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The formation of ammonia from glutamine and glutamate by mitochondria from rat liver and kidney
ARCHIVES
OF
BIOCHEMISTRY
AND
The Formation
127, 718-724 (1968)
BIOPHYSICS
of Ammonia
by Mitochondria
from from
Glutamine
and Glutamate
Rat Liver and Kidney
F. J. R. HIRD AND M. A. MARGINSON Russell Grimwade
School of Biochemistry,
University
of Melbourne,
Parkuille,
Victoria 3052, Australia
Received March 20, 1968; accepted May 21, 1968 The metabolism of glutamate and glutamine by mitochondria from rat liver and kidney has been investigated. Kidney mitochondria have been shown to be considerably more active both in rates of oxidation and in ammonia formation. The rapid oxidative formation of ammonia from both glutamine nitrogen atoms by kidney mitochondria could not adequately be explained in terms of glutaminase I (hydrolysis) or glutaminase II (transamination followed by hydrolysis) activity. The evidence obtained suggested additional pathway(s) but there is the possibility that permeability factors could be involved
The metabolism of L-glutamine in mammalian tissues is thought to be initiated in two different ways (1): 1. Hydrolysis by glutaminase I (L-glutamine amidohydrolase, E.C. 3.5.1.2.) to glutamate and ammonia:
The glutaminase II system is present in the soluble fraction of rat liver and to a lesser extent of kidney (7); it has also been shown to be present in liver mitochondria (8). But the restriction of its occurrence to liver and kidney seems to be from the transaminase component since L-glutamine + Hz0 + L-glutamate + ammonia. the w-amidase has been shown to be presGlutaminase I has been characterized ent in many tissues (9). Investigations, up to the present, have as an enzyme distributed in favor of the mitochondrion (2-4)) occurring in many been mainly directed toward hydrolytic organs (3), needing high levels of phos- and transamination reactions involving phate for maximum activity (5) and being glutamine nitrogen. There is, however, rapidly inactivated in the absence of the possibility of oxidative processes being involved both in a deamination rephosphate (6). 2. Transamination with a keto acid by action and the concomitant metabolism glutamine transaminase (L-glutamine : 2- of the carbon skeleton. This paper preoxoacid aminotransferase, E. C. 2.6.1.15)) sents results which implicate oxidative to form an amino acid and cy-ketoglu- processes in the production of ammonia taramate, with hydrolysis of this keto- from glutamine by kidney mitochondria amide to ammonia and cu-ketoglutarate by and which characterize glutamine as a an w-amidase (w-amidodicarboxylate ami- rapidly oxidizable substrate for these dohydrolase, E.C. 3.5.1.3.), the system of mitochondria. transamination followed by amide hydrolyMATERIALS AND METHODS sis being known as glutaminase II: L-Glutamine
+ keto acid = a-ketoglutaramate + amino acid.
a-Ketoglutaramate
+ Hz0 4 a-ketoglutarate
+ ammonia.
Reagents. L-Glutamine was obtained from Mann Research Laboratories, New York; L-glutamic acid and a-ketoglutaric acid from British Drug Houses Ltd., Dorset; L-malic acid from Nutritional Biochemicals Corporation, Cleveland, Ohio; yeast hexo718
MITOCHONDRIAL
METABOLISM
kinase and ATP from Sigma Chemical Co., St. Louis, Missouri, and ADP from C. F. Boehringer and Soehne G.m.b.H. Mannheim, Germany. All other reagents were of A.R. grade. The yeast hexokinase was freed from ammonia by dialysis against 0.3 M glucose (10). Preparation of mitochondria. Mitochondrial suspensions from rat liver and kidney were prepared as previously described (10). Incubation of mitochondria. The amperometric experiments were carried out at 30” (11). Other incubations were carried out at 38” in 25.ml Erlenmeyer flasks and in Warburg flasks attached to manometers (the aerobic experiments) or to an oxygen-free nitrogen manifold (the anaerobic experiments). The incubation mixtures were as previously described (10). Estimation of ammonia. Ammonia was estimated as described previously (10). Certain corrections were applied when glutamine was present during the Conway distillation. Under the conditions of this distillation, (90 minutes at 30” in half-saturated K2COa), glutamine was shown to decompose to the extent of 5 mole f( to ammonia and, presumably to pyrrolidone carboxylate. This rate of decomposition was not affected by glutamine concentration between 0 and 10 mM, therefore it was possible by use of simple equations, to correct for it between upper and lower limits. These limits would, of course, be dependent on the form of the nitrogen remaining (glutamate plus aspartate or as glutamine) and would be at a maximum (5”r) at low levels of ammonia production. Actual corrections for glutamine metabolism have been made at an average of the upper and lower values. Estimation of protein. Mitochondrial protein was prepared for estimation according to the method of Aldridge (12). Ethanol : water : ether (9: 6: 5) 10 ml, was added to 0.30 ml mitochondrial suspension, mixed well and allowed to stand until the protein had flocculated. The protein was recovered by centrifugation and dissolved in a suitable volume of M NaOH. The concentration of protein was then measured using the method of Ellman bovine serum albumin as a (13), with crystalline standard.
the addition of glutamate gives a marked increase in the rate of oxygen consumption. The observations presented in Figs. 1 and 2 are consistent, therefore, with the oxidation of glutamine being dependdent on its prior conversion to glutamate. In contrast, however, Fig. 3 shows that the rate of oxygen uptake of rat kidwhich are oxidizing ney mitochondria, (at saturating concentration) glutamate (10 mM) is substantially increased by the addition of glutamine. A qualitatively similar picture is obtained with kidney mitochondria if the order of the two substrates is reversed; i.e., the rate of oxygen uptake is substantially increased if glutamate is added when the system is already oxidizing a saturating glutamine The kidney mitochonconcentration. drial system apparently possesses independent systems for the oxidation of glutamate and of glutamine. Heart mitochondria in the presence of ADP and inorganic phosphate do not give any evidence for the oxidation of glutamine and preparations of brain mitochondria resemble those from liver. Kidney mitochondria oxidize glutaMITOCHONDRIA 1
+ ADP (1.6~
moles)
0.46
( IOmM)
RESULTS
Oxidation of glutamate and glutamine. Figure 1 indicates that the addition of Lglutamine to rat liver mitochondria, which are already oxidizing L-glutamate, does not alter significantly the rate of oxygen uptake by the system. Figure 2 shows that when glutamine is added first there is a delay before a steady rate of oxidation is reached and that at this stage
719
OF GLUTAMINE
0
I
1
I
I
I
2
3
4
TIME
(min.)
FIG. 1. Oxygen uptake of a rat liver mitochondrial preparation in the presence of excess ADP and after the addition of glutamate to 10 mM and then of glutamine to 10 mM. Incubation mixture of 30 mM sucrose, 21 mM KCl, 2.5 mM MgCb, 1.2 mM EDTA, 33 mM Tris-HCl, 40 mM phosphate, pH 7.4; 3 mg mitochondrial protein, 30”.
720
HIRD AND MARGINSON ~ITOCHONORIA
+ AOP
(1.6)~
moles)
0.46
0
0
2
I
3
4
TIME
bnin.1
5
6
7
FIG. 2. Oxygen uptake of a rat liver mitochondrial preparation in the presence of excess ADP and after the addition of glutamine (10 mM) and then of glutamate (10 mM). Incubation mixture as for Fig. 1; 3 mg mitochondrial protein, 30”. MITOCHONDRIA
+ADP
(1.6)rmoles)
1
0.46
L-GLU
t-i
(IOmM)
1 L-GLU
0
(NH~)
(10mM)
L
I
I
I
I
I
0
I
2
3
4
5
TIME
(min.)
FIG. 3. Oxygen
uptake of a rat kidney mitochondrial preparation in the presence of excess ADP and after the addition of glutamate (10 mM) followed by glutamine (10 mM). Incubation mixture as for Fig. 1; 2.3 mg mitochondrial protein, 30”.
mine and glutamate to give ADP/O ratios of between 2.8-3.3; the oxidation of glutamine also is dependent on phosphorylation of ADP and this characterizes it to be not of the simple L-amino acid oxidase type. The difference between liver and kidney mitochondria is further emphasized
by the results shown in Table I. In these long term experiments, the oxygen uptake was followed manometrically at 38”. Under the conditions of the experiment the oxygen uptake in the presence of both glutamate and glutamine was linear. The rate of oxygen uptake of kidney mitochondria in the presence of glutamine is more than twice that of kidney mitochondria in the presence of glutamate. It is apparent that glutamine is an extremely good substrate for kidney mitochondria. These long-term experiments also show that glutamine is oxidized very slowly by liver mitochondria. From a large number of experiments (amperometric) with closely agreeing figures, the average rates of oxygen consumption by kidney mitochondria (in mpmole OJmg protein/min) were 60, 133, 145, and 148 at concentrations of glutamine of 1, 2, 5, and 10 m&q respectively. The system is therefore close to being saturated at 2 mM glutamine. Related figures obtained for glutamate in the kidney systems were 17, 53, and 55 for 1, 5, and 10 mM, respectively. The long-term manometric experiments (Table II) also support the short term amperometric experiments (Fig. 3) in that they show that the addition of glutamate to a kidney system saturated with glutamine, brings about an increase in oxygen uptake. Formation of ammonia from glutamate and glutamine. Mitochondria from liver produced less ammonia from glutamine than from glutamate and as the amount was not substantially different in the absence of oxygen, it is probable that simple hydrolysis of glutamine accounts for the ammonia produced. The behaviour of kidney mitochondria was notably difthey produced about ferent . Although the same amount of ammonia from glutamate as did liver mitochondria, they produced 22 pmole of ammonia from 15 pmole of glutamine. This high level of ammonia production was substantially lowered in the absence of oxygen. HOWever, the anaerobic deamination of glutamine by kidney mitochondria is very high when compared with the anaerobic
MITOCHONDRIAL
METABOLISM TABLE
FORM~TIOW OF AMMONI.~ DURING BY I~AT
Rate of oxygen uptake in m~mole 02/mg protein/min
Air Air Air NY
Endogenous Glutamate (5 mM) Glutamine (5 mM) Glutamine (5 m&f) protein,
2.4 mg/flask;
TABLE
II
incubation
OXYGEN UPTIKE ;\ND AMMONII PRODUCTION By KIDNEY MITOCHOXDRI.\ OXIDIZING GLUT-MINE, GLUT~~.MATE .IND GLC-TAMINIC PLUS GLUT.W.ITF:
The incubation was for GO min at 38” with 1.1 mg mitochondrial protein; all rates of oxygen lIptake remained linear. The rates of ammonia prodrlced were not linear in the lat,ter part of the experimental period. Incltbation mixtures as for -Fig. 5. Rate of oxygen Ammonia produced (rmole by uptake 3 ml reac(mrmole/mg tion mixture) proteinjmin)
Substrate
Endogenous 5 mM gllltamine 10 mM glutamine 5 my glutamate 10 rnhl gllltamate 5 rn>f glutamine glutamate 5 mM glutamine rnM glrltamate
I
OXID:ITION OF L-GLUT:~MSTE .~ND KIDNEY MITOCHONDRI~~
Gas phase
Substrate
a Mitochondrial
LIVER
+ 5 rnM
0 143 145 33 55 If.50
0.06 14.5 18.8 0.63 1.04 11.4
+ 10
IF0
9.6
deamination of glutamine by liver mitochondria or when compared with the aerobic deamination of glutamate by mitochondria from either tissue. It is clear that a substantial proportion of the ammonia derived from incubating glutamine with kidney mitochondria must reflect oxidative processes. Not only did removal of oxygen from the system cause a marked fall in ammonia production, but the actual amount of ammonia produced was 7 pmole greater than the maximum which can be derived from amide hydrolysis of glutamine. As this amount of ammonia is five times that produced from the amino group of glutamate, it is clear that processes are operating
721
OF GLUTAMINE
AND
L-GLUT.IMINE
Ammonia formation rmole/‘3 ml after 40 min incubation at 38”
Liver
Kidney
Lirer
Kidney
2.3 33 2.6
5.7 G7 152
0.06 1.11 0.60 0.63
0.12 1.40 22.4 8.3
mixtures
as for Fig. 5.
which differ from the simple oxidation of glutamate which has been derived from glutamine. In an attempt to compare further the deamination of glutamate with that of glutamine, the effect of adding malonate and glutamate to the system was studied. It has previously been shown that the deamination of glutamate by liver mitochondria is greatly stimulated by malonate and abolished by adding a source of oxaloacetate (14, 15). With kidney mitochondria, malonate was shown to have either no effect or to stimulate slightly the rate of ammonia formation from both glutamine and glutamate after about 2030 min incubation. The addition of malonate, however, diminished the oxygen uptake by about 50 and 709; in the case of glutamine and glutamate, respectively. The joint observations show that ammonia production by kidney mitochondria from glutamine is malonate insensitive and that part of the oxidative metabolism of the carbon skeleton (presumably through succinate) is malonate sensitive. The addition of glutamate lowers the aerobic production of ammonia from glutamine (see Table II). The site of inhibition is not known. The relation between ammonia production and oxygen uptake. Although glutamine concentration after incubation was not directly determined it proved possible to form some estimate of glutamine utilization from the ammonia production in long term experiments with low concentrations of glutamine (l-2 mM). Figure 4 is a time-progress
722
HIRD
0
25
50 TIME
75
AND
100
(min:)
FIG. 4. Oxidation of glutamine by rat kidney mitochondria, showing ammonia production (A) and the oxygen uptake which paralleled it ( A). Incubation mixtures as for Fig. 1 and in addition Lglutamine (2 mM), ATP (mM), glucose (23 mM), hexokinase in excess of 100 K.M. units, 2.8 mg mitochondrial protein, 38”, final volume 3.0 ml.
curve for ammonia production from 2 mM glutamine compared with the oxygen uptake by the same mitochondrial preparation. At the end of the experimental period, the mitochondria had taken up 40 patom oxygen and had produced 10.6 pmole ammonia from the addition of 6 pmole glutamine. This figure indicates that between 4.6 (assuming 6 pmole of amide removed hydrolytically) and 6.0 pmole glutamine had been “oxidatively” removed. The ratio of ratom oxygen taken up to pmole glutamine oxidized was therefore in the range of 8.7-6.7. Assuming that the oxidation of glutamine, like that of glutamate proceeds to either oxaloacetate or aspartate, this ratio should be between 3 and 4. Expressed in another way, in this experiment a maximum of 1.4 rmole of aspartate and 4.6 pmole of oxaloacetate could have been produced as end products of glutamine oxidation. As this would indicate a maximum oxygen uptake of 22.6 patom, a minimum of 17.4
MARGINSON
patom of oxygen uptake remains unaccounted for. To investigate this anomaly, the oxidation of glutamate (mM), glutamine (mM), and malate (mM) by kidney mitochondria was compared. The ratio of patom oxygen taken up in 50 min to rmole substrate added was 3.0 for glutamate, 6.7 for glutamine and 8.0 for malate. The glutamate figure is consistent with the reaction glutamate + aspartate with little further metabolism of the aspartate so formed. However, the very high figures for glutamine and malate suggest the further utilization of oxaloacetate either to form pyruvate or as a mediator for the metabolism of endogenous precursors of acetyl CoA. This observation suggests that glutamate may not be an intermediate in the oxidation of glutamine. The influence of wketoglutarate on ammonia production. Since glutaminase II has been reported to be present in both mitochondria and cell sap (89, it is conceivable that the rapid oxidation of glutamine could be due to production of a-ketoglutarate which would act as the amino group acceptor for the glutaminase II system. If this system were responsible for the initial steps of glutamine oxidation, then it would be expected that addition of a keto acid, for example LYto mitochondria in the ketoglutarate, presence of glutamine would, under anmarkedly stimulate aerobic conditions, the production of ammonia. The results of such an experiment are shown in Fig. 5. Consistent with the results presented in Table I, the production of ammonia from glutamine was markedly diminished under anaerobic conditions. When CYketoglutarate is added (l-10 mM) there is a further marked diminution in ammonia production. Although there is some indication of a restoration of the anaerobic level at high concentrations of cu-ketoglutarate (20 mM), these observations are not compatible with a glutaminase II system being active in the mitochondrial preparation at kidney
MITOCHONDRIAL 20
A.
‘.‘\‘\ ‘\
AEROBIC
‘1
16
METABOLISM
‘\ ‘\ f to - m
‘\
‘\
12
‘-A ‘\
5 n
;
‘\
li
4
‘.
;> ANAEROBIC
o0
5
IO
CONCENTRATION d-KETOGLUTARATE
I5
20
OF (mM
1
FIG. 5. Effect
of a-ketoglutarate concentration on the aerobic and anaerobic production of ammonia from glutamine by rat kidney mitochondria. Incubation mixtures as for Fig. 4 except that glutamine concentration was 5 mM and each flask contained 3 mg mitochondrial protein. The anaerobic incubations were carried out in flasks attached to an oxygen-free nitrogen manifold.
concentrations of cY-ketoglutarate which are likely to obtain in uiuo. The marked effect of low concentrations of cY-ketoglutarate in depressing ammonia formation is of interest and could be attributed to a Krebs-Cohen dismutation (16): 2 a-Ketoglutarate
+ NH< -
glutamate + succinate + Con.
DISCUSSION
The rate of oxidation of glutamate and of glutamine by mitochondria prepared from kidney tissue is very much greater than the rate obtained with liver mitochondria; in the case of L-glutamine, the ratio is approximately 60 : 1. Ammonia production from glutamine is also much greater ( X 37) with kidney mitochondria. It is clear that there are great differences between the mitochondria from these tissues and that glutamine is potentially a major substrate for kidney tissue. From the results obtained, no simple interpretation of the relative importance of the various pathways of oxidation or of
OF GLUTAMINE
723
ammonia production is possible. It does appear however that glutamine and glutamate are metabolized independently. No evidence was found which would support prior transamination of glutamine with subsequent hydrolysis of the a-ketoglutaramate (glutaminase II) as a major method of mitochondrial oxidation of glutamine. A dehydrogenation of glutamine which removed the a-amino group formand ammonia ing a-ketoglutaramate therefore arose as a possibility. However, direct evidence supporting such an oxidative step has not yet been In preliminary experiments, obtained. sonically disrupted mitochondria prepared from rat kidneys did not give any evidence spectroscopically of a reduction of NAD or of NADP in the presence of glutamine. On the other hand, NAD was reduced in the presence of glutamate indicating the survival of glutamate dehydrogenase (E.C. 1.4.1.3) in the preparation. The observed differences between the rates of oxidation of glutamate and of glutamine by intact mitochondria are perhaps explicable in terms of permeability but in amperometric experiments with kidney mitochondria there is an almost “instantaneous” response in rate of oxygen uptake to the addition of small increments of glutamate or of glutamine in the presence of excess ADP. However, there still remains the possibility that the metabolism of glutamine is taking place by the known hydrolytic and oxidative pathways via glutamate, but that glutamate liberated within a certain mitochondrial compartment, readily accessible to glutamine but not to glutamate, is oxidized very much more readily than, and separately from, glutamate in other parts of the mitochondrion. Another alternative, not yet explored, is that there is more than one population of mitochondria involved in the kidney preparations. We have, however, no observations which would help evaluate an explanation in these terms. Pitts and co-workers (17, 18) have studied glutamine metabolism in the kid-
724
HIRD
AND
neys of acidotic dogs and their experiments have clearly shown that in uivo there is a substantial conversion of both the amide and the amino nitrogen to ammonia, with the former predominating. Although these results were obtained with acidotic animals, they clearly establish the significance of glutamine nitrogen in the maintenance of acid-base balance in the animal, In addition, attention has recently been drawn (19, 20) to the possibility that glutamine carbon atoms may play a role in kidney gluconeogenesis. The results presented in the present paper would indicate that L-glutamine is a major substrate for isolated kidney mitochondria and further serve to emphasize the potential importance of glutamine in kidney metabolism. ACKNOWLEDGMENTS We thank I. W. D. Croker for valuable assistance and the Australian Research Grants Committee for financial support. REFERENCES 1. MEISTER, A. Physiol. Reu. 36, 103 (1956). 2. ERRERA, M. J. Biol. Chem. 178, 483 (1949).
MARGINSON 3. ERRERA, M. AND GREENSTEIN, J. P. J. Biol. Chem. 178, 495 (1949). 4. SHEPHERD, J. A. AND KALNITSKY, G. J. Biol. &em. 192, l(l951). 5. CARTER, C. E. AND GREENSTEIN, J. P. J. Nat. Cancer Inst. 7, 433 (1947). 6. KLINGMAN, J. D. AND HANDLER, P. J. Biol. Chem. 232, 369 (1958). 7. PRICE, V. E. AND GREENSTEIN, J. P. J. Nat. Cuncer h-t. 7, 275 (1947). 8. KATUNUMA, N., HUZINO, A. AND TOMINO, I. Aduan. Enzyme Regulation 5, 55 (1967). 9. MEISTER, A. J. Biol. Chem. 200, 571 (1953). 10. HIRD, F. J. R. AND MARGINSON, M. A. Arch. Biochem. Biophys. 115, 247 (1966). 11. HIRD, F. J. R. AND WEIDEMANN, M. J. Biohem. J. 98, 378 (1966). 12. ALDRIDGE, W. N. Biochem. J. 67, 423 (1957). 13. ELLMAN, G. L. Anal. Biochem. 3,40 (1962). 14. Hmn, F. J. R. AND MARGINSON, M. A. Nature 201, 1224 (1964). 15. HIRD, F. J. R. AND MORTON, D. J. Biochim. Biophys. Acta 85, 353 (1964). 16. KREBS, H. A. AND COHEN, P. P. Biochem. J. 33, 1895 (1939). 17. PITTS, R. F. The Physiologist 9, 97 (1966). 18. STONE, W. J. AND Prrrs, R. F. J. Clin. Invest. 46, 1141 (1967). 19. GOODMAN, A. D., Fusz, R. E. AND CAHILL, G. F. J. Clin. Inuest. 45, 612 (1966). 20. LOTSPEICH, W. D. Science 155, 1066 (1967).
OF
BIOCHEMISTRY
AND
The Formation
127, 718-724 (1968)
BIOPHYSICS
of Ammonia
by Mitochondria
from from
Glutamine
and Glutamate
Rat Liver and Kidney
F. J. R. HIRD AND M. A. MARGINSON Russell Grimwade
School of Biochemistry,
University
of Melbourne,
Parkuille,
Victoria 3052, Australia
Received March 20, 1968; accepted May 21, 1968 The metabolism of glutamate and glutamine by mitochondria from rat liver and kidney has been investigated. Kidney mitochondria have been shown to be considerably more active both in rates of oxidation and in ammonia formation. The rapid oxidative formation of ammonia from both glutamine nitrogen atoms by kidney mitochondria could not adequately be explained in terms of glutaminase I (hydrolysis) or glutaminase II (transamination followed by hydrolysis) activity. The evidence obtained suggested additional pathway(s) but there is the possibility that permeability factors could be involved
The metabolism of L-glutamine in mammalian tissues is thought to be initiated in two different ways (1): 1. Hydrolysis by glutaminase I (L-glutamine amidohydrolase, E.C. 3.5.1.2.) to glutamate and ammonia:
The glutaminase II system is present in the soluble fraction of rat liver and to a lesser extent of kidney (7); it has also been shown to be present in liver mitochondria (8). But the restriction of its occurrence to liver and kidney seems to be from the transaminase component since L-glutamine + Hz0 + L-glutamate + ammonia. the w-amidase has been shown to be presGlutaminase I has been characterized ent in many tissues (9). Investigations, up to the present, have as an enzyme distributed in favor of the mitochondrion (2-4)) occurring in many been mainly directed toward hydrolytic organs (3), needing high levels of phos- and transamination reactions involving phate for maximum activity (5) and being glutamine nitrogen. There is, however, rapidly inactivated in the absence of the possibility of oxidative processes being involved both in a deamination rephosphate (6). 2. Transamination with a keto acid by action and the concomitant metabolism glutamine transaminase (L-glutamine : 2- of the carbon skeleton. This paper preoxoacid aminotransferase, E. C. 2.6.1.15)) sents results which implicate oxidative to form an amino acid and cy-ketoglu- processes in the production of ammonia taramate, with hydrolysis of this keto- from glutamine by kidney mitochondria amide to ammonia and cu-ketoglutarate by and which characterize glutamine as a an w-amidase (w-amidodicarboxylate ami- rapidly oxidizable substrate for these dohydrolase, E.C. 3.5.1.3.), the system of mitochondria. transamination followed by amide hydrolyMATERIALS AND METHODS sis being known as glutaminase II: L-Glutamine
+ keto acid = a-ketoglutaramate + amino acid.
a-Ketoglutaramate
+ Hz0 4 a-ketoglutarate
+ ammonia.
Reagents. L-Glutamine was obtained from Mann Research Laboratories, New York; L-glutamic acid and a-ketoglutaric acid from British Drug Houses Ltd., Dorset; L-malic acid from Nutritional Biochemicals Corporation, Cleveland, Ohio; yeast hexo718
MITOCHONDRIAL
METABOLISM
kinase and ATP from Sigma Chemical Co., St. Louis, Missouri, and ADP from C. F. Boehringer and Soehne G.m.b.H. Mannheim, Germany. All other reagents were of A.R. grade. The yeast hexokinase was freed from ammonia by dialysis against 0.3 M glucose (10). Preparation of mitochondria. Mitochondrial suspensions from rat liver and kidney were prepared as previously described (10). Incubation of mitochondria. The amperometric experiments were carried out at 30” (11). Other incubations were carried out at 38” in 25.ml Erlenmeyer flasks and in Warburg flasks attached to manometers (the aerobic experiments) or to an oxygen-free nitrogen manifold (the anaerobic experiments). The incubation mixtures were as previously described (10). Estimation of ammonia. Ammonia was estimated as described previously (10). Certain corrections were applied when glutamine was present during the Conway distillation. Under the conditions of this distillation, (90 minutes at 30” in half-saturated K2COa), glutamine was shown to decompose to the extent of 5 mole f( to ammonia and, presumably to pyrrolidone carboxylate. This rate of decomposition was not affected by glutamine concentration between 0 and 10 mM, therefore it was possible by use of simple equations, to correct for it between upper and lower limits. These limits would, of course, be dependent on the form of the nitrogen remaining (glutamate plus aspartate or as glutamine) and would be at a maximum (5”r) at low levels of ammonia production. Actual corrections for glutamine metabolism have been made at an average of the upper and lower values. Estimation of protein. Mitochondrial protein was prepared for estimation according to the method of Aldridge (12). Ethanol : water : ether (9: 6: 5) 10 ml, was added to 0.30 ml mitochondrial suspension, mixed well and allowed to stand until the protein had flocculated. The protein was recovered by centrifugation and dissolved in a suitable volume of M NaOH. The concentration of protein was then measured using the method of Ellman bovine serum albumin as a (13), with crystalline standard.
the addition of glutamate gives a marked increase in the rate of oxygen consumption. The observations presented in Figs. 1 and 2 are consistent, therefore, with the oxidation of glutamine being dependdent on its prior conversion to glutamate. In contrast, however, Fig. 3 shows that the rate of oxygen uptake of rat kidwhich are oxidizing ney mitochondria, (at saturating concentration) glutamate (10 mM) is substantially increased by the addition of glutamine. A qualitatively similar picture is obtained with kidney mitochondria if the order of the two substrates is reversed; i.e., the rate of oxygen uptake is substantially increased if glutamate is added when the system is already oxidizing a saturating glutamine The kidney mitochonconcentration. drial system apparently possesses independent systems for the oxidation of glutamate and of glutamine. Heart mitochondria in the presence of ADP and inorganic phosphate do not give any evidence for the oxidation of glutamine and preparations of brain mitochondria resemble those from liver. Kidney mitochondria oxidize glutaMITOCHONDRIA 1
+ ADP (1.6~
moles)
0.46
( IOmM)
RESULTS
Oxidation of glutamate and glutamine. Figure 1 indicates that the addition of Lglutamine to rat liver mitochondria, which are already oxidizing L-glutamate, does not alter significantly the rate of oxygen uptake by the system. Figure 2 shows that when glutamine is added first there is a delay before a steady rate of oxidation is reached and that at this stage
719
OF GLUTAMINE
0
I
1
I
I
I
2
3
4
TIME
(min.)
FIG. 1. Oxygen uptake of a rat liver mitochondrial preparation in the presence of excess ADP and after the addition of glutamate to 10 mM and then of glutamine to 10 mM. Incubation mixture of 30 mM sucrose, 21 mM KCl, 2.5 mM MgCb, 1.2 mM EDTA, 33 mM Tris-HCl, 40 mM phosphate, pH 7.4; 3 mg mitochondrial protein, 30”.
720
HIRD AND MARGINSON ~ITOCHONORIA
+ AOP
(1.6)~
moles)
0.46
0
0
2
I
3
4
TIME
bnin.1
5
6
7
FIG. 2. Oxygen uptake of a rat liver mitochondrial preparation in the presence of excess ADP and after the addition of glutamine (10 mM) and then of glutamate (10 mM). Incubation mixture as for Fig. 1; 3 mg mitochondrial protein, 30”. MITOCHONDRIA
+ADP
(1.6)rmoles)
1
0.46
L-GLU
t-i
(IOmM)
1 L-GLU
0
(NH~)
(10mM)
L
I
I
I
I
I
0
I
2
3
4
5
TIME
(min.)
FIG. 3. Oxygen
uptake of a rat kidney mitochondrial preparation in the presence of excess ADP and after the addition of glutamate (10 mM) followed by glutamine (10 mM). Incubation mixture as for Fig. 1; 2.3 mg mitochondrial protein, 30”.
mine and glutamate to give ADP/O ratios of between 2.8-3.3; the oxidation of glutamine also is dependent on phosphorylation of ADP and this characterizes it to be not of the simple L-amino acid oxidase type. The difference between liver and kidney mitochondria is further emphasized
by the results shown in Table I. In these long term experiments, the oxygen uptake was followed manometrically at 38”. Under the conditions of the experiment the oxygen uptake in the presence of both glutamate and glutamine was linear. The rate of oxygen uptake of kidney mitochondria in the presence of glutamine is more than twice that of kidney mitochondria in the presence of glutamate. It is apparent that glutamine is an extremely good substrate for kidney mitochondria. These long-term experiments also show that glutamine is oxidized very slowly by liver mitochondria. From a large number of experiments (amperometric) with closely agreeing figures, the average rates of oxygen consumption by kidney mitochondria (in mpmole OJmg protein/min) were 60, 133, 145, and 148 at concentrations of glutamine of 1, 2, 5, and 10 m&q respectively. The system is therefore close to being saturated at 2 mM glutamine. Related figures obtained for glutamate in the kidney systems were 17, 53, and 55 for 1, 5, and 10 mM, respectively. The long-term manometric experiments (Table II) also support the short term amperometric experiments (Fig. 3) in that they show that the addition of glutamate to a kidney system saturated with glutamine, brings about an increase in oxygen uptake. Formation of ammonia from glutamate and glutamine. Mitochondria from liver produced less ammonia from glutamine than from glutamate and as the amount was not substantially different in the absence of oxygen, it is probable that simple hydrolysis of glutamine accounts for the ammonia produced. The behaviour of kidney mitochondria was notably difthey produced about ferent . Although the same amount of ammonia from glutamate as did liver mitochondria, they produced 22 pmole of ammonia from 15 pmole of glutamine. This high level of ammonia production was substantially lowered in the absence of oxygen. HOWever, the anaerobic deamination of glutamine by kidney mitochondria is very high when compared with the anaerobic
MITOCHONDRIAL
METABOLISM TABLE
FORM~TIOW OF AMMONI.~ DURING BY I~AT
Rate of oxygen uptake in m~mole 02/mg protein/min
Air Air Air NY
Endogenous Glutamate (5 mM) Glutamine (5 mM) Glutamine (5 m&f) protein,
2.4 mg/flask;
TABLE
II
incubation
OXYGEN UPTIKE ;\ND AMMONII PRODUCTION By KIDNEY MITOCHOXDRI.\ OXIDIZING GLUT-MINE, GLUT~~.MATE .IND GLC-TAMINIC PLUS GLUT.W.ITF:
The incubation was for GO min at 38” with 1.1 mg mitochondrial protein; all rates of oxygen lIptake remained linear. The rates of ammonia prodrlced were not linear in the lat,ter part of the experimental period. Incltbation mixtures as for -Fig. 5. Rate of oxygen Ammonia produced (rmole by uptake 3 ml reac(mrmole/mg tion mixture) proteinjmin)
Substrate
Endogenous 5 mM gllltamine 10 mM glutamine 5 my glutamate 10 rnhl gllltamate 5 rn>f glutamine glutamate 5 mM glutamine rnM glrltamate
I
OXID:ITION OF L-GLUT:~MSTE .~ND KIDNEY MITOCHONDRI~~
Gas phase
Substrate
a Mitochondrial
LIVER
+ 5 rnM
0 143 145 33 55 If.50
0.06 14.5 18.8 0.63 1.04 11.4
+ 10
IF0
9.6
deamination of glutamine by liver mitochondria or when compared with the aerobic deamination of glutamate by mitochondria from either tissue. It is clear that a substantial proportion of the ammonia derived from incubating glutamine with kidney mitochondria must reflect oxidative processes. Not only did removal of oxygen from the system cause a marked fall in ammonia production, but the actual amount of ammonia produced was 7 pmole greater than the maximum which can be derived from amide hydrolysis of glutamine. As this amount of ammonia is five times that produced from the amino group of glutamate, it is clear that processes are operating
721
OF GLUTAMINE
AND
L-GLUT.IMINE
Ammonia formation rmole/‘3 ml after 40 min incubation at 38”
Liver
Kidney
Lirer
Kidney
2.3 33 2.6
5.7 G7 152
0.06 1.11 0.60 0.63
0.12 1.40 22.4 8.3
mixtures
as for Fig. 5.
which differ from the simple oxidation of glutamate which has been derived from glutamine. In an attempt to compare further the deamination of glutamate with that of glutamine, the effect of adding malonate and glutamate to the system was studied. It has previously been shown that the deamination of glutamate by liver mitochondria is greatly stimulated by malonate and abolished by adding a source of oxaloacetate (14, 15). With kidney mitochondria, malonate was shown to have either no effect or to stimulate slightly the rate of ammonia formation from both glutamine and glutamate after about 2030 min incubation. The addition of malonate, however, diminished the oxygen uptake by about 50 and 709; in the case of glutamine and glutamate, respectively. The joint observations show that ammonia production by kidney mitochondria from glutamine is malonate insensitive and that part of the oxidative metabolism of the carbon skeleton (presumably through succinate) is malonate sensitive. The addition of glutamate lowers the aerobic production of ammonia from glutamine (see Table II). The site of inhibition is not known. The relation between ammonia production and oxygen uptake. Although glutamine concentration after incubation was not directly determined it proved possible to form some estimate of glutamine utilization from the ammonia production in long term experiments with low concentrations of glutamine (l-2 mM). Figure 4 is a time-progress
722
HIRD
0
25
50 TIME
75
AND
100
(min:)
FIG. 4. Oxidation of glutamine by rat kidney mitochondria, showing ammonia production (A) and the oxygen uptake which paralleled it ( A). Incubation mixtures as for Fig. 1 and in addition Lglutamine (2 mM), ATP (mM), glucose (23 mM), hexokinase in excess of 100 K.M. units, 2.8 mg mitochondrial protein, 38”, final volume 3.0 ml.
curve for ammonia production from 2 mM glutamine compared with the oxygen uptake by the same mitochondrial preparation. At the end of the experimental period, the mitochondria had taken up 40 patom oxygen and had produced 10.6 pmole ammonia from the addition of 6 pmole glutamine. This figure indicates that between 4.6 (assuming 6 pmole of amide removed hydrolytically) and 6.0 pmole glutamine had been “oxidatively” removed. The ratio of ratom oxygen taken up to pmole glutamine oxidized was therefore in the range of 8.7-6.7. Assuming that the oxidation of glutamine, like that of glutamate proceeds to either oxaloacetate or aspartate, this ratio should be between 3 and 4. Expressed in another way, in this experiment a maximum of 1.4 rmole of aspartate and 4.6 pmole of oxaloacetate could have been produced as end products of glutamine oxidation. As this would indicate a maximum oxygen uptake of 22.6 patom, a minimum of 17.4
MARGINSON
patom of oxygen uptake remains unaccounted for. To investigate this anomaly, the oxidation of glutamate (mM), glutamine (mM), and malate (mM) by kidney mitochondria was compared. The ratio of patom oxygen taken up in 50 min to rmole substrate added was 3.0 for glutamate, 6.7 for glutamine and 8.0 for malate. The glutamate figure is consistent with the reaction glutamate + aspartate with little further metabolism of the aspartate so formed. However, the very high figures for glutamine and malate suggest the further utilization of oxaloacetate either to form pyruvate or as a mediator for the metabolism of endogenous precursors of acetyl CoA. This observation suggests that glutamate may not be an intermediate in the oxidation of glutamine. The influence of wketoglutarate on ammonia production. Since glutaminase II has been reported to be present in both mitochondria and cell sap (89, it is conceivable that the rapid oxidation of glutamine could be due to production of a-ketoglutarate which would act as the amino group acceptor for the glutaminase II system. If this system were responsible for the initial steps of glutamine oxidation, then it would be expected that addition of a keto acid, for example LYto mitochondria in the ketoglutarate, presence of glutamine would, under anmarkedly stimulate aerobic conditions, the production of ammonia. The results of such an experiment are shown in Fig. 5. Consistent with the results presented in Table I, the production of ammonia from glutamine was markedly diminished under anaerobic conditions. When CYketoglutarate is added (l-10 mM) there is a further marked diminution in ammonia production. Although there is some indication of a restoration of the anaerobic level at high concentrations of cu-ketoglutarate (20 mM), these observations are not compatible with a glutaminase II system being active in the mitochondrial preparation at kidney
MITOCHONDRIAL 20
A.
‘.‘\‘\ ‘\
AEROBIC
‘1
16
METABOLISM
‘\ ‘\ f to - m
‘\
‘\
12
‘-A ‘\
5 n
;
‘\
li
4
‘.
;> ANAEROBIC
o0
5
IO
CONCENTRATION d-KETOGLUTARATE
I5
20
OF (mM
1
FIG. 5. Effect
of a-ketoglutarate concentration on the aerobic and anaerobic production of ammonia from glutamine by rat kidney mitochondria. Incubation mixtures as for Fig. 4 except that glutamine concentration was 5 mM and each flask contained 3 mg mitochondrial protein. The anaerobic incubations were carried out in flasks attached to an oxygen-free nitrogen manifold.
concentrations of cY-ketoglutarate which are likely to obtain in uiuo. The marked effect of low concentrations of cY-ketoglutarate in depressing ammonia formation is of interest and could be attributed to a Krebs-Cohen dismutation (16): 2 a-Ketoglutarate
+ NH< -
glutamate + succinate + Con.
DISCUSSION
The rate of oxidation of glutamate and of glutamine by mitochondria prepared from kidney tissue is very much greater than the rate obtained with liver mitochondria; in the case of L-glutamine, the ratio is approximately 60 : 1. Ammonia production from glutamine is also much greater ( X 37) with kidney mitochondria. It is clear that there are great differences between the mitochondria from these tissues and that glutamine is potentially a major substrate for kidney tissue. From the results obtained, no simple interpretation of the relative importance of the various pathways of oxidation or of
OF GLUTAMINE
723
ammonia production is possible. It does appear however that glutamine and glutamate are metabolized independently. No evidence was found which would support prior transamination of glutamine with subsequent hydrolysis of the a-ketoglutaramate (glutaminase II) as a major method of mitochondrial oxidation of glutamine. A dehydrogenation of glutamine which removed the a-amino group formand ammonia ing a-ketoglutaramate therefore arose as a possibility. However, direct evidence supporting such an oxidative step has not yet been In preliminary experiments, obtained. sonically disrupted mitochondria prepared from rat kidneys did not give any evidence spectroscopically of a reduction of NAD or of NADP in the presence of glutamine. On the other hand, NAD was reduced in the presence of glutamate indicating the survival of glutamate dehydrogenase (E.C. 1.4.1.3) in the preparation. The observed differences between the rates of oxidation of glutamate and of glutamine by intact mitochondria are perhaps explicable in terms of permeability but in amperometric experiments with kidney mitochondria there is an almost “instantaneous” response in rate of oxygen uptake to the addition of small increments of glutamate or of glutamine in the presence of excess ADP. However, there still remains the possibility that the metabolism of glutamine is taking place by the known hydrolytic and oxidative pathways via glutamate, but that glutamate liberated within a certain mitochondrial compartment, readily accessible to glutamine but not to glutamate, is oxidized very much more readily than, and separately from, glutamate in other parts of the mitochondrion. Another alternative, not yet explored, is that there is more than one population of mitochondria involved in the kidney preparations. We have, however, no observations which would help evaluate an explanation in these terms. Pitts and co-workers (17, 18) have studied glutamine metabolism in the kid-
724
HIRD
AND
neys of acidotic dogs and their experiments have clearly shown that in uivo there is a substantial conversion of both the amide and the amino nitrogen to ammonia, with the former predominating. Although these results were obtained with acidotic animals, they clearly establish the significance of glutamine nitrogen in the maintenance of acid-base balance in the animal, In addition, attention has recently been drawn (19, 20) to the possibility that glutamine carbon atoms may play a role in kidney gluconeogenesis. The results presented in the present paper would indicate that L-glutamine is a major substrate for isolated kidney mitochondria and further serve to emphasize the potential importance of glutamine in kidney metabolism. ACKNOWLEDGMENTS We thank I. W. D. Croker for valuable assistance and the Australian Research Grants Committee for financial support. REFERENCES 1. MEISTER, A. Physiol. Reu. 36, 103 (1956). 2. ERRERA, M. J. Biol. Chem. 178, 483 (1949).
MARGINSON 3. ERRERA, M. AND GREENSTEIN, J. P. J. Biol. Chem. 178, 495 (1949). 4. SHEPHERD, J. A. AND KALNITSKY, G. J. Biol. &em. 192, l(l951). 5. CARTER, C. E. AND GREENSTEIN, J. P. J. Nat. Cancer Inst. 7, 433 (1947). 6. KLINGMAN, J. D. AND HANDLER, P. J. Biol. Chem. 232, 369 (1958). 7. PRICE, V. E. AND GREENSTEIN, J. P. J. Nat. Cuncer h-t. 7, 275 (1947). 8. KATUNUMA, N., HUZINO, A. AND TOMINO, I. Aduan. Enzyme Regulation 5, 55 (1967). 9. MEISTER, A. J. Biol. Chem. 200, 571 (1953). 10. HIRD, F. J. R. AND MARGINSON, M. A. Arch. Biochem. Biophys. 115, 247 (1966). 11. HIRD, F. J. R. AND WEIDEMANN, M. J. Biohem. J. 98, 378 (1966). 12. ALDRIDGE, W. N. Biochem. J. 67, 423 (1957). 13. ELLMAN, G. L. Anal. Biochem. 3,40 (1962). 14. Hmn, F. J. R. AND MARGINSON, M. A. Nature 201, 1224 (1964). 15. HIRD, F. J. R. AND MORTON, D. J. Biochim. Biophys. Acta 85, 353 (1964). 16. KREBS, H. A. AND COHEN, P. P. Biochem. J. 33, 1895 (1939). 17. PITTS, R. F. The Physiologist 9, 97 (1966). 18. STONE, W. J. AND Prrrs, R. F. J. Clin. Invest. 46, 1141 (1967). 19. GOODMAN, A. D., Fusz, R. E. AND CAHILL, G. F. J. Clin. Inuest. 45, 612 (1966). 20. LOTSPEICH, W. D. Science 155, 1066 (1967).