional formation of glutamate and aspartate) from pyruvate in the absence of FC; in the FC-poisoned homogenate supplemented with gluInhib. % 100
I
80
XPU
60
20 0
xKG 2
FIG. 1. Effects of increasing (PU) and wketoglutarate (KG) formation, in per cent.)
5
IO
15 IL9 FC
amounts of FC on the amination in rat liver homogenate. (Inhibition
of pyruvate of NH2 N
of Fluorocitrate
TABLE
I
2.
-
-
FCK
FCE
-
6.2 ml. FCE
FCE
1 ml. FCE
-
Fluorbcitrate
-i
I
/ :
Other
supplements
Found
after With
incubation, pyruvate
&l/g.
-1Z 0 -3t
+2: -‘
I
,:i -2021+153j )' +q+123
-6’+3: -15 -1t
+li )ii t237 +132’+41 ;'i k105,+153;-13i-2(
‘176l+119/+30 +31 +43 /
_- II--,--,-I!
1208 +85~+46 b168 +200;-44
A due to pyruvate, PM/g.
+115 +130%
-49 -65% -
-122 -6%l
-122 -61%
$21 1-G -SO%,
+25%
-18
T
-34
--78-50-
-64
-18
I
--90-26
-23
-145 / -72 -30 - 82% - 60% --
-ri-T
-40 -.Wz
-98 -88%
in
percent
h due to FC (with pyruvate), b&/g., and
on the Synthesis of Amino Acids from Pyruvate in Rat Liver Homogenate (FCE, enzymic FC; FCS, synthetic FC) All samples contain homogenate (= 200 mg. liver), (NH,)&Ol , ATP, Mg++, K+, and EDTA as indicated General; Procedure; Na pyruvate, 80 PM per sample; other supplements as shown in table; incubation time, 1 hr.
Effect
P p
FLUOROCITRATE
AND
i _f 1 f__-
AMINO
ACID
I T .-
I
.-
SYNTHESIS
1N LIVER
639
(ilO
RRAUNSTEIN
AND
AZARKH
tamate, the inhibitory effect of FC on the formation of total NH% 3 j21-51%) is much smaller than with pyruvatc alone, whereas the forma-
t ion of alanine is even markedly increased (by 48- 130 70) by the addition of FC, which also induces the disappeamnce of considerable amounts of glutamic acid. Supplements of 10 pmoles glutamate or cw-ketoglutarate (,Expt. 2) result in similar but smaller effects. With a supplement of citrate (20 or 10 pmoles; Table I, Expts. 4 and <5) significant increases of total NH2 N are observed in the control test without pyruvate (due to the synthesis of glutamate) ; in the samples with pyruvate t’he addition of citrate induces a very large extrasyllthesix of NH2 N, alanine, glutamate, and aspartate, greatly exceeding the clalclllated additive effect [cf. simila,r results in experiments with liver slicncs, reported by Kritxmann (3)]. In the pyruvate samples supplemented with citrate, FC markedly inhibits total NH2 N formation (by 50-TO%), since it prevents the format’ion, or accumulation, of glutamate and aspartate; yet the absoluk: values of KHZ N formation are much higher than w&h pyruvate + I?(’ without citrate. This is due to the fact that) IX! causes no substantial inhibition, or even an augmentation, of the increased alanine formation in the pyruva,te + cit’rate tests. The c$fccts of supplementat,ion with 10 pmoles Lu-ketoglutarate (Expt. 2) RI’P similar t.o those obtained with caitrate, but somewhat less marked. The experimental data here reported clearly indicate, in full agrechment with our former findings, that the amination of pyruvatc ill mammalian (and avian) liver tissue proceeds by way of transreaminatiolt. This process is profoundly impaired when the formation of ketoglutaratc is inhibited with IX, or w&h fluoroacet,ate as in our previous esperimerits,” and can be restored by the addition of ketoglutarate or it,s prc(‘ursors (glut’amate, citrate) to t’he FC-inhibited homogenates. The restitution effects on total NH2 S formation arc more &I~OIIstrative with glutamate, since it, does not act, as a direct, acceptor of amino nitrogen, while those on alanine synthesis can tletter he nlade evident with ketoglutarate or citrate, hec~ause thty are not. dire{+ SlI, donors for transamination. Owing to the complicated interactlions bet.wecn the metaholites in the z Prof. R. A. Peters (personal communication) admits the possibility that I’(’ is indeed beiny formed from Auoroacetate in vitro in liver tissue as in the tissue of other organs [in contrast to the failure of the liver irk ciao t,o form FC” from itrjetted fluoroacetate (IS)].
FLUOROCITRATE
AND
AMINO
ACID
SYNTHESIS
IN
LIVER
641
incubation systems, there are certain difficulties in interpreting some of the effects shown in Table I. Most of the intricacies can, however, readily be accounted for on the basis of the following considerations. 1. It will be noted that there is no perfect balance between the A values for total NH2 N and for the sum of alanine, glutamate, and aspartate. This is due to the changes of other (mainly endogenous) sources of NH2 N which have not been estimated, and partly to the relatively large analytical errors in determining small amounts of the single amino acids by quantitative paper chromatography (about f5 pmoles/g. for each value). 2. The blocking of citrate oxidation is never complete, even with high concentrations of FC; about 30%, or more, of the citrate fail to be trapped and can be oxidized [cf. the data of Peters et al. (14)] and aminated (Table I, Exp. 5) in the presence of FC. This explains the partial restitution by citrate of transreamination in homogenates inhibited with FC. 3. It is, evidently, the rate of oxidation of pyruvate to ketoglutarate via the tricarboxylic acid cycle which limits the over-all rate of amino acid synthesis from pyruvate in surviving liver tissue. That is why ketoglutarate or its precursors increase the synthesis of NH2 N and of the individual amino acids from pyruvate even in the absence of FC. 4. In the experiments here reported and in others, we regularly observed that the addition of citrate favors the formation of aspartate from ammonia and pyruvate; this effect is suppressed by FC. It seems possible that oxidation of isocitrate to ketoglutarate by the “isocitric enzyme” (20) results in liberation of CO2 at a particularly appropriate topographical site or in an activated form (e.g., a carbonate-enzyme complex), so that it is more readily utilized for the carboxylation of pyruvate than the ordinary carbonate of the cell content and suspension medium. Increased amounts of oxalacetate would thus become available for transreamination to aspartate and for the synthesis of citrate. The suggested mechanism of CO2 “recycling” would be of importance in speeding up the over-all rate of the normally integrated oxidation of pyruvate in the respiratory tricarboxylic acid cycle. It might also account for the high efficiency of supplementation with citrate (usually higher than with ketoglutarate) in stimulating the synthesis of amino acids from pyruvate in noninhibited liver tissue. 5. The oxidation of citrate (and of its precursors) most probably exerts a sparing action (by competition for the respiratory electron- and
642
BRAUNSTEIN
AND AZARKH
hydrogen-transporting systems) on the simultaneous oxidation of ketoglutarate and glutamate. FC interferes with this sparing effect and, by accelerating the oxidation of ketoglutarate and slowing down the carboxylation and oxidation of pyruvate (vide supra), causes a shift in transamination equilibra which favors the disappearance of glutamate and accumulation of alanine in t,he FC-containing test systems (see Table I). SUMMARY
The effects of synthetic and enzymic fluorocitrate (FC) on the synthesis of amino acids from ammonia and pyruvate in stabilized homogenates of rat liver are similar to the effect of fluoroacetate; this provides evidence in support of the capacity of liver tissue in vitro to form FC from fluoroacetate. FC greatly inhibits the total formation of NH2 N and the synthesis of alanine from pyruvate in liver homogenate. The increased synthesis of NH2 N from pyruvate in samples supplemented with small amounts of glutamate (or ketoglutarate) is inhibited by FC to a lesser extent, whereas the synthesis of alanine is even increased by FC in the presence of pyruvate and glutamate. Owing to the incomplete blocking of the oxidation of citrate by PC, citrate is active in restoring the synthesis of alanine, and t,o some ext,cnt of total NH2 N, from pyruvate in FC-poisoned homogenate. The experimental results provide additional proof that the amination of pyruvate in liver proceeds by the indirect path of bransreamination via glutamic acid. The intricate metabolic interactions in the test systems are discussed, and the suggestion is made that recycling of CO2 liberated by the “isocitric enzyme” may be of importance in speeding up both the over-all oxidation of pyruvate in the normal respiratory cycle and its indirect amination in liver. REFERENCES 1. BRAUNSTEIN, A. E., Advances in Protein Chem. 3, 1 (1947); “The Paths of Assimilation and Dissimilation of Nitrogen in Animals,” 12th Annual A. Bach Lecture. Acad. Press of U.S.S.R., Moscow, 1957; Advances in Enzymol. 19, in press (1957). 2. AZARHH, R. M., BRAUNSTEIN, A. E., AND KLUGE, I. V., VZZZth Congr. Physiol., Biochem. and Pharmacol. U.S.S.R., Kiev, May 1955; Abstr. Communs., Moscow, 1966, pp. 11, 89, 303.
FLUOROCTIRATE
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
AND
AMINO
ACID
SYNTHESIS
IN
LIVER
643
KRITZMANN, M. G., J. Biol. Chem. 167,77 (1947). BERIOZOV, T. T., Doklady Akad. Nauk S.S.S.R. 86, 603 (1952); 96,623 (1953). VOLOVNIK, B. YA., Biokhimiya 20, 490 (1955). SINITSINA, A. L., Biokhimiya 19,89 (1954). KLUOE, I. V., Biokhimiya 21, 516 (1956). WISS, O., Helv. Chim. Acta 31, 1189 (1948). KAPLANSKY, S. YA., et al., Biokhimiya 10, 296, 401 (1945); 21, 119 (1956). CEDRANOOLO, F., “Le Transaminazioni.” Giorn. Biochim. Italo-FrancoElvet., Ed. Consil. Nazion. di Ricercha, Roma, 1954. KRETOVICH, V. L., “Fundamentals of Plant Biochemistry” (Russ.), 2nd ed., “Soviet Science” State Publ. House, Moscow, 1956. RYMAN, B. E., AND WALSH, E. F., Biochem. J. 56,191 (1954). QUASTEL, J. H., AND SCHOLEFIELD, P. G., J. Biol. Chem. 214,245 (1955). BUFFA, P., PETERS, R. A., AND WAKELIN, R. W., Biochem. J. 46, 467 (1951) ; (cf. Proc. Roy. Sot. (London). B140.497 1953). MORRISON, J. F., AND PETERS, R. A., Biochem. J. 56, 473 (1954). GAL, E. M., PETERS, R. A., AND WAKELIN, R. W., Biochem. J. 64, 161 (1956). CANZANELLI, A., RAPPORT, D., AND GUILD, R., J. Biol. Chem. 133,291 (1950). COCKING, E. C., AND YEMM, E. W., Biochem. J. 66, xii (1954). GIRI, K. W., AND RAO, N. A. N., J. Indian Inst. Sci. 36,343 (1953). MOYLE, J., AND DIXON, M., Biochem. J. 63, 549, 552 (1956).
OF BIOCHEMISTRY
AND
BIOPHYSICS
69, 634-643 (19%)
The Effect of Fluorocitrate on the Synthesis of Amino Acids from Ammonia and a-Keto Acids in Rat Liver Homogenate A. E. Braunstein
and R. M. Azarkh
From the Institute of Biological and Medical Chemistry, .Icademy of Medical Sciences of the Union of Soviet Socialist Republics, Noscow, IrS2T.R.
Received January 15, 1957
The importance of indirect amination by the joint action of glutamica dehydrogenase and aminopherases in t,he biosynt,hesis of amino acids from their keto analogs and ammonia had first been suggested in 19X8-N by Euler and by Braunstein. In this laboratory and elsewhere, much txperiment’al evidence has been obtained in support of the predominant role of this mechanism, or ‘%ransreamination” (1, 2), in the conversion of ammonia to amino acids by animals (l-7)) microorganisms, and plants. It is almost generally accepted, at present, that glutamic acid is the primary product of assimilation of inorganic nitrogen in animals, plants, and most bacterial species. But several authors still hold the opinion that alanine and aspartic acid, and possibly other amino acids, are formed from their keto analogs by direct amination, rather than via glut’amic. acid, in the tissues of animals (8-10) and of plants (II). It has recently been shown in this laboratory that the rates of amination of keto acids, except a-ketoglutaric acid, by surviving liver or kidney tissue of rats and birds are greatly lowered when the activit,y of aminopherases in the tissue is reduced, either as a result of ppridoxme deficiency (6) or by the blocking of pyridoxal enzymes with rarbonyl reagents (1, 2). Transamination is depressed to very low levels in the liver of rats depleted in pyridoxal phosphate 11y repeated administration of heavy doses of isoniazid (1). Homogenates of such livers readily aminate ketoglutarate, but they synthesize almost no alanine and only small amounts of aminodicarboxylic acids from pyruvate and ammonia : 634
FLUOROCITRATE AND AMINO ACID SYNTHESIS IN LIVER
635
their capacity to form alanine is restored by supplementation of the incubation mixtures with purified glutamic-alanine aminopherase (1). it has also been shown that steric inversion of the n-isomers of essential mine acids is impaired in the living pyridoxine-deficient rat, owing to Jure of reamination of the cr-keto acids (4, 5). In full accord with the theory of transreamination, it could be demonitnbted (1,2) that the synthesis of amino acids from pyruvate or oxalacete in mammalian or avian liver and kidney tissue preparations is o,j.eatly depressed if the oxidative formation of ketoglutarate via the D citric acid cycle is blocked with the aid of appropriate specific inhibitors, e.g., with fluoroacetate (14), cocaine (12), or mesotartrate (13). The direct amination of ketoglutarate is not impaired, as a rule, under these conditions. In such experiments the amination of pyruvate and oxalacetate could be restored, as expected, by supplementation of the incubation samples with glutamate or with such intermediates of the citric acid cycle, the conversion of which to ketoglutarate had not been blocked (1, 2). We have extensively used fluoroacetate in experiments on the synthesis of amino acids in liver homogenates. The effects of fluoroacetate were considered as due to the enzymic formation of fluorocitrate, resulting in specific inhibition of the conversion of citric acid to ketoglutarate [owing to blocking of aconitase (15)]. Doubt has been cast on this interpretation by a recent investigation from R. A. Peters’ laboratory (16), tending to show that in liver fluoroacetate is not transformed into fluorocitrate. We have therefore made some further experiments on the amination of keto acids in rat liver homogenates, using concentrates of biosynthetic fluorocitric acid (14) or pure synthetic fluorocitrate.’ These experiments are reported below. Their results were quite similar to those obtained formerly with fluoroacetate; they confirm the view that the synthesis of amino acids from ammonia and keto acids, other than ketoglutarate, in animal tissues proceeds by way of transreamination. EXPERIMENTAL
General Procedure and Analytical
Methods
Determinations were made of the increase in total NH2 nitrogen and in individual amino acids upon incubation of stabilized rat liver homogenates with a-keto acids and ammonium carbonate. The livers were taken from well-fed young male albino rats weighing SO-120 g. 1 We wish to express our gratitude gift of synthetic sodium fluorocitrate.
to Professor R. A. Peters for the generous
Gxi
BRAUNSTEIN
AND
AZARKH
As formerly found by the authors, the rates of a.mino acid synthesis in rat tissues are markedly affected by the age and origin (strain) of the animals, by their nutritional state (a generous supply of protein and vitamins rather than of carbohydrate [cf. (17)l seems to be especially important.), and by seasonal influencer (low rates of amination in spring and early summer). In experiments with homogenates it is important to st,abilize the mitochondri:l (i.e., the enzyme systems of the respiratory tricarboxylic cycle) wit,h ATP, Mg+. I<+, and ethylenediamine tetraacetate (EDTA), and carefully to adjust the pH of the incubation mixture (optimal range: pH 7.0-7.2). The experimental procedure was as follows: the rat, livers were cooled in iced 1.15% KC1 solution and homogenized 2 min. in a Potter-Elvehjem glass homogcsizer with 6.5 vol. of buffer solution (pH 7.2) containing0.1 M K phosphate. 1.15% KCl, and 4 X low3 M disodium EDTA. Theexperimental samples contained, in a total volume of 3 ml. : 1.5 ml. homogen ate (= 200 mg. liver tissue), 2 X 1OW M ATP, 2 X lo-” M MgS04 , 3 X lo+ dl (KH4)&03 The keto acids (Ka pyruvate or Sa ketoglutarate) were added at :I level of 80 pmoles per sample. Before the addition of ammonium carbonate anti keto acid the samples were left in cont,act wit,h fluorocitrat.e (resp. with plain bllf fer) at room temperature for 5-10 min. The samples were incubated with shaking in 0% for 60-80 min. at 3X”, and fiard with trichloroacetic acid. Total amino N was determined spectrophotometrically by the ninhydrin filtrates, madr method of Cocking and Yemm (18) in aliquots of the deprot,einized alkaline, freed of NH8 in z~acuo, and suitably diluted (1:50). Unidimensional paper chromatograms (run in water-saturated phenol, with HCl in the atmosphere) of the undiluted filtrates were used for t.he quantitative estimation of &nine, ~Zutamate, and aspartate, according to Giri and Rao (191. with separate calibration graphs for each amino acid. The amounts of amino N and of the individual amino acids formed from thr added keto acids were calculed by subtraction of the respective values determined in corresponding cont,rol samples incubated with all supplements except keto acid. The analytical results are expressed as micromoles/g. fresh liver tissue. The concentrate of enzymic fluorocitrate was obtained by incubation of fluoro acetate and fumarate with guinea pig kidney homogenate, fractional precipitation of Pb salts, and elution of the tricarboxylate zone from paper chromatograms (14). The concentrate probably contained less t,han 1% fiuorocitric acid. PRESEKTATION
In
AND
Discussion
OF E:XPERIMEXTAL
REYXLTS
the experiments with enzymic fluorocitrate (ICE), arbit,rary (0.2-1.0 ml.) of the aqueous solut,iolls of the tricarhoxylic acid fraction eluted from paper chromatograms were used. Their content of fluorocitrate (FC) and of citrate has not been determined; but when cornparing t,he effects of FCE with those of synthetic fluorocit,rate (KX), the presence of significant amounts of citrate in t,he former preparatiorl should be taken into account. amounts
FLUOROCITRATE
AND
AMINO
ACID
SYNTHESIS
IN
LIVER
637
The effects of different concentrations of FCS (2-15 pg. per test sample) on the synthesis of amino acids from ammonia and pyruvate or ketoglutarate in rat liver homogenate are charted in Fig. 1, the curves representing the percentage degree of inhibition of total NH2 N formation. It will be seen that the amination of pyruvate is inhibited to the extent of 46 % by 2 pg. FCS and of 75 % by 10 pg. FCS, no further increase of inhibition being obtained with higher concentrations of fluorocitrate. With cr-ketoglutarate, a very slight inhibition (lo-11 %) of amino acid synthesis is observed, which is constant at all tested FCS concentrations (cf. Table I, Expt. 3). This slight inhibition effect is probably due to the increased rate of oxidation of ketoglutarate in the FC-poisoned homogenate (see below). In the following experiments FCS was employed at a level of 10 pg. per test sample. Table I summarizes the data of a number of experiments concerning the effects of FCE and FCS on the amination of pyruvate by stabilized rat liver homogenate. It can be seenthat the total NH2 N formation from NH, and pyruvate is reduced by 62-87 % in the presence of FCE or FCS; the synthesis of alanine is inhibited to a lesser extent (usually 45-65 %), owing to the total suppression by FC of the formation of glutamate and aspartate (or to the conversion of these amino acids to alanine). Experiments l-3 show that a supplement of 20 pmoles glutamic acid causes a substantial increas of NH2 N and alanine formation (and some additional formation of glutamate and aspartate) from pyruvate in the absence of FC; in the FC-poisoned homogenate supplemented with gluInhib. % 100
I
80
XPU
60
20 0
xKG 2
FIG. 1. Effects of increasing (PU) and wketoglutarate (KG) formation, in per cent.)
5
IO
15 IL9 FC
amounts of FC on the amination in rat liver homogenate. (Inhibition
of pyruvate of NH2 N
of Fluorocitrate
TABLE
I
2.
-
-
FCK
FCE
-
6.2 ml. FCE
FCE
1 ml. FCE
-
Fluorbcitrate
-i
I
/ :
Other
supplements
Found
after With
incubation, pyruvate
&l/g.
-1Z 0 -3t
+2: -‘
I
,:i -2021+153j )' +q+123
-6’+3: -15 -1t
+li )ii t237 +132’+41 ;'i k105,+153;-13i-2(
‘176l+119/+30 +31 +43 /
_- II--,--,-I!
1208 +85~+46 b168 +200;-44
A due to pyruvate, PM/g.
+115 +130%
-49 -65% -
-122 -6%l
-122 -61%
$21 1-G -SO%,
+25%
-18
T
-34
--78-50-
-64
-18
I
--90-26
-23
-145 / -72 -30 - 82% - 60% --
-ri-T
-40 -.Wz
-98 -88%
in
percent
h due to FC (with pyruvate), b&/g., and
on the Synthesis of Amino Acids from Pyruvate in Rat Liver Homogenate (FCE, enzymic FC; FCS, synthetic FC) All samples contain homogenate (= 200 mg. liver), (NH,)&Ol , ATP, Mg++, K+, and EDTA as indicated General; Procedure; Na pyruvate, 80 PM per sample; other supplements as shown in table; incubation time, 1 hr.
Effect
P p
FLUOROCITRATE
AND
i _f 1 f__-
AMINO
ACID
I T .-
I
.-
SYNTHESIS
1N LIVER
639
(ilO
RRAUNSTEIN
AND
AZARKH
tamate, the inhibitory effect of FC on the formation of total NH% 3 j21-51%) is much smaller than with pyruvatc alone, whereas the forma-
t ion of alanine is even markedly increased (by 48- 130 70) by the addition of FC, which also induces the disappeamnce of considerable amounts of glutamic acid. Supplements of 10 pmoles glutamate or cw-ketoglutarate (,Expt. 2) result in similar but smaller effects. With a supplement of citrate (20 or 10 pmoles; Table I, Expts. 4 and <5) significant increases of total NH2 N are observed in the control test without pyruvate (due to the synthesis of glutamate) ; in the samples with pyruvate t’he addition of citrate induces a very large extrasyllthesix of NH2 N, alanine, glutamate, and aspartate, greatly exceeding the clalclllated additive effect [cf. simila,r results in experiments with liver slicncs, reported by Kritxmann (3)]. In the pyruvate samples supplemented with citrate, FC markedly inhibits total NH2 N formation (by 50-TO%), since it prevents the format’ion, or accumulation, of glutamate and aspartate; yet the absoluk: values of KHZ N formation are much higher than w&h pyruvate + I?(’ without citrate. This is due to the fact that) IX! causes no substantial inhibition, or even an augmentation, of the increased alanine formation in the pyruva,te + cit’rate tests. The c$fccts of supplementat,ion with 10 pmoles Lu-ketoglutarate (Expt. 2) RI’P similar t.o those obtained with caitrate, but somewhat less marked. The experimental data here reported clearly indicate, in full agrechment with our former findings, that the amination of pyruvatc ill mammalian (and avian) liver tissue proceeds by way of transreaminatiolt. This process is profoundly impaired when the formation of ketoglutaratc is inhibited with IX, or w&h fluoroacet,ate as in our previous esperimerits,” and can be restored by the addition of ketoglutarate or it,s prc(‘ursors (glut’amate, citrate) to t’he FC-inhibited homogenates. The restitution effects on total NH2 S formation arc more &I~OIIstrative with glutamate, since it, does not act, as a direct, acceptor of amino nitrogen, while those on alanine synthesis can tletter he nlade evident with ketoglutarate or citrate, hec~ause thty are not. dire{+ SlI, donors for transamination. Owing to the complicated interactlions bet.wecn the metaholites in the z Prof. R. A. Peters (personal communication) admits the possibility that I’(’ is indeed beiny formed from Auoroacetate in vitro in liver tissue as in the tissue of other organs [in contrast to the failure of the liver irk ciao t,o form FC” from itrjetted fluoroacetate (IS)].
FLUOROCITRATE
AND
AMINO
ACID
SYNTHESIS
IN
LIVER
641
incubation systems, there are certain difficulties in interpreting some of the effects shown in Table I. Most of the intricacies can, however, readily be accounted for on the basis of the following considerations. 1. It will be noted that there is no perfect balance between the A values for total NH2 N and for the sum of alanine, glutamate, and aspartate. This is due to the changes of other (mainly endogenous) sources of NH2 N which have not been estimated, and partly to the relatively large analytical errors in determining small amounts of the single amino acids by quantitative paper chromatography (about f5 pmoles/g. for each value). 2. The blocking of citrate oxidation is never complete, even with high concentrations of FC; about 30%, or more, of the citrate fail to be trapped and can be oxidized [cf. the data of Peters et al. (14)] and aminated (Table I, Exp. 5) in the presence of FC. This explains the partial restitution by citrate of transreamination in homogenates inhibited with FC. 3. It is, evidently, the rate of oxidation of pyruvate to ketoglutarate via the tricarboxylic acid cycle which limits the over-all rate of amino acid synthesis from pyruvate in surviving liver tissue. That is why ketoglutarate or its precursors increase the synthesis of NH2 N and of the individual amino acids from pyruvate even in the absence of FC. 4. In the experiments here reported and in others, we regularly observed that the addition of citrate favors the formation of aspartate from ammonia and pyruvate; this effect is suppressed by FC. It seems possible that oxidation of isocitrate to ketoglutarate by the “isocitric enzyme” (20) results in liberation of CO2 at a particularly appropriate topographical site or in an activated form (e.g., a carbonate-enzyme complex), so that it is more readily utilized for the carboxylation of pyruvate than the ordinary carbonate of the cell content and suspension medium. Increased amounts of oxalacetate would thus become available for transreamination to aspartate and for the synthesis of citrate. The suggested mechanism of CO2 “recycling” would be of importance in speeding up the over-all rate of the normally integrated oxidation of pyruvate in the respiratory tricarboxylic acid cycle. It might also account for the high efficiency of supplementation with citrate (usually higher than with ketoglutarate) in stimulating the synthesis of amino acids from pyruvate in noninhibited liver tissue. 5. The oxidation of citrate (and of its precursors) most probably exerts a sparing action (by competition for the respiratory electron- and
642
BRAUNSTEIN
AND AZARKH
hydrogen-transporting systems) on the simultaneous oxidation of ketoglutarate and glutamate. FC interferes with this sparing effect and, by accelerating the oxidation of ketoglutarate and slowing down the carboxylation and oxidation of pyruvate (vide supra), causes a shift in transamination equilibra which favors the disappearance of glutamate and accumulation of alanine in t,he FC-containing test systems (see Table I). SUMMARY
The effects of synthetic and enzymic fluorocitrate (FC) on the synthesis of amino acids from ammonia and pyruvate in stabilized homogenates of rat liver are similar to the effect of fluoroacetate; this provides evidence in support of the capacity of liver tissue in vitro to form FC from fluoroacetate. FC greatly inhibits the total formation of NH2 N and the synthesis of alanine from pyruvate in liver homogenate. The increased synthesis of NH2 N from pyruvate in samples supplemented with small amounts of glutamate (or ketoglutarate) is inhibited by FC to a lesser extent, whereas the synthesis of alanine is even increased by FC in the presence of pyruvate and glutamate. Owing to the incomplete blocking of the oxidation of citrate by PC, citrate is active in restoring the synthesis of alanine, and t,o some ext,cnt of total NH2 N, from pyruvate in FC-poisoned homogenate. The experimental results provide additional proof that the amination of pyruvate in liver proceeds by the indirect path of bransreamination via glutamic acid. The intricate metabolic interactions in the test systems are discussed, and the suggestion is made that recycling of CO2 liberated by the “isocitric enzyme” may be of importance in speeding up both the over-all oxidation of pyruvate in the normal respiratory cycle and its indirect amination in liver. REFERENCES 1. BRAUNSTEIN, A. E., Advances in Protein Chem. 3, 1 (1947); “The Paths of Assimilation and Dissimilation of Nitrogen in Animals,” 12th Annual A. Bach Lecture. Acad. Press of U.S.S.R., Moscow, 1957; Advances in Enzymol. 19, in press (1957). 2. AZARHH, R. M., BRAUNSTEIN, A. E., AND KLUGE, I. V., VZZZth Congr. Physiol., Biochem. and Pharmacol. U.S.S.R., Kiev, May 1955; Abstr. Communs., Moscow, 1966, pp. 11, 89, 303.
FLUOROCTIRATE
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
AND
AMINO
ACID
SYNTHESIS
IN
LIVER
643
KRITZMANN, M. G., J. Biol. Chem. 167,77 (1947). BERIOZOV, T. T., Doklady Akad. Nauk S.S.S.R. 86, 603 (1952); 96,623 (1953). VOLOVNIK, B. YA., Biokhimiya 20, 490 (1955). SINITSINA, A. L., Biokhimiya 19,89 (1954). KLUOE, I. V., Biokhimiya 21, 516 (1956). WISS, O., Helv. Chim. Acta 31, 1189 (1948). KAPLANSKY, S. YA., et al., Biokhimiya 10, 296, 401 (1945); 21, 119 (1956). CEDRANOOLO, F., “Le Transaminazioni.” Giorn. Biochim. Italo-FrancoElvet., Ed. Consil. Nazion. di Ricercha, Roma, 1954. KRETOVICH, V. L., “Fundamentals of Plant Biochemistry” (Russ.), 2nd ed., “Soviet Science” State Publ. House, Moscow, 1956. RYMAN, B. E., AND WALSH, E. F., Biochem. J. 56,191 (1954). QUASTEL, J. H., AND SCHOLEFIELD, P. G., J. Biol. Chem. 214,245 (1955). BUFFA, P., PETERS, R. A., AND WAKELIN, R. W., Biochem. J. 46, 467 (1951) ; (cf. Proc. Roy. Sot. (London). B140.497 1953). MORRISON, J. F., AND PETERS, R. A., Biochem. J. 56, 473 (1954). GAL, E. M., PETERS, R. A., AND WAKELIN, R. W., Biochem. J. 64, 161 (1956). CANZANELLI, A., RAPPORT, D., AND GUILD, R., J. Biol. Chem. 133,291 (1950). COCKING, E. C., AND YEMM, E. W., Biochem. J. 66, xii (1954). GIRI, K. W., AND RAO, N. A. N., J. Indian Inst. Sci. 36,343 (1953). MOYLE, J., AND DIXON, M., Biochem. J. 63, 549, 552 (1956).