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Catabolism of threonine in mammals by coupling of l -threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase
496
Biochimica et Biophysica Acta, 544 (1978) 496--503 © Elsevier/North-Holland Biomedical Press
BBA 28769
CATABOLISM OF THREONINE IN MAMMALS BY COUPLING OF L-THREONINE 3.DEHYDROGENASE WITH 2-AMINO-3-OXOBUTYRATE-CoA LIGASE
ROBERT A. DALE
Department of Biochemistry, West Middlesex Hospital, Isleworth, Middlesex TW7 6AF
(U.K.) (Received May 29th, 1978)
Summary There is doubt about the L-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of L-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. L-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing L-threonine without and with CoA and oxaloacetate; L-[U-14C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from L-threonine to 10--14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised L-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate {precursor of aminoacetone), which is produced from L-threonine by L-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.
Introduction Degradation of dietary threonine to glycine and acetate in a mammal was first shown by Meltzer and Sprinson [1] who fed rats with labelled threonine and observed labelled glycine and acetyl radicals in the urine. It seemed feasible that threonine aldolase (EC 2.1.2.1) catalysed the reaction, but doubt was
497 expressed about this mode of degradation [2]. Threonine was found to be catabolised to glycine and CoASAc by Arthrobacter 9759 [3], the metabolic pathway involving coupling of L-threonine 3-dehydrogenase (EC 1.1.1.103), which produces 2-amino-3-oxobutyrate, with 2-amino-3-oxobutyrate-CoA ligase ("aminoacetone synthase") which catalyses the formation of glycine and CoASAc from its substrates. The knowledge that guinea-pig liver contains an active threonine dehydrogenase and "aminoacetone synthase" [4] led the author to investigate the possibility that in mammals there is a catabolic pathway of threonine similar to that in Arthrobacter 9759. Materials and Methods
Materiah. L-[U-14C]Threonine was obtained from The Radiochemical Centre, Amersham, U.K. and aconitase (EC 4.2.1.3), cytochrome c (type 111) and alcohol dehydrogenase (EC 1.1.1.1) were from Sigma London Chemical Co. Ltd., Kingston-upon-Thames, U.K. Aminoacetone was a gift from Professor A. Neuberger. All other chemicals, reagents and materials were of analytical reagent grade or of the highest quality available. Mitochondria Mitochondria were prepared for aminoacetone and citrate assays from livers of guinea-pigs [5] and were resuspended in 2 vols. 2 mM potassium phosphate, pH 7.4, frozen at approx. --70°C and stored at --20°C. The disrupted mitochondria were separated into supematant (matrix) and pellet (membranes) after centrifuging at 32 000 X g for 5 rain at 0°C. The pellet was restored to the volume of the mitochondria by re-suspending in 10 mM potassium phosphate, pH 7.6, for marker enzymes or in 40 mM Tris-HC1, pH 8.2, for L-threonine 3-dehydrogenase and 2-amino-3-oxobutyrate-CoA ligase. Outer membranes were removed by the digitonin method [5] and the residues of the mitochondria were resuspended and frozen as described above (3 preparations) together with non-digitonin-treated mitochondria. Mitochondria prepared for autoradiography, some aminoacetone assays and threonine aldolase assays (guinea-pig liver and kidney) [6] were centrifuged each time at 8500 X g for 10 rain. The mitochondria used for autoradiography and aminoacetone assays were resuspended and stored as described above. Mitochondria for threonine aldolase assay were stored at --20°C and prior to assay they were resuspended in 3 vols. 5 mM sodium phosphate, pH 7.4, frozen and thawed; supematant was obtained by centrifuging for 15 rain at 32 000 X g. The supernatant of the first mitochondrial centrifuging was centrifuged for 15 rain at 34 000 X g and the supernatant (cytosol) was stored at --20°C. Enzyme assays. Protein was assayed with crystalline bovine serum albumin as standard [7]. L-Threonine 3-dehydrogenase from guinea-pig liver was assayed in a volume of 0.8 ml containing 37.5 mM Tris-HC1 buffer, pH 8.2, 2.5 mM MgC12, 1.25 mM L-threonine and 50 ~1 of once-frozen and thawed disrupted mitochondria; 1 mM NAD was used to start the reaction, but was omitted from controls. The mixture was incubated for 20 or 30 min (Table I) at 37°C, being shaken 120 times/min. (30 mM sodium phosphate buffer, pH 7.7, was used instead of Tris-HC1 buffer for 14C experiments and 2 experiments in Table I). Reactions were stopped by adding 0.25 ml of 5% HC104. After centrifuging, the supernatants were removed and assayed for aminoacetone
498 [8,9], which is formed by spontaneous decarboxylation of 2-amino-3-oxobutyrate [10], and citrate [11]. The presence of 2-amino-3-oxobutyrate-CoA ligase was assessed by including 1.0 mM CoA [3] and 1.1 or 2.2 mM oxaloacetate (for citrate formation via endogenous citrate synthase) in the reaction mixtures for the L-threonine 3-dehydrogenase assay and by observing the decrease in aminoacetone formed and the appearance of CoASAc as citrate. The supernatants for citrate assays were neutralised with KOH and stored at --20°C. The products of 2-amino-3oxobutyrate-CoA ligase (glycine and CoASAc) were further investigated by autoradiograms (see below) by adding 5 pCi of L-[U-~4C]threonine to reaction mixtures for the L-threonine 3-dehydrogenase and ligase assays (3 liver experiments, 1 kidney experiment). A portion of each supematant was used to assay aminoacetone and the remaining supernatant was neutralised with KOH and stored at --20°C for chromatography. Succinate-cytochrome c reductase was assayed as described by Werner and Neupert [12] and malate (EC 1.1.1.37) and isocitrate (EC 1.1.1.42) dehydrogenases were assayed by the method of Sottocasa et al. [13]. 0.5 mM MgCI2 was used instead of 0.1 mM MnC12 for the isocitrate dehydrogenase assay. Threonine aldolase was assayed by measuring the rate of formation of acetaldehyde. Cytoplasmic enzyme was assayed at 33°C in 2 ml containing 20 mM Tris-HC1 buffer, pH 7.4, 10 uM pyridoxal phosphate, 120 pM NADH, 50 gg of alcohol dehydrogenase, and 0.1 ml of cytosol. The reaction was started by adding either 20 mM DL-allothreonine or 80 mM L-threonine and the decrease in absorbance at 340 nm was observed. Mitochondrial enzyme was assayed in stoppered tubes in 2 ml containing 20 mM Tris-HC1 buffer, pH 7.4, 10 pM pyridoxal phosphate, 20 mM DL-allothreonine or 80 mM L-threonine and 0.1 ml of mitochondrial suspension or supernatant. Substrate was omitted from controls. After 10--30 rain incubation at 37°C reactions were stopped by chilling the tubes in ice and adding 0.1 ml of 3 M trichloroacetic acid. After centrifuging, the supernatant was removed and assayed for acetaldehyde [ 14,15]. Chromatography and autoradiography. Neutralised supernatants and relevant markers were applied to duplicate 3 MM sheets, one for staining and one for autoradiography. One-way chromatography for aminoacetone [16], threonine and glycine [17] and citrate [18] and two-way chromatography for threonine, glycine and citrate [18] were then carried out. Autoradiography was performed with a Kodak RP-X-Omat film. Citrate assay. Citrate was assayed by aconitase [11] which was activated as described by Sigma Chemical Co. The neutralised supernatants, after thawing, were kept at 0°C to precipitate KC104. Results
Aminoacetone formation from L-threonine 3-dehydrogenase Table I shows the m o u n t s of aminoacetone formed from L-threonine and NAD under different conditions. Incubation of aminoacetone (35 nmol) in control reaction mixtures (6 experiments) resulted in a mean recovery of 96%. The addition of CoA reduced the formation of aminoacetone (mean) to 10--14%. The addition of oxaloacetate (1.1 or 2.2 mM), without CoA, diminished
499 TABLE I F O R M A T I O N O F C I T R A T E ( V I A C o A S A c ) IN P L A C E O F A M I N O A C E T O N E BY ADDITION OF CoA AND/OR OXALOACETATE
FROM L-THREONINE
The assay conditions are described under Materials and Methods. Each experiment relates to a different mitochondrial preparation. The citrate produced from 2.2 mM oxaloacetate was not corrected for the a p p r o x . 1 0 % d e c r e a s e in A 3 4 0 n m . T h e r e s u l t s a r e e x p r e s s e d as m e a n w i t h t h e r a n g e in p a r e n t h e s i s . Conditions
Sodium phosphate (pH 7.7)
Incubation time (rain)
Number of experiments
n m o l p r o d u c t / 5 0 ~ul d i s r u p t e d mitochondria Aminoacetone
Citrate
30
2
--
--
133, 138 15, 16
---
192 86 20 14
(158--220) (62--121) (15--25) (10--21)
0 115 (71--168) 0 236 (200--254)
250 I18 34 29
(215--269) (84--153) (22--56) (17--46)
0 64 (56--71) 0 280 (258--302)
NAD NAD + CoA Tris-HCl ( p H 8 . 2 )
20
3
NAD NAD + oxaloaeetate (2.2 raM) NAD + CoA NAD + CoA + oxaloacetate (2.2 raM) Trls-HCI ( p H 8 . 2 ) NAD NAD + oxaloacetate (I.I mM) NAD + CoA NAD + CoA + oxaloaeetate (1.1 mM)
30
5
the formation of aminoacetone (mean) to about 47%. The addition of CoA in several pilot experiments with guinea-pig kidney mitochondria reduced the formation of aminoacetone to 40--50%.
Identification of products formed in place of aminoacetone via 2-amino-3-oxobu tyrate-CoA ligase (a) Chromatograms and autoradiograms. Reaction mixture supernatants from liver and kidney mitochondria incubated with L-[U-14C]threonine but without CoA and oxaloacetate produced strong identical aminoacetone spots on chromatograms and autoradiograms; glycine spots were just detectable and there were no citrate spots. Similar incubations with added CoA and oxaloacetate produced faint aminoacetone spots and well-defined identical glycine spots on the chromatograms and autoradiograms; the citrate spots on the autoradiograms were identical to the chromatographic markers. No citrate spots were obtained from kidney mitochondria (b) Citrate assay. (Table I). The citrate formed in the presence of CoA and/ or oxaloacetate is shown in parallel with the decrease in the formation of aminoacetone. In the last 5 experiments in the presence of CoA, decreasing the concentration of oxaloacetate to 0.6 mM reduced the citrate formed to 169 nmol (mean) whereas increasing the concentration of oxalacetate to 2.2 mM made little difference to the citrate formed, namely 258 nmol (mean). In the absence of CoA, increasing the concentration of oxaloacetate from 1.1 to 2.2 mM (last 5 experiments) increased the citrate formed from a mean of 64 nmol to 110 nmol. The mean and range of citrate formed from disrupted mitochondria and supernatants (Table II) were 245 (223--276) nmol and 272 (179--
41
13 ( ( 8 - - 2 3 )
3.6 ( 3 . 3 - - 4 . 0 )
0
3.8 (3.4---4.2)
20 ( 1 7 - - 2 5 )
93 (74--112)
91 ( 7 2 - - 1 0 8 )
Malate d e h y d r o g e n a s e
3.3
14
14
(2.5--4.6)
(10--20)
(11--21)
Isocitrate dehydrogenase
* T h e m i t o c h o n d r i a , s u p e r n a t a n t a n d p e l l e t w e r e e a c h a d d e d t o H 2 0 , Tris-HCl b u f f e r , Mg 2+ a n d L - t h r e o n i n e a t 0 ° C u n t i l all o t h e r r e a g e n t s w e r e a d d e d in o r d e r t o prevent the yield of a m i n o a c e t o n e decreasing m o r e than 20--30% due to the delay b e t w e e n thawing and incubation.
(23--61)
(7--38)
21
129
Supematant *
Pellet * (resuspended)
(98--157)
29 ( 1 4 - - 5 3 )
185 (140--230)
Succinate-cytochrome c reductase
C o A ligase
Mitochondria * (disrupted)
Marker enzymes
2-Amino-3oxobutyrate-
L - T h r e o n i n e 3debydrogenase
-Fraction
T h e results w e r e o b t a i n e d f r o m 5 m i t o c h o n d r i a l p r e p a r a t i o n s . T h e a s s a y c o n d i t i o n s are d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . 2 - A m i n o - 3 - o x o b u t y r a t e - C o A llgase was a s s a y e d in t h e p r e s e n c e o f C o A a n d 1.1 m M o x a l o a c e t a t e . T h e a c t i v i t i e s o f t h e m a r k e r e n z y m e s are e x p r e s s e d a s / ~ m o l / m i n p e r m l o f e a c h f r a c t i o n . T h e activities o f the e n z y m e s u n d e r i n v e s t i g a t i o n are e x p r e s s e d as n m o l a m i n o a c e t o n e f o r m e d / 3 0 r a i n p e r 50/~l o f e a c h f r a c t i o n . ( T h e r e a c t i o n r a t e w a s n o t l i n e a r w i t h t i m e o r t h e v o l u m e o f m i t o c h o n d r i a l f r a c t i o n , as a s s a y e d . ) T h e r e s u l t s a r e e x p r e s s e d as m e a n w i t h t h e r a n g e in p a r e n t h e s i s .
SUBMITOCHONDRIAL DISTRIBUTION OF L-THREONINE 3-DEHYDROGENASE AND 2-AMINO-3-OXOBUTYRATE-CoA LIGASE
T A B L E II
L •
O O
501 314) nmol, respectively; a trace of citrate only was produced by the pellet. Citrate was barely detectable (6 experiments) after incubation of 300 nmol of citrate with mitochondria in the absence of oxaloacetate and L-threonine.
Submitochondrial sites o f L-threonine 3.dehydrogenase and 2-amino-3-oxobu tyrate-CoA ligase (Table II). The marker enzymes, malate and isocitrate dehydrogenases, had similar activities in the disrupted unspun mitochondria and in the supernatant, whereas the mean activities in the pellet were about 22--24% of those in the unspun mitochondria. The mean activities of L-threonine 3-dehydrogenase in the supematant and pellet were about 70% and 22%, respectively, of the activity in the unspun disrupted mitochondria. The relative activities of 2-amino3-oxobutyrate-CoA ligase in the supematant and unspun disrupted mitochondria were similar, as indicated by the decrease in aminoacetone formed to about 16% (mean); the activity in the pellet was less, the aminoacetone being reduced to about 33% (mean). Removal of outer membranes (3 preparations) reduced the L-threonine 3-dehydrogenase activity to about 90%; addition of CoA to the reaction mixtures reduced the aminoacetone formed to about 10%. The results from the matrix markers, malate and isocitrate dehydrogenases, were similar to L-threonine 3-dehydrogenase results in mitochondria treated and not treated with digitonin. Threonine aldolase The activities of the cytosol and solubilised mitochondrial enzyme from guinea-pig liver with 20 mM DL-allothreonine were, respectively, 11.0 and 8.4 nmol/min per mg protein, being similar to those from rabbit liver, namely, 7.7 nmol/min per mg protein [17]. The activities obtained with 80 mM L-threonine were approximately 13% and 5%, respectively, of the cytosol and mitochondrial activities obtained with D L-allothreonine; the activities were scarcely detectable when the concentration of L-threonine was reduced to 1. 25 mM. The activities of the aldolases from the kidney cytosol and mitochondria with 20 mM DL-allothreonine were approximately 33% and 45%, respectively, of the liver enzymes; there was no detectable activity with 1.25 mM L-threonine. There was no evidence of [14C]glycine in autoradiograms obtained from control L-[U-14C]threonine reaction mixtures, although mitochondrial aldolase was active in such media with D L-allothreonine. Discussion
The large decrease in the formation of aminoacetone (and its precursor 2-amino-3-oxobutyrate) from L-threonine in the presence of CoA and oxaloacetate and the simultaneous appearance in the same reaction mixture of CoASAc (as citrate) and glycine support the view that 2-amino-3-oxobutyrateCoA ligase exists in mammalian liver mitochondria and catalyses the CoAdependent cleavage of 2-amino-3-oxobutyrate into glycine and CoASAc. These reactions involve the coupling of L-threonine 3-dehydrogenase and the ligase, as in micro-organisms [3,19], and could be a new pathway for the catabolism of threonine and for energising the tricarboxylic acid cycle via CoASAc. The
502 formation of appreciable amounts of citrate and a considerable decrease of aminoacetone in the presence of L-threonine and oxaloacetate (without added CoA) appears to indicate conversion of endogenous CoASAc [20] (and oxaloacetate) to citrate and CoA via citrate synthase and further supports the existence of 2-amino-3-oxobutyrate-CoA ligase. It is suggested that there may be coupling of citrate synthase and the ligase which could be a novel pathway for the cycling of CoA-CoASAc. Glycine formation from L-threonine by threonine aldolase in mammals seems unlikely because there was no detectable aldolase activity with 1.25 mM L-threonine, nor was there evidence in autoradiograms of glycine in control supernatants. Also there is no evidence of allothreonine supporting growth in mammals [21] or occurring as a natural substance [2], nor is there evidence of threonine epimerase in rat liver [22]. L-Threonine 3-dehydrogenase and 2-amino-3-oxobutyrate-CoA ligase are matrix enzymes, as indicated by the observations that the removal of the outer membranes made little difference to their activities and most of their activities and those of the marker enzymes were in the mitochondrial supernatant. The delay between thawing and incubation during the investigation of the sub-mitochondrial sites of L-threonine 3-dehydrogenase and the ligase decreased production of aminoacetone in disrupted mitochondria and supernatants. The decrease in aminoacetone could be due to monoamine oxidase (EC 1.4.3.4) which Urata and Granick [4] regarded as the cause of losses of up to 50% of aminoacetone. The citrate assay as indicated by internal standards was a b o u t 10% lower than normal when the concentration of oxaloacetate in the reaction mixture was increased from 1.1 to 2.2 mM. This decrease was due to excess oxaloacetate (presumably n o t metabolised) which was found to restrict the full increase in Aaaon m from the isocitrate dehydrogenase-NADP reaction.
Acknowledgement The help from the X-ray Department with autoradiography is gratefully acknowledged.
References 1 2 3 4 5 6 7 8 9 10 11 12 13
Meltzer, H.L. and Sprinson, D.B. (1952) J. Biol. Chem. 197, 461--474 Karasek, M.A. and Greenberg, D.M. (1957) J. Biol. Chem. 227, 191--205 McGilvray, D. and Morris, J.G. (1969) Biochem. J. 112, 657---671 Urata, G. and Graniek, S. (1963) J. Biol. Chem. 238, 811--820 Greenawalt, J.W. (1974) Methods E n z y m o l . 31A, 310--323 Johnson, D. and Lardy, H. (1967) Methods Enzymol. 10, 94--96 Gornall, A.G., BardawlU, C.J. and David, M.M. (1949) J. Biol. Chem. 177. 751--766 Granick, S. (1966) J. BioL Chem. 241, 1359--1375 Dale, R.A. (1969) Biochem. J. 114. 499--507 Laver, W.G., Neuberger, A. and Scott, J.J, (1959) J. Chem. Soc. p. 1483 ViUafranca, J.J. and Mildvan, A.S. (1971) J. Biol. Chem. 246, 772---779 Werner, S, and Neupert, W. (1972) Eur. J. Biochem. 25, 379--396 Sottocasa, G.L., Kuylenstierna, B., Ernster, L. and Bergstrand, A. (1967) Methods E n z y m o l . 10, 448-463 14 Paz. M.A., Blumenfeld, O.O., Rojkind, M., Henson, E., Furfine, C. and Gallop, P.M. (1965) Arch. Biochem. Biophys. 109, 548--559
503 15 Turner, M.K. and Mervyn, L. (1971) in The Cobalamins (Arnstein, H.R.V. and Wrighton, R.J., ed&), pp. 35--40, Churchill Livingstone, Edinburgh and London 16 EUiott, W.H. (1960) Biochero. J. 74, 90--94 17 Schirch, L, and Gross, T. (1963) J. Biol. Chem. 243, 5 6 5 1 - - 5 6 5 5 13 Nordmann, J. and Nordmann, R. (1969) in Chromatographic and Electrophoretic Techniques (Smith, I., ed.), Vol. 1, pp. 342--363, William Helnemann Medical Books Ltd., London 19 Bell, S.C. and Turner, J.M. (1976) Biochem. J. 156, 449--458 20 Garland, P.B., Shepherd, D. and Yates, D.W. (1965) Biochem. J. 97, 5 8 7 - - 5 9 4 21 West, H.D. and Carter, H.E. (1938) J. Biol. Chem. 122, 611--617 22 Malkin, L.I. and Greenberg, D.M. (1964) Biochim. Biophys. Acta 35, 117--131
Biochimica et Biophysica Acta, 544 (1978) 496--503 © Elsevier/North-Holland Biomedical Press
BBA 28769
CATABOLISM OF THREONINE IN MAMMALS BY COUPLING OF L-THREONINE 3.DEHYDROGENASE WITH 2-AMINO-3-OXOBUTYRATE-CoA LIGASE
ROBERT A. DALE
Department of Biochemistry, West Middlesex Hospital, Isleworth, Middlesex TW7 6AF
(U.K.) (Received May 29th, 1978)
Summary There is doubt about the L-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of L-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. L-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing L-threonine without and with CoA and oxaloacetate; L-[U-14C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from L-threonine to 10--14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised L-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate {precursor of aminoacetone), which is produced from L-threonine by L-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.
Introduction Degradation of dietary threonine to glycine and acetate in a mammal was first shown by Meltzer and Sprinson [1] who fed rats with labelled threonine and observed labelled glycine and acetyl radicals in the urine. It seemed feasible that threonine aldolase (EC 2.1.2.1) catalysed the reaction, but doubt was
497 expressed about this mode of degradation [2]. Threonine was found to be catabolised to glycine and CoASAc by Arthrobacter 9759 [3], the metabolic pathway involving coupling of L-threonine 3-dehydrogenase (EC 1.1.1.103), which produces 2-amino-3-oxobutyrate, with 2-amino-3-oxobutyrate-CoA ligase ("aminoacetone synthase") which catalyses the formation of glycine and CoASAc from its substrates. The knowledge that guinea-pig liver contains an active threonine dehydrogenase and "aminoacetone synthase" [4] led the author to investigate the possibility that in mammals there is a catabolic pathway of threonine similar to that in Arthrobacter 9759. Materials and Methods
Materiah. L-[U-14C]Threonine was obtained from The Radiochemical Centre, Amersham, U.K. and aconitase (EC 4.2.1.3), cytochrome c (type 111) and alcohol dehydrogenase (EC 1.1.1.1) were from Sigma London Chemical Co. Ltd., Kingston-upon-Thames, U.K. Aminoacetone was a gift from Professor A. Neuberger. All other chemicals, reagents and materials were of analytical reagent grade or of the highest quality available. Mitochondria Mitochondria were prepared for aminoacetone and citrate assays from livers of guinea-pigs [5] and were resuspended in 2 vols. 2 mM potassium phosphate, pH 7.4, frozen at approx. --70°C and stored at --20°C. The disrupted mitochondria were separated into supematant (matrix) and pellet (membranes) after centrifuging at 32 000 X g for 5 rain at 0°C. The pellet was restored to the volume of the mitochondria by re-suspending in 10 mM potassium phosphate, pH 7.6, for marker enzymes or in 40 mM Tris-HC1, pH 8.2, for L-threonine 3-dehydrogenase and 2-amino-3-oxobutyrate-CoA ligase. Outer membranes were removed by the digitonin method [5] and the residues of the mitochondria were resuspended and frozen as described above (3 preparations) together with non-digitonin-treated mitochondria. Mitochondria prepared for autoradiography, some aminoacetone assays and threonine aldolase assays (guinea-pig liver and kidney) [6] were centrifuged each time at 8500 X g for 10 rain. The mitochondria used for autoradiography and aminoacetone assays were resuspended and stored as described above. Mitochondria for threonine aldolase assay were stored at --20°C and prior to assay they were resuspended in 3 vols. 5 mM sodium phosphate, pH 7.4, frozen and thawed; supematant was obtained by centrifuging for 15 rain at 32 000 X g. The supernatant of the first mitochondrial centrifuging was centrifuged for 15 rain at 34 000 X g and the supernatant (cytosol) was stored at --20°C. Enzyme assays. Protein was assayed with crystalline bovine serum albumin as standard [7]. L-Threonine 3-dehydrogenase from guinea-pig liver was assayed in a volume of 0.8 ml containing 37.5 mM Tris-HC1 buffer, pH 8.2, 2.5 mM MgC12, 1.25 mM L-threonine and 50 ~1 of once-frozen and thawed disrupted mitochondria; 1 mM NAD was used to start the reaction, but was omitted from controls. The mixture was incubated for 20 or 30 min (Table I) at 37°C, being shaken 120 times/min. (30 mM sodium phosphate buffer, pH 7.7, was used instead of Tris-HC1 buffer for 14C experiments and 2 experiments in Table I). Reactions were stopped by adding 0.25 ml of 5% HC104. After centrifuging, the supernatants were removed and assayed for aminoacetone
498 [8,9], which is formed by spontaneous decarboxylation of 2-amino-3-oxobutyrate [10], and citrate [11]. The presence of 2-amino-3-oxobutyrate-CoA ligase was assessed by including 1.0 mM CoA [3] and 1.1 or 2.2 mM oxaloacetate (for citrate formation via endogenous citrate synthase) in the reaction mixtures for the L-threonine 3-dehydrogenase assay and by observing the decrease in aminoacetone formed and the appearance of CoASAc as citrate. The supernatants for citrate assays were neutralised with KOH and stored at --20°C. The products of 2-amino-3oxobutyrate-CoA ligase (glycine and CoASAc) were further investigated by autoradiograms (see below) by adding 5 pCi of L-[U-~4C]threonine to reaction mixtures for the L-threonine 3-dehydrogenase and ligase assays (3 liver experiments, 1 kidney experiment). A portion of each supematant was used to assay aminoacetone and the remaining supernatant was neutralised with KOH and stored at --20°C for chromatography. Succinate-cytochrome c reductase was assayed as described by Werner and Neupert [12] and malate (EC 1.1.1.37) and isocitrate (EC 1.1.1.42) dehydrogenases were assayed by the method of Sottocasa et al. [13]. 0.5 mM MgCI2 was used instead of 0.1 mM MnC12 for the isocitrate dehydrogenase assay. Threonine aldolase was assayed by measuring the rate of formation of acetaldehyde. Cytoplasmic enzyme was assayed at 33°C in 2 ml containing 20 mM Tris-HC1 buffer, pH 7.4, 10 uM pyridoxal phosphate, 120 pM NADH, 50 gg of alcohol dehydrogenase, and 0.1 ml of cytosol. The reaction was started by adding either 20 mM DL-allothreonine or 80 mM L-threonine and the decrease in absorbance at 340 nm was observed. Mitochondrial enzyme was assayed in stoppered tubes in 2 ml containing 20 mM Tris-HC1 buffer, pH 7.4, 10 pM pyridoxal phosphate, 20 mM DL-allothreonine or 80 mM L-threonine and 0.1 ml of mitochondrial suspension or supernatant. Substrate was omitted from controls. After 10--30 rain incubation at 37°C reactions were stopped by chilling the tubes in ice and adding 0.1 ml of 3 M trichloroacetic acid. After centrifuging, the supernatant was removed and assayed for acetaldehyde [ 14,15]. Chromatography and autoradiography. Neutralised supernatants and relevant markers were applied to duplicate 3 MM sheets, one for staining and one for autoradiography. One-way chromatography for aminoacetone [16], threonine and glycine [17] and citrate [18] and two-way chromatography for threonine, glycine and citrate [18] were then carried out. Autoradiography was performed with a Kodak RP-X-Omat film. Citrate assay. Citrate was assayed by aconitase [11] which was activated as described by Sigma Chemical Co. The neutralised supernatants, after thawing, were kept at 0°C to precipitate KC104. Results
Aminoacetone formation from L-threonine 3-dehydrogenase Table I shows the m o u n t s of aminoacetone formed from L-threonine and NAD under different conditions. Incubation of aminoacetone (35 nmol) in control reaction mixtures (6 experiments) resulted in a mean recovery of 96%. The addition of CoA reduced the formation of aminoacetone (mean) to 10--14%. The addition of oxaloacetate (1.1 or 2.2 mM), without CoA, diminished
499 TABLE I F O R M A T I O N O F C I T R A T E ( V I A C o A S A c ) IN P L A C E O F A M I N O A C E T O N E BY ADDITION OF CoA AND/OR OXALOACETATE
FROM L-THREONINE
The assay conditions are described under Materials and Methods. Each experiment relates to a different mitochondrial preparation. The citrate produced from 2.2 mM oxaloacetate was not corrected for the a p p r o x . 1 0 % d e c r e a s e in A 3 4 0 n m . T h e r e s u l t s a r e e x p r e s s e d as m e a n w i t h t h e r a n g e in p a r e n t h e s i s . Conditions
Sodium phosphate (pH 7.7)
Incubation time (rain)
Number of experiments
n m o l p r o d u c t / 5 0 ~ul d i s r u p t e d mitochondria Aminoacetone
Citrate
30
2
--
--
133, 138 15, 16
---
192 86 20 14
(158--220) (62--121) (15--25) (10--21)
0 115 (71--168) 0 236 (200--254)
250 I18 34 29
(215--269) (84--153) (22--56) (17--46)
0 64 (56--71) 0 280 (258--302)
NAD NAD + CoA Tris-HCl ( p H 8 . 2 )
20
3
NAD NAD + oxaloaeetate (2.2 raM) NAD + CoA NAD + CoA + oxaloacetate (2.2 raM) Trls-HCI ( p H 8 . 2 ) NAD NAD + oxaloacetate (I.I mM) NAD + CoA NAD + CoA + oxaloaeetate (1.1 mM)
30
5
the formation of aminoacetone (mean) to about 47%. The addition of CoA in several pilot experiments with guinea-pig kidney mitochondria reduced the formation of aminoacetone to 40--50%.
Identification of products formed in place of aminoacetone via 2-amino-3-oxobu tyrate-CoA ligase (a) Chromatograms and autoradiograms. Reaction mixture supernatants from liver and kidney mitochondria incubated with L-[U-14C]threonine but without CoA and oxaloacetate produced strong identical aminoacetone spots on chromatograms and autoradiograms; glycine spots were just detectable and there were no citrate spots. Similar incubations with added CoA and oxaloacetate produced faint aminoacetone spots and well-defined identical glycine spots on the chromatograms and autoradiograms; the citrate spots on the autoradiograms were identical to the chromatographic markers. No citrate spots were obtained from kidney mitochondria (b) Citrate assay. (Table I). The citrate formed in the presence of CoA and/ or oxaloacetate is shown in parallel with the decrease in the formation of aminoacetone. In the last 5 experiments in the presence of CoA, decreasing the concentration of oxaloacetate to 0.6 mM reduced the citrate formed to 169 nmol (mean) whereas increasing the concentration of oxalacetate to 2.2 mM made little difference to the citrate formed, namely 258 nmol (mean). In the absence of CoA, increasing the concentration of oxaloacetate from 1.1 to 2.2 mM (last 5 experiments) increased the citrate formed from a mean of 64 nmol to 110 nmol. The mean and range of citrate formed from disrupted mitochondria and supernatants (Table II) were 245 (223--276) nmol and 272 (179--
41
13 ( ( 8 - - 2 3 )
3.6 ( 3 . 3 - - 4 . 0 )
0
3.8 (3.4---4.2)
20 ( 1 7 - - 2 5 )
93 (74--112)
91 ( 7 2 - - 1 0 8 )
Malate d e h y d r o g e n a s e
3.3
14
14
(2.5--4.6)
(10--20)
(11--21)
Isocitrate dehydrogenase
* T h e m i t o c h o n d r i a , s u p e r n a t a n t a n d p e l l e t w e r e e a c h a d d e d t o H 2 0 , Tris-HCl b u f f e r , Mg 2+ a n d L - t h r e o n i n e a t 0 ° C u n t i l all o t h e r r e a g e n t s w e r e a d d e d in o r d e r t o prevent the yield of a m i n o a c e t o n e decreasing m o r e than 20--30% due to the delay b e t w e e n thawing and incubation.
(23--61)
(7--38)
21
129
Supematant *
Pellet * (resuspended)
(98--157)
29 ( 1 4 - - 5 3 )
185 (140--230)
Succinate-cytochrome c reductase
C o A ligase
Mitochondria * (disrupted)
Marker enzymes
2-Amino-3oxobutyrate-
L - T h r e o n i n e 3debydrogenase
-Fraction
T h e results w e r e o b t a i n e d f r o m 5 m i t o c h o n d r i a l p r e p a r a t i o n s . T h e a s s a y c o n d i t i o n s are d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . 2 - A m i n o - 3 - o x o b u t y r a t e - C o A llgase was a s s a y e d in t h e p r e s e n c e o f C o A a n d 1.1 m M o x a l o a c e t a t e . T h e a c t i v i t i e s o f t h e m a r k e r e n z y m e s are e x p r e s s e d a s / ~ m o l / m i n p e r m l o f e a c h f r a c t i o n . T h e activities o f the e n z y m e s u n d e r i n v e s t i g a t i o n are e x p r e s s e d as n m o l a m i n o a c e t o n e f o r m e d / 3 0 r a i n p e r 50/~l o f e a c h f r a c t i o n . ( T h e r e a c t i o n r a t e w a s n o t l i n e a r w i t h t i m e o r t h e v o l u m e o f m i t o c h o n d r i a l f r a c t i o n , as a s s a y e d . ) T h e r e s u l t s a r e e x p r e s s e d as m e a n w i t h t h e r a n g e in p a r e n t h e s i s .
SUBMITOCHONDRIAL DISTRIBUTION OF L-THREONINE 3-DEHYDROGENASE AND 2-AMINO-3-OXOBUTYRATE-CoA LIGASE
T A B L E II
L •
O O
501 314) nmol, respectively; a trace of citrate only was produced by the pellet. Citrate was barely detectable (6 experiments) after incubation of 300 nmol of citrate with mitochondria in the absence of oxaloacetate and L-threonine.
Submitochondrial sites o f L-threonine 3.dehydrogenase and 2-amino-3-oxobu tyrate-CoA ligase (Table II). The marker enzymes, malate and isocitrate dehydrogenases, had similar activities in the disrupted unspun mitochondria and in the supernatant, whereas the mean activities in the pellet were about 22--24% of those in the unspun mitochondria. The mean activities of L-threonine 3-dehydrogenase in the supematant and pellet were about 70% and 22%, respectively, of the activity in the unspun disrupted mitochondria. The relative activities of 2-amino3-oxobutyrate-CoA ligase in the supematant and unspun disrupted mitochondria were similar, as indicated by the decrease in aminoacetone formed to about 16% (mean); the activity in the pellet was less, the aminoacetone being reduced to about 33% (mean). Removal of outer membranes (3 preparations) reduced the L-threonine 3-dehydrogenase activity to about 90%; addition of CoA to the reaction mixtures reduced the aminoacetone formed to about 10%. The results from the matrix markers, malate and isocitrate dehydrogenases, were similar to L-threonine 3-dehydrogenase results in mitochondria treated and not treated with digitonin. Threonine aldolase The activities of the cytosol and solubilised mitochondrial enzyme from guinea-pig liver with 20 mM DL-allothreonine were, respectively, 11.0 and 8.4 nmol/min per mg protein, being similar to those from rabbit liver, namely, 7.7 nmol/min per mg protein [17]. The activities obtained with 80 mM L-threonine were approximately 13% and 5%, respectively, of the cytosol and mitochondrial activities obtained with D L-allothreonine; the activities were scarcely detectable when the concentration of L-threonine was reduced to 1. 25 mM. The activities of the aldolases from the kidney cytosol and mitochondria with 20 mM DL-allothreonine were approximately 33% and 45%, respectively, of the liver enzymes; there was no detectable activity with 1.25 mM L-threonine. There was no evidence of [14C]glycine in autoradiograms obtained from control L-[U-14C]threonine reaction mixtures, although mitochondrial aldolase was active in such media with D L-allothreonine. Discussion
The large decrease in the formation of aminoacetone (and its precursor 2-amino-3-oxobutyrate) from L-threonine in the presence of CoA and oxaloacetate and the simultaneous appearance in the same reaction mixture of CoASAc (as citrate) and glycine support the view that 2-amino-3-oxobutyrateCoA ligase exists in mammalian liver mitochondria and catalyses the CoAdependent cleavage of 2-amino-3-oxobutyrate into glycine and CoASAc. These reactions involve the coupling of L-threonine 3-dehydrogenase and the ligase, as in micro-organisms [3,19], and could be a new pathway for the catabolism of threonine and for energising the tricarboxylic acid cycle via CoASAc. The
502 formation of appreciable amounts of citrate and a considerable decrease of aminoacetone in the presence of L-threonine and oxaloacetate (without added CoA) appears to indicate conversion of endogenous CoASAc [20] (and oxaloacetate) to citrate and CoA via citrate synthase and further supports the existence of 2-amino-3-oxobutyrate-CoA ligase. It is suggested that there may be coupling of citrate synthase and the ligase which could be a novel pathway for the cycling of CoA-CoASAc. Glycine formation from L-threonine by threonine aldolase in mammals seems unlikely because there was no detectable aldolase activity with 1.25 mM L-threonine, nor was there evidence in autoradiograms of glycine in control supernatants. Also there is no evidence of allothreonine supporting growth in mammals [21] or occurring as a natural substance [2], nor is there evidence of threonine epimerase in rat liver [22]. L-Threonine 3-dehydrogenase and 2-amino-3-oxobutyrate-CoA ligase are matrix enzymes, as indicated by the observations that the removal of the outer membranes made little difference to their activities and most of their activities and those of the marker enzymes were in the mitochondrial supernatant. The delay between thawing and incubation during the investigation of the sub-mitochondrial sites of L-threonine 3-dehydrogenase and the ligase decreased production of aminoacetone in disrupted mitochondria and supernatants. The decrease in aminoacetone could be due to monoamine oxidase (EC 1.4.3.4) which Urata and Granick [4] regarded as the cause of losses of up to 50% of aminoacetone. The citrate assay as indicated by internal standards was a b o u t 10% lower than normal when the concentration of oxaloacetate in the reaction mixture was increased from 1.1 to 2.2 mM. This decrease was due to excess oxaloacetate (presumably n o t metabolised) which was found to restrict the full increase in Aaaon m from the isocitrate dehydrogenase-NADP reaction.
Acknowledgement The help from the X-ray Department with autoradiography is gratefully acknowledged.
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